專利名稱：篩選在瘧原蟲中抑制gpi生物合成的化合物的方法
技術(shù)領(lǐng)域：
本發(fā)明涉及篩選在瘧原蟲中抑制GPI生物合成的化合物的方法。
背景技術(shù)：
瘧疾是由寄生性原蟲引起的最常見(jiàn)的傳染性人類疾病。該疾病由于感染瘧原蟲所致，以蚊子，岡比亞瘧紋(Anopheles gambiae)為媒介。每年約有50億病例感染瘧疾，包括超過(guò)兩百萬(wàn)的致死例(Gardner等，Nature419498-511，2003)。目前，40％的世界人口生活在瘧疾流行區(qū)，在該區(qū)域據(jù)說(shuō)每3名新生兒中就有一名死于瘧疾。
已知糖基磷脂酰肌醇(GPI)在寄生蟲生長(zhǎng)和感染過(guò)程中起著重要的作用，所述寄生蟲包括瘧原蟲。在這些寄生蟲的細(xì)胞表面上有許多GPI-錨定蛋白。GPI-錨定蛋白包括如在寄生蟲侵入紅細(xì)胞時(shí)起作用的MSP-1蛋白。GPI-錨定蛋白起寄生蟲抗原的作用，在宿主中引發(fā)免疫應(yīng)答。因此，它們長(zhǎng)期以來(lái)是疫苗開(kāi)發(fā)的研究對(duì)象。
GPI不僅作為將蛋白拴到細(xì)胞膜的錨，而且是瘧原蟲細(xì)胞膜的豐富糖脂成分。最近的研究揭示GPI有毒，并能促成致死癥狀。GPI誘導(dǎo)炎性細(xì)胞因子如TNF-α和粘附分子的表達(dá)。結(jié)果，受染紅細(xì)胞粘附毛細(xì)管，阻塞血管(隔離作用(sequestration))，尤其是腦血管。它能引起進(jìn)一步的炎癥反應(yīng)，而炎癥反應(yīng)認(rèn)為能導(dǎo)致腦病。最近，Schofield等報(bào)道抗-GPI抗體降低利用鼠科瘧原蟲伯氏瘧原蟲(Plasmodium berghei)創(chuàng)建的體內(nèi)感染模型系統(tǒng)的致死率，并且該抗體在體外抑制由惡性瘧原蟲(Plasmodium falciparum)引起的后期炎癥反應(yīng)(Schofield L等，Nature 418785-789，2002)。此發(fā)現(xiàn)說(shuō)明GPI是瘧疾感染致死率的主要因素。
還報(bào)道肌醇?；瘜?duì)于甘露糖與GPI的結(jié)合來(lái)說(shuō)是必需的(Gerold，P.等，Biochem.J.344731-738，1999)，由過(guò)量葡糖胺引起的肌醇?；囊种茣?huì)抑制惡性瘧原蟲的成熟(Naik，R.S.等，J.Biol.Chem.2782036-2042，2003)。因此，選擇地抑制GPI生物合成尤其是肌醇?；幕衔锟梢允欠浅Ｓ杏玫目汞懰幬?。
發(fā)明的公開(kāi)本發(fā)明的目的是提供抑制GPI生物合成的抗瘧藥物。更具體地說(shuō)，本發(fā)明提供了編碼GWT1蛋白的瘧原蟲DNA，所述蛋白是參與GPI生物合成的蛋白質(zhì)(GPI合酶)。本發(fā)明還提供了在篩選抗瘧藥物的方法中利用該DNA的方法。本發(fā)明還提供了編碼瘧原蟲GPI生物合成蛋白的DNA的簡(jiǎn)并突變體DNA。該簡(jiǎn)并突變體DNA比天然DNA具有更低的AT含量。本發(fā)明還提供了在篩選抗瘧藥物的方法中利用該簡(jiǎn)并突變體DNA的方法。
最早發(fā)現(xiàn)GWT1基因編碼真菌GPI-錨定蛋白合酶(WO 02/04626)，其在從酵母到人類的生物體中是保守的。本發(fā)明人證實(shí)GWT1同源物(惡性瘧原蟲(P.falciparum)GWT1的PfGWT1；約氏瘧原蟲(P.yoelii yoelii)GWT1的PyGWT1)包括在惡性瘧原蟲和約氏瘧原蟲的全基因組序列內(nèi)(Gardner等，Nature 419498-511，2003；Carlton等，Nature 419512-519，2003)。本發(fā)明人還發(fā)現(xiàn)GWT1基因產(chǎn)物在GPI生物合成途徑中起GlcN-PI?；D(zhuǎn)移酶的作用。PfGWT1基因產(chǎn)物期望具有相似的活性，這樣抑制其活性的化合物就可能是有價(jià)值的抗瘧藥物。
在WO 02/04626中，本發(fā)明人發(fā)現(xiàn)了一組抑制真菌GWT1基因產(chǎn)物的活性的化合物。抑制PfGWT1基因產(chǎn)物的活性的化合物有望成為抗瘧藥物。
在本發(fā)明中，本發(fā)明人成功地對(duì)幾乎是PfGWT1全長(zhǎng)的區(qū)域進(jìn)行了分離。利用瘧原蟲如惡性瘧原蟲的GWT1基因產(chǎn)物，抗瘧藥物的篩選可以通過(guò)結(jié)合試驗(yàn)，葡糖胺(酰基)磷脂酰肌醇(PI-GlcN)?；D(zhuǎn)移酶試驗(yàn)，或利用GPI-錨定蛋白檢測(cè)系統(tǒng)來(lái)進(jìn)行。從所述篩選方法獲得的化合物可能是有價(jià)值的抗瘧藥物。更進(jìn)一步地，本發(fā)明人揭示簡(jiǎn)并突變體DNA(編碼瘧原蟲GPI生物合成蛋白的DNA簡(jiǎn)并突變體)，該DNA比天然DNA具有更低AT含量，補(bǔ)償GWT1基因-缺陷型真菌的表型。因此，利用GPI合酶基因-缺陷型真菌的表型作為指標(biāo)，該真菌中導(dǎo)入了(比瘧原蟲中編碼GPI生物合成蛋白的DNA)具有更低AT含量的簡(jiǎn)并突變體DNA，就有可能篩選出抑制在瘧原蟲中參與GPI生物合成的蛋白活性的化合物。
更具體地，本發(fā)明提供了下述[1]到[25][1](a)到(d)中任一項(xiàng)的DNA，該DNA編碼具有GlcN-PI?；D(zhuǎn)移酶活性的瘧原蟲蛋白。
(a)編碼包含SEQ ID NO2或4所示氨基酸序列的蛋白的DNA，(b)包含SEQ ID NO1或3所示核苷酸序列的DNA，(c)在嚴(yán)謹(jǐn)條件與包含SEQ ID NO1或3所示核苷酸序列的DNA雜交的DNA，和(d)編碼包含SEQ ID NO2或4所示氨基酸序列，且其中已添加、缺失、取代和/或插入一個(gè)或多個(gè)氨基酸所得的蛋白的DNA；[2]由項(xiàng)[1]的DNA編碼的蛋白；[3]插入項(xiàng)[1]的DNA的載體；[4]在可表達(dá)狀態(tài)包含項(xiàng)[1]的DNA或者項(xiàng)[3]的載體的轉(zhuǎn)化體；[5]一種抗瘧藥物，其包含抑制項(xiàng)[2]的蛋白的活性的化合物作為活性成分；[6]項(xiàng)[5]的抗瘧藥物，其中抑制項(xiàng)[2]的蛋白的活性的化合物是選自下述化合物(1)-(5)中的至少一種化合物
和 [7]一種篩選具有抗瘧活性的化合物的方法，其包括下述步驟(1)使項(xiàng)[2]的蛋白與試樣和具有與該蛋白結(jié)合的活性的標(biāo)記化合物接觸，(2)檢測(cè)與該蛋白結(jié)合的標(biāo)記化合物，和(3)選出減少與該蛋白結(jié)合的標(biāo)記化合物的量的試樣；[8]項(xiàng)[7]的方法，其中具有與蛋白結(jié)合的活性的標(biāo)記化合物是通過(guò)對(duì)選自項(xiàng)[6]的化合物(1)-(5)中的至少一種進(jìn)行標(biāo)記而獲得的；[9]一種篩選具有抗瘧活性的化合物的方法，其包括下述步驟(1)使試樣與項(xiàng)[2]的蛋白接觸，(2)檢測(cè)GlcN-(酰基)PI，和(3)選出減少GlcN-(?；?PI水平的試樣；[10]一種篩選具有抗瘧活性的化合物的方法，其包括下述步驟(1)使試樣與過(guò)量表達(dá)項(xiàng)[2]的蛋白的細(xì)胞接觸，(2)測(cè)定GPI-錨定蛋白轉(zhuǎn)運(yùn)到該細(xì)胞的細(xì)胞壁的量，和
(3)選出減少步驟(2)中所測(cè)定的GPI-錨定蛋白轉(zhuǎn)運(yùn)到該細(xì)胞的細(xì)胞壁的量的試樣；[11]一種治療瘧疾的方法，包括施用抑制項(xiàng)[2]的蛋白的活性的化合物；[12]項(xiàng)[11]的方法，其中抑制項(xiàng)[2]的蛋白的活性的化合物是項(xiàng)[5]的化合物；[13]一種編碼具有補(bǔ)償GPI合酶基因-缺陷型酵母表型的活性的蛋白的DNA，該DNA是編碼在瘧原蟲中參與GPI生物合成的蛋白的DNA的簡(jiǎn)并突變體，其比天然DNA具有更低的AT含量；[14]一種編碼具有補(bǔ)償GPI合酶基因-缺陷型酵母表型的活性的蛋白的DNA，該DNA是編碼在瘧原蟲中參與GPI生物合成的蛋白的DNA的簡(jiǎn)并突變體，其具有少于70％的AT含量；[15]項(xiàng)[13]或[14]的DNA，其中該DNA選自下組(a)編碼包含SEQ ID NOs2和4以及SEQ ID NOs6-47中奇數(shù)序列鑒定號(hào)中任一所示氨基酸序列的蛋白的DNA，(b)包含SEQ ID NOs1和3以及SEQ ID NOs6-47中偶數(shù)序列鑒定號(hào)中任一所示核苷酸序列的DNA，(c)在嚴(yán)謹(jǐn)條件下與包含SEQ ID NOs1和3以及SEQ ID NOs6-47中偶數(shù)序列鑒定號(hào)中任一所示核苷酸序列的DNA雜交的DNA，和(d)編碼包含SEQ ID NOs2和4以及SEQ ID NOs6-47中奇數(shù)序列鑒定號(hào)中任一所示氨基酸序列，且在所述氨基酸序列中添加，缺失，取代和/或插入一個(gè)或多個(gè)氨基酸而得到的蛋白的DNA；[16]包含核苷酸序列SEQ ID NO5的DNA；[17]一種載體，其中插入項(xiàng)[13]-[16]中任一的DNA；[18]在可表達(dá)狀態(tài)包含項(xiàng)[13]-[16]中任一DNA或者項(xiàng)[17]的載體的轉(zhuǎn)化體；[19]項(xiàng)[18]的轉(zhuǎn)化體，其為GPI合酶基因-缺陷型真菌；[20]項(xiàng)[18]的轉(zhuǎn)化體，其為GPI合酶基因-缺陷型酵母；[21]一種制備由項(xiàng)[13]-[16]中任一DNA編碼的蛋白的方法，該方法包括培養(yǎng)項(xiàng)[18]-[20]中的任一轉(zhuǎn)化體，并從該轉(zhuǎn)化體或其培養(yǎng)上清液收集表達(dá)的蛋白的步驟；[22]一種篩選具有抗瘧活性的化合物的方法，該方法包括以下步驟
(1)將試樣與表達(dá)項(xiàng)[13]-[16]中任一DNA的GPI合酶基因-缺陷型真菌接觸；(2)分析該真菌的生長(zhǎng)，和(3)選出抑制該真菌生長(zhǎng)的被試化合物；[23]一種篩選具有抗瘧活性的化合物的方法，該方法包括以下步驟(1)將試樣與表達(dá)項(xiàng)[13]-[16]中任一DNA的GPI合酶基因-缺陷型真菌接觸，(2)檢測(cè)GPI-錨定蛋白轉(zhuǎn)運(yùn)到真菌細(xì)胞壁的量，和(3)選出使步驟(2)中所檢測(cè)的GPI-錨定蛋白轉(zhuǎn)運(yùn)到真菌細(xì)胞壁的量減少的試樣，[24]一種篩選具有抗瘧活性的化合物的方法，該方法包括以下步驟(1)將項(xiàng)[13]-[16]中任一DNA導(dǎo)入GPI合酶基因-缺陷型真菌，并使該DNA編碼的蛋白表達(dá)，(2)獲得步驟(1)中表達(dá)的蛋白，(3)使所得蛋白與試樣和具有與該蛋白結(jié)合的活性的標(biāo)記化合物接觸，(4)檢測(cè)與該蛋白結(jié)合的標(biāo)記化合物，和(5)選出減少與該蛋白結(jié)合的標(biāo)記化合物的量的試樣；和[25]一種篩選具有抗瘧活性的化合物的方法，該方法包括以下步驟(1)將(i)編碼具有補(bǔ)償GWT1-缺陷型酵母表型的活性的蛋白的DNA，其中所述DNA是編碼瘧原蟲GWT1蛋白的DNA的簡(jiǎn)并突變體，該DNA比天然DNA具有更低的AT含量，或者(ii)插入DNA簡(jiǎn)并突變體且表達(dá)由該簡(jiǎn)并突變體DNA編碼的蛋白的載體，導(dǎo)入GWT1-缺陷型真菌，(2)獲得步驟(1)中表達(dá)的蛋白，(3)使所得蛋白與試樣接觸，(4)檢測(cè)GlcN-(?；?PI，和(5)選出減少GlcN-(酰基)PI水平的被試化合物。
本發(fā)明第一次分離出了編碼惡性瘧原蟲GWT1蛋白的DNA(PfGWT1)。編碼PfGWT1蛋白的DNA的核苷酸序列示于SEQ ID NO1，PfGWT1蛋白的氨基酸序列顯示于SEQ ID NO2。另外，編碼間日瘧原蟲(Plasmodium vivax)GWT1蛋白(PvGWT1)的DNA的核苷酸序列示于SEQ ID NO3，PvGWT1蛋白的氨基酸序列顯示于SEQ ID NO4。
GWT1蛋白參與糖基磷脂酰肌醇(GPI)的生物合成，糖基磷脂酰肌醇對(duì)瘧原蟲的生長(zhǎng)和感染性是必需的。因此，抑制瘧原蟲GWT1蛋白活性的化合物可以用作抗瘧藥物。所述抗瘧藥物可以利用此瘧原蟲GWT1蛋白來(lái)進(jìn)行篩選。
本發(fā)明提供了編碼瘧原蟲GWT1蛋白的DNA。所述DNA包括編碼包含SEQ ID NO2或4氨基酸序列的蛋白的DNA，和包含SEQ ID NO1或3核苷酸序列的DNA。
本發(fā)明還提供了與包含SEQ ID NO2或4氨基酸序列的蛋白功能等同的蛋白的編碼DNA。在此，表述“功能等同”是指具有與目的蛋白的生物特性相同的生物特性，所述目的蛋白包含氨基酸序列SEQ ID NO2或4(PfGWT1或PvGWT1蛋白)。PfGWT1和PvGWT1蛋白的生物特性包括GlcN-PI?；D(zhuǎn)移酶活性。GlcN-PI?；D(zhuǎn)移酶活性可采用Costello和Orlean(J.Biol.Chem.(1992)2678599-8603)或Franzot和Doering(Biochem.J.(1999)34025-32)所報(bào)道的方法來(lái)進(jìn)行測(cè)定。
與包含SEQ ID NO2或4氨基酸序列的蛋白功能等同的蛋白的編碼DNA包括在嚴(yán)謹(jǐn)條件中與包含SEQ ID NO1或3核苷酸序列的DNA雜交的DNA，和編碼包含SEQ ID NO2或4氨基酸序列的蛋白的DNA，所述氨基酸中添加、缺失、取代和/或插入一個(gè)或多個(gè)氨基酸。
本發(fā)明的DNA可以采用本領(lǐng)域技術(shù)人員所熟知的方法來(lái)進(jìn)行分離。所述方法的實(shí)例包括利用雜交(Southern E.M.，J.Mol.Biol.98503-517，1975)和聚合酶鏈?zhǔn)椒磻?yīng)(PCR)(Saiki R.K.等，Science 2301350-1354，1985；SaikiR.K.等，Science 239487-491，1988)。更具體地，對(duì)于本領(lǐng)域技術(shù)人員來(lái)說(shuō)，利用SEQ ID NO1或3的DNA或其部分作為探針，或通過(guò)利用與包含SEQID NO1或3核苷酸序列的DNA特異性雜交的DNA作為引物，從瘧原蟲中分離出與包含SEQ ID NO1或3核苷酸序列的DNA高度同源的DNA是常規(guī)實(shí)驗(yàn)。另外，通過(guò)雜交或PCR技術(shù)分離出的DNA，以及與包含SEQ IDNO1或3核苷酸序列的DNA雜交的DNA也包括在本發(fā)明的DNA之中。所述DNA包括含有SEQ ID NO2或4氨基酸序列的蛋白的瘧原蟲同源物的編碼DNA。瘧原蟲同源物包括惡性瘧原蟲，間日瘧原蟲，三日瘧原蟲(Plasmodium malariae)和卵形瘧原蟲(Plasmodium ovale)同源物，其包含SEQID NO2或4的氨基酸序列。
優(yōu)選，在嚴(yán)謹(jǐn)雜交條件利用雜交反應(yīng)分離出上述DNA。如本文所用，表達(dá)“嚴(yán)謹(jǐn)雜交條件”是指，例如在65℃于4xSSC中進(jìn)行雜交，然后在65℃于0.1xSSC中洗滌1小時(shí)?？蛇x的嚴(yán)謹(jǐn)條件是在42℃于含50％甲酰胺的4xSSC中進(jìn)行雜交?；蛘?，在65℃于PerfectHybTM(TOYOBO)溶液中雜交2.5小時(shí)，然后如下洗滌(1)在25℃，于含0.05％SDS的2xSSC中洗滌5分鐘；(2)在25，于含0.05％SDS的2xSSC中洗滌15分鐘；及(3)在50℃，于含0.1％SDS的0.1x SSC中洗滌20分鐘。如此分離的DNA有望編碼與SEQ ID NO2或4氨基酸序列在氨基酸水平具有高度同源性的多肽。在此，“高度同源性”是指與完整氨基酸序列有至少70％或更高的序列同一性，優(yōu)選80％或更高，更優(yōu)選90％或更高，最優(yōu)選95％或更高。
氨基酸序列水平或核苷酸序列水平的同一性程度可以采用Karlin和Altschul的BLAST算法(Karlin S.和Altschul S.F，Proc.Natl.Acad.Sci.USA.872264-2268，1990；Karlin S.和Altschul S.F，Proc.Natl.Acad.Sci.USA.905873-5877，1993)來(lái)確定。已開(kāi)發(fā)了基于BLAST算法的程序(稱為BLASTN和BLASTX)(Altschul S.F.等，J.Mol.Biol.215403，1990)。當(dāng)利用BLASTN對(duì)核苷酸序列進(jìn)行分析時(shí)，例如，將參數(shù)設(shè)為得分＝1 00，字長(zhǎng)＝12。另外，當(dāng)利用BLASTX對(duì)氨基酸序列進(jìn)行分析時(shí)，例如，將參數(shù)設(shè)為得分＝50，字長(zhǎng)＝3。當(dāng)使用BLAST和Gapped BLAST程序時(shí)，使用各程序的默認(rèn)參數(shù)。所述分析的具體步驟是已知的(請(qǐng)參見(jiàn)美國(guó)國(guó)家生物技術(shù)信息中心(NationalInstitute of Biotechnology Information)網(wǎng)站http://www.ncbi.nlm.nih.gov)。
本發(fā)明的DNA包含基因組DNA、cDNA和化學(xué)合成DNA?；蚪MDNA或cDNA可以根據(jù)本領(lǐng)域技術(shù)人員所熟知的常規(guī)方法來(lái)加以制備。例如，基因組DNA可以如下制備(i)提取瘧原蟲基因組DNA；(ii)(利用如質(zhì)粒、噬菌體、粘粒、BAC或PAC作載體)構(gòu)建基因組文庫(kù)；(iii)涂布該文庫(kù)；然后(iv)利用探針進(jìn)行菌落雜交或噬菌體雜交，所述探針是在編碼本發(fā)明的瘧原蟲GWT1蛋白的DNA(例如SEQ ID NO1或3)的基礎(chǔ)上獲得的。另外，利用編碼本發(fā)明瘧原蟲GWT1蛋白的DNA(例如SEQ ID NO1或3)的特異性引物，通過(guò)PCR制備基因組DNA。另一方面，cDNA可以如下制備(i)在提取的瘧原蟲mRNA基礎(chǔ)上合成cDNA；(ii)將該合成cDNA插入載體如λZAP中構(gòu)建cDNA文庫(kù)；(iii)涂布該cDNA文庫(kù)；和(iv)如上所述，進(jìn)行菌落雜交或噬菌體雜交。另外，也可以利用PCR制備eDNA。
本發(fā)明還提供了與包含SEQ ID NO2或4氨基酸序列的蛋白結(jié)構(gòu)相似的蛋白的編碼DNA。所述DNA包括那些編碼蛋白的核苷酸序列的DNA，所述蛋白包含其中取代，缺失，插入和/或添加一個(gè)或多個(gè)氨基酸殘基的氨基酸序列。對(duì)于上述蛋白中的氨基酸突變位點(diǎn)和數(shù)量并無(wú)限制，只要該突變蛋白仍保留原始蛋白的功能，如Mark，D.F.等，Proc.Natl.Acad.Sci.USA(1984)81，5662-5666；Zoller，M.J.&Smith，M.，Nucleic Acids Research(1982)10，6487-6500；Wang，A.等，Science 224，143 1-1433；Dalbadie-McFarland，G.等，Proc.Natl.Acad.Sci.USA(1982)79，6409-6413中所述。突變氨基酸的百分?jǐn)?shù)一般為全部氨基酸殘基的10％或更少，優(yōu)選5％或更少，更優(yōu)選1％或更少。此外，突變氨基酸的數(shù)量通常為30個(gè)氨基酸或更少，優(yōu)選15個(gè)氨基酸或更少，更優(yōu)選5個(gè)氨基酸或更少，還更優(yōu)選3個(gè)氨基酸或更少，甚至更優(yōu)選2個(gè)氨基酸或更少。
優(yōu)選的突變氨基酸殘基是在其突變(已知為保守氨基酸取代法)后保持側(cè)鏈性質(zhì)的氨基酸殘基。氨基酸側(cè)鏈性質(zhì)的例子是疏水性(A，I，L，M，F(xiàn)，P，W，Y，V)和親水性(R，D，N，C，E，Q，G，H，K，S，T)。側(cè)鏈包括脂肪族側(cè)鏈(G，A，V，L，I，P)；含羥基側(cè)鏈(S，T，Y)；含硫原子側(cè)鏈(C，M)；含羧酸和酰胺側(cè)鏈(D，N，E，Q)；堿性側(cè)鏈(R，K，H)；和芳香族側(cè)鏈(H，F(xiàn)，Y，W)。
包含瘧原蟲GWT1蛋白的融合蛋白是其中添加一個(gè)或多個(gè)氨基酸殘基的蛋白實(shí)例。融合蛋白可利用本領(lǐng)域技術(shù)人員所熟知的技術(shù)來(lái)進(jìn)行制備。例如，對(duì)此特定技術(shù)沒(méi)有限制，將編碼本發(fā)明瘧原蟲GWT1蛋白的DNA與編碼另一肽或蛋白的DNA聯(lián)合，使它們的閱讀框匹配。本發(fā)明的蛋白可以與許多已知肽形成融合蛋白。所述肽包括FLAG(Hopp，T.P.等，Biotechnology(1988)6，1204-1210)，6xHis，10xHis，流感凝集素(HA)，人c-myc片段，VSP-GP片段，p18HIV片段，T7-標(biāo)簽，HSV-標(biāo)簽，E-標(biāo)簽，SV40T抗原片段，lck標(biāo)簽，α-微管蛋白片段，B-標(biāo)簽和蛋白C(Protein C)片段。與本發(fā)明蛋白融合的蛋白例子包括谷胱甘肽-S-轉(zhuǎn)移酶(GST)，HA，免疫球蛋白恒定區(qū)，β-半乳糖苷酶和麥芽糖結(jié)合蛋白(MBP)。
除利用上述雜交和PCR技術(shù)外，本領(lǐng)域技術(shù)人員可通過(guò)下述方法制得上述DNA，該方法包括例如定點(diǎn)誘變而在該DNA中引入突變(Kramer W.and Fritz H-J.，Methods Enzymol.154350，1987)。蛋白的氨基酸序列還可以由于編碼該蛋白的核苷酸序列發(fā)生突變而使其自然突變。此外，簡(jiǎn)并突變體DNA，其核苷酸突變并不會(huì)引起蛋白中的氨基酸發(fā)生突變(簡(jiǎn)并突變體)也包括在本發(fā)明內(nèi)。另外，本發(fā)明還包括由本發(fā)明上述DNA編碼的蛋白。
本發(fā)明提供了包括本發(fā)明DNA的載體，包含本發(fā)明DNA或載體的轉(zhuǎn)化體，和利用這些轉(zhuǎn)化體制備本發(fā)明蛋白的方法。
本發(fā)明的載體并無(wú)限制，只要它能穩(wěn)定地保留插入載體內(nèi)的DNA。例如，當(dāng)大腸桿菌作宿主時(shí)，優(yōu)選用pBluescript載體(Stratagene)作為克隆載體。當(dāng)使用載體來(lái)制備本發(fā)明的蛋白時(shí)，表達(dá)載體是特別有用的。表達(dá)載體并無(wú)特別的限制，只要它能在體外，在大腸桿菌中，在培養(yǎng)細(xì)胞中和在體內(nèi)表達(dá)蛋白。表達(dá)載體的優(yōu)選例子包括用于體外表達(dá)的pBEST載體(Promega Corporation)，用于在大腸桿菌中表達(dá)的pET載體(Novagen)，用于在培養(yǎng)細(xì)胞中表達(dá)的pME18S-FL3載體(GenBank登錄號(hào)AB009864)，和用于體內(nèi)表達(dá)的pME18S載體(Mol.Cell Biol.8466-472，1988)。可采用常規(guī)方法將本發(fā)明的DNA插入載體內(nèi)，例如采用限制性酶位點(diǎn)進(jìn)行連接酶反應(yīng)(Current Protocols in Molecular Biology，Ausubel等編輯，John Wiley&Sons，Inc.1987，Section 11.4-11.11)。
導(dǎo)入本發(fā)明載體的宿主細(xì)胞并無(wú)特別地限制，并且可以根據(jù)發(fā)明目的使用各種宿主細(xì)胞。例如，用于表達(dá)蛋白的細(xì)胞包括但不限于細(xì)菌細(xì)胞(如鏈球菌(Streptococcus)，葡萄球菌(Staphylococcus)，大腸桿菌，鏈霉菌(Streptomyces)，枯草桿菌(Bacillus subtilis)，真菌細(xì)胞(如酵母，曲霉(Aspergillus))，昆蟲細(xì)胞(如果蠅(Drosophila)S2，夜蛾(Spodoptera)SF9)，動(dòng)物細(xì)胞(如CHO，COS，HeLa，C127，3T3，BHK，HEK293，Bowes黑素瘤細(xì)胞)，以及植物細(xì)胞。將載體轉(zhuǎn)染入宿主細(xì)胞可以采用常規(guī)方法進(jìn)行，如磷酸鈣沉淀，電穿孔(Current protocols in Molecular Biology，Ausubel等編輯，John Wiley&Sons，Inc.1987，Section 9.1-9.9)，Lipofectamine法(GIBCO-BRL)和顯微注射。
通過(guò)將合適的分泌信號(hào)摻入目的蛋白內(nèi)，可以將在宿主細(xì)胞中表達(dá)的蛋白分泌到內(nèi)質(zhì)網(wǎng)的腔，細(xì)胞周圍空腔(cavity)或細(xì)胞外環(huán)境內(nèi)。這些信號(hào)對(duì)于目的蛋白來(lái)講可以是內(nèi)源的或外源的。
當(dāng)本發(fā)明的蛋白分泌到培養(yǎng)基中時(shí)，從該培養(yǎng)基中收集蛋白。如果本發(fā)明的蛋白是在細(xì)胞內(nèi)產(chǎn)生的，就將該細(xì)胞裂解，然后收集蛋白。
利用本領(lǐng)域熟知的方法可從重組的細(xì)胞培養(yǎng)物中對(duì)本發(fā)明蛋白進(jìn)行收集和純化，所述方法包括但不限于，硫酸銨或乙醇沉淀，酸提取，陰離子或陽(yáng)離子交換層析，磷酸纖維素層析，疏水作用層析，親和層析，羥磷灰石層析和外源凝集素(lectin)層析。
包括本發(fā)明DNA的化合物是分離的化合物。在此，術(shù)語(yǔ)“分離的”是指從原來(lái)的環(huán)境中分離出來(lái)(例如，如果是自然存在，即為天然環(huán)境)。樣品中的目的化合物非常豐富，和/或樣品中的目的化合物是部分或基本純化的，則該樣品中的化合物是“分離的”化合物。在本文中，術(shù)語(yǔ)“基本純化的”是指化合物從天然環(huán)境分離的狀態(tài)，而且該狀態(tài)中已除去了至少60％，優(yōu)選75％，最優(yōu)選90％的其它共存的天然成分。
本發(fā)明提供了抗瘧藥物，該藥物抑制瘧原蟲GWT1基因產(chǎn)物的活性。抑制瘧原蟲GWT1基因產(chǎn)物活性的優(yōu)選化合物是WO 02/04626所述的化合物，和包括化合物(1)至(5) 化合物(1)1-(4-丁基芐基)異喹啉 化合物(2)4-[4-(1-異喹啉基甲基)苯基]-3-丁炔-1-醇 化合物(3)5-丁基-2-(1-異喹啉基甲基)苯酚
化合物(4)2-(4-溴-2-氟芐基)-3-甲氧基吡啶 化合物(5)N-[2-(4-丁基芐基)-3-吡啶基]-N-甲胺象這樣，抑制瘧原蟲GWT1基因產(chǎn)物的活性的化合物或其鹽或其水合物可以給藥于哺乳動(dòng)物(優(yōu)選人)。還可以利用常規(guī)方法將它配制成片劑，粉劑，微粒劑，顆粒劑，包衣片劑，膠囊，糖漿，錠劑，吸入劑，栓劑，注射劑，軟膏劑，眼膏，滴眼劑，滴鼻劑，滴耳劑，糊劑，洗劑等，然后給藥。
為配制藥物，可以使用藥物制劑常用的助劑(例如填充劑，粘合劑，滑潤(rùn)劑，著色劑，調(diào)味劑，以及需要時(shí)的穩(wěn)定劑，乳化劑，吸收促進(jìn)劑，表面活性劑，pH調(diào)節(jié)劑，防腐劑和抗氧化劑)。藥物制劑可以用常規(guī)方法，通過(guò)將通常用作藥劑成分的各組分混合來(lái)制備。
例如，口服制劑可以如下制備將本發(fā)明的化合物或其藥學(xué)上可接受的鹽與填充劑，必要時(shí)還有粘合劑，崩解劑，潤(rùn)滑劑，著色劑，調(diào)味劑等組合，并通過(guò)常規(guī)方法將該混合物配制成粉劑，微粒劑，顆粒劑，片劑，包衣片劑，膠囊等。
這些組分的例子包括動(dòng)物脂肪和植物油，如大豆油，牛脂(tallow)，以及合成甘油酯；烴，如液體石蠟，角鯊烯，以及固體石蠟；酯油，如肉豆蔻酸辛基十二醇酯和肉豆蔻酸異丙酯；高級(jí)醇，如十六醇十八醇混合物以及二十二醇；硅氧烷樹(shù)脂；硅油；表面活性劑，如聚氧乙烯脂肪酸酯，山梨聚糖脂肪酸酯，甘油脂肪酸酯，聚氧乙烯山梨聚糖脂肪酸酯，聚氧乙烯硬化的蓖麻油，以及聚氧乙烯聚氧丙烯嵌段共聚物；水溶性大分子，如羥乙基纖維素，聚丙烯酸，羧基乙烯基聚合物，聚乙二醇，聚乙烯基吡咯烷酮，以及甲基纖維素；低級(jí)醇，如乙醇和異丙醇；多元醇，如甘油，丙二醇，二丙二醇，以及山梨醇；糖，如葡萄糖和蔗糖；無(wú)機(jī)粉末，如硅酸酐，硅酸鋁鎂，以及硅酸鋁；和純水。填充劑的實(shí)例子包括乳糖，玉米淀粉，精制白糖，葡萄糖，甘露醇，山梨醇，結(jié)晶纖維素，以及二氧化硅。粘合劑的例子包括聚乙烯醇，聚乙烯醚，甲基纖維素，乙基纖維素，阿拉伯樹(shù)膠，黃芪膠，明膠，蟲膠，羥丙基甲基纖維素，羥丙基纖維素，聚乙烯吡咯烷酮，聚丙二醇聚氧乙烯嵌段聚合物，甲葡胺(meglumine)等。崩解劑的例子包括淀粉，瓊脂，粉狀明膠，結(jié)晶纖維素，碳酸鈣，碳酸氫鈉，檸檬酸鈣，糊精，果膠，以及羧甲基纖維素鈣。潤(rùn)滑劑是硬脂酸鎂，滑石，聚乙二醇，二氧化硅，硬化的植物油等。著色劑的例子為那些允許加入藥物中的著色劑。調(diào)味劑是可可粉，l-薄荷醇，芳香分散劑，薄荷油，冰片，肉桂粉等。對(duì)于這些片劑和顆粒劑當(dāng)然可以使用糖衣，必要時(shí)還可以使用其它適宜的包衣。
此外，液體制劑如糖漿和注射劑可以采用常規(guī)方法來(lái)制備。在該方法中，通過(guò)將pH調(diào)節(jié)劑，增溶劑，等滲劑等，以及必要時(shí)的增溶助劑，穩(wěn)定劑等，加至本發(fā)明的化合物或其藥學(xué)上可接受的鹽中，來(lái)制備。
制備外用制劑的方法沒(méi)有限制，而且可以通過(guò)常規(guī)方法來(lái)制備。也就是說(shuō)，用于制劑的基礎(chǔ)材料可以選自藥物、準(zhǔn)藥物(quasi-drug)、化妝品等常用的各種材料。具體地，可使用的基礎(chǔ)材料是，如動(dòng)物脂肪和植物油，礦物油，酯油，蠟，高級(jí)醇，脂肪酸，硅油，表面活性劑，磷脂，醇，多元醇，水溶性大分子，粘土礦和純化水。必要時(shí)，可以添加pH調(diào)節(jié)劑，抗氧化劑，螯合劑，防腐劑和抗真菌劑，著色物質(zhì)，香料等。但是本發(fā)明的外用制劑的基礎(chǔ)材料并不限于此。再者，如果需要，還可以混入具有分化誘導(dǎo)作用的組分，血流促進(jìn)劑，殺真菌劑，消炎劑，細(xì)胞活化劑，維生素，氨基酸，保濕劑，以及角質(zhì)裂解劑。上述基礎(chǔ)材料按外用制劑常用濃度的量加入。
如本發(fā)明所述，術(shù)語(yǔ)“鹽”優(yōu)選包括，如與無(wú)機(jī)或有機(jī)酸的鹽，與無(wú)機(jī)或有機(jī)堿的鹽，或與酸性或堿性氨基酸的鹽。尤其優(yōu)選是藥學(xué)上可接受的鹽。酸和堿以0.1-5個(gè)酸或堿分子與1個(gè)化合物分子的適當(dāng)比形成鹽。
與無(wú)機(jī)酸的鹽的優(yōu)選例子是與鹽酸，氫溴酸，硫酸，硝酸和磷酸的鹽。與有機(jī)酸的鹽優(yōu)選包括與醋酸，琥珀酸，延胡索酸，馬來(lái)酸，酒石酸，檸檬酸，乳酸，硬脂酸，苯甲酸，甲磺酸，對(duì)甲苯磺酸的鹽。
與無(wú)機(jī)堿的鹽的優(yōu)選例子是堿金屬鹽如鈉鹽和鉀鹽；堿土金屬鹽如鈣鹽和鎂鹽；鋁鹽，和銨鹽。與有機(jī)堿的鹽優(yōu)選包括與二乙胺，二乙醇胺，甲葡胺和N，N’-二芐基乙二胺的鹽。
與酸性氨基酸的鹽的優(yōu)選例子是與天冬氨酸和谷氨酸的鹽，與堿性氨基酸的鹽優(yōu)選包括與精氨酸，賴氨酸和鳥氨酸的鹽。
施用本發(fā)明的化合物或其鹽或其水合物時(shí)，對(duì)其形式?jīng)]有特別的限制，它們可以根據(jù)常規(guī)方法經(jīng)口或經(jīng)非胃腸道途徑給藥。它們可以配制成如片劑，粉劑，微粒劑，膠囊，糖漿，錠劑，吸入劑，栓劑，注射劑，軟膏劑，眼膏，滴眼劑，滴鼻劑，滴耳劑，糊劑和洗劑劑型。本發(fā)明的藥物組合物的劑量可以根據(jù)癥狀程度，患者年齡，性別和體重，劑型，鹽的類型，疾病的具體類型等適當(dāng)?shù)剡x擇。
將治療有效量的本發(fā)明的化合物給藥于患者。在此，“治療有效量”是指產(chǎn)生所需藥理效果并且有效地恢復(fù)或或緩解所要治療的患者之癥狀的藥劑量。所述劑量根據(jù)疾病的類型，癥狀的程度，患者的體重、年齡、性別、對(duì)制劑的敏感性而顯著地不同。但通常，成人的日劑量為約0.03mg-1000mg，優(yōu)選0.1mg-500mg，更優(yōu)選0.1mg-100mg，并且每日給藥一次或幾次，或者每若干天給藥一次或幾次。注射劑量一般為約1-3000μg/kg，優(yōu)選約3-1000μg/kg。
此外，本發(fā)明涉及利用瘧原蟲GWT1基因產(chǎn)物篩選抗瘧藥物的方法。所述篩選方法包括但不限于[1]結(jié)合試驗(yàn)，該試驗(yàn)篩選與標(biāo)記化合物競(jìng)爭(zhēng)結(jié)合瘧原蟲GWT1基因產(chǎn)物的化合物；[2]GlcN-PI?；D(zhuǎn)移酶分析系統(tǒng)，該系統(tǒng)篩選能抑制瘧原蟲GWT1基因產(chǎn)物的GlcN-PI酰基轉(zhuǎn)移酶活性的化合物；和[3]GPI-錨定蛋白檢測(cè)系統(tǒng)，其中在細(xì)胞，優(yōu)選真菌細(xì)胞中表達(dá)瘧原蟲GWT1基因產(chǎn)物，然后檢測(cè)細(xì)胞表面上的GPI-錨定蛋白。本發(fā)明并不限于這些方法，而且包括利用瘧原蟲GWT1基因產(chǎn)物篩選抗瘧藥物的任何方法。上面列出的方法[1]-[3]在下面進(jìn)行詳細(xì)描述。
篩選與標(biāo)記化合物競(jìng)爭(zhēng)結(jié)合瘧原蟲GWT1基因產(chǎn)物的化合物的結(jié)合試驗(yàn)本發(fā)明的兩種方法公開(kāi)如下，即(1)制備瘧原蟲GWT1基因產(chǎn)物(下文中稱為瘧原蟲GWT1蛋白)的方法和(2)涉及標(biāo)記化合物的結(jié)合實(shí)驗(yàn)法(下文中稱為結(jié)合試驗(yàn))
(1)制備瘧原蟲GWT1蛋白的方法瘧原蟲GWT1蛋白是從細(xì)胞膜級(jí)分，優(yōu)選從真菌細(xì)胞，更優(yōu)選從釀酒酵母(S.cerevisiae)細(xì)胞中制得的，其中引入了SEQ ID NO2的瘧原蟲GWT1蛋白的編碼DNA。優(yōu)選將所述DNA導(dǎo)入GWT1基因-缺陷型細(xì)胞中。在結(jié)合試驗(yàn)中，可以在未進(jìn)行任何其它處理的條件下使用所制的膜級(jí)分，或者在使用前對(duì)膜級(jí)分進(jìn)行進(jìn)一步的純化。利用釀酒酵母的方法將在下面進(jìn)行詳細(xì)描述。
(a)瘧原蟲GWT1基因的導(dǎo)入本發(fā)明使用的瘧原蟲GWT1基因可以是天然存在的基因，或優(yōu)選是基于SEQ ID NO2或4的氨基酸序列進(jìn)行合成的。瘧原蟲GWT1基因富含腺嘌呤和胸腺嘧啶。因此，可以推斷利用常規(guī)的基因重組技術(shù)將很難對(duì)該基因進(jìn)行操作，且該基因在酵母，細(xì)胞等中的表達(dá)效率將會(huì)很低(inefficient)。因此，優(yōu)選對(duì)核苷酸序列進(jìn)行設(shè)計(jì)，將其中與每個(gè)相應(yīng)氨基酸對(duì)應(yīng)的密碼子替換為那些認(rèn)為可以在酵母，細(xì)胞等中能有效表達(dá)的密碼子，在該設(shè)計(jì)序列的基礎(chǔ)上進(jìn)行DNA合成，制備人工的瘧原蟲GWT1基因，然后在下述實(shí)驗(yàn)中使用該基因。
瘧原蟲GWT1的表達(dá)質(zhì)粒是通過(guò)將瘧原蟲GWT1基因插入到釀酒酵母表達(dá)載體內(nèi)來(lái)加以制備的，例如，通過(guò)將適合的啟動(dòng)子和終止子，如pKT10-衍生的GAPDH啟動(dòng)子和GAPDH終止子，插到Y(jié)Ep352的多克隆位點(diǎn)內(nèi)來(lái)制備表達(dá)載體(Tanaka等，Mol.Cell Biol.，104303-4313，1990)。將釀酒酵母(如G2-10菌株)培養(yǎng)在合適的培養(yǎng)基(如YPD培養(yǎng)基(酵母提取物-聚蛋白胨-葡萄糖培養(yǎng)基))中，并在適宜的溫度(如30℃)振蕩，在對(duì)數(shù)生長(zhǎng)晚期收獲細(xì)胞。洗滌后，采用如乙酸鋰法將GWT1-表達(dá)質(zhì)粒導(dǎo)入釀酒酵母細(xì)胞中。該方法描述于User Manual of YEAST MAKERTMYeast Transformation System(BD Biosciences Clontech)。瘧原蟲GWT1-過(guò)表達(dá)菌株和攜帶陰性對(duì)照載體的菌株可通過(guò)將轉(zhuǎn)化細(xì)胞于30℃，在SD(ura-)培養(yǎng)基上培養(yǎng)兩天而獲得。
適合除釀酒酵母之外的真菌種類的表達(dá)載體和基因轉(zhuǎn)移法見(jiàn)下述報(bào)道適合粟酒裂殖酵母(Schizosaccharomyces Pombe，S.pombe)的表達(dá)載體如pcL和其轉(zhuǎn)移法由Igarashi等描述(Nature 35380-83，1991)，適合白色念珠菌(C.Albicans)的表達(dá)載體如pRM10和其轉(zhuǎn)移法由Pla J.等描述(Yeast，121677-1702，1996)；適合煙曲霉(A.Fumigatus)的表達(dá)載體如pAN7-1和其轉(zhuǎn)移法由Punt P.J.等描述(GENE，56117-124，1987)；適合新生隱球菌(C.Neoformans)的表達(dá)載體如pPM8及其轉(zhuǎn)化法由Monden P.等描述(FEMSMicrobiol.Lett.，18741-45，2000)。
(b)制備膜級(jí)分的方法在適合的培養(yǎng)基(如SD(ura-)液體培養(yǎng)基)中培養(yǎng)，并在適合的溫度(如30℃)振蕩導(dǎo)入了瘧原蟲GWT1基因的釀酒酵母細(xì)胞。在對(duì)數(shù)生長(zhǎng)中期時(shí)收獲真菌細(xì)胞，然后用合適量(如真菌細(xì)胞的3倍體積)的勻漿緩沖液(如50mMTris-HCl，pH 7.5，10mM EDTA，CompleteTM(Roche))懸浮。將合適量的玻璃珠(如真菌細(xì)胞的4倍體積)加至懸浮液中。渦旋混合物，并置于冰上。重復(fù)此操作數(shù)次將真菌細(xì)胞破碎。
將1毫升的勻漿緩沖液加入所得的裂解液中。離心混合物；如2,500rpm離心5分鐘，使玻璃珠和未碎的真菌細(xì)胞沉淀。將上清液移到另一試管內(nèi)。離心該試管，如13,500rpm離心10分鐘，使含有細(xì)胞器的膜級(jí)分(總膜級(jí)分)沉淀。用1ml結(jié)合緩沖液(如0.1M磷酸鹽緩沖液，pH 7.0，0.05％Tween 20，CompleteTM(Roche))懸浮沉淀物，然后離心，如2,500rpm離心1分鐘，除去未懸浮的物質(zhì)。然后離心上清液，如15,000rpm離心5分鐘。沉淀物用150-650μl的結(jié)合緩沖液重懸浮來(lái)制備膜級(jí)分。
采用適合粟酒裂殖酵母的Yoko-o等的方法(Eur.J.Biochem.257630-637，1998)；適合白色念珠菌的Sentandreu M等的方法(J.Bacteriol.，180282-289，1998)；適合煙曲霉的Mouyna I等的方法(J.Biol.Chem.，27514882-14889，2000)；和適合新生隱球菌的Thompson JR等的方法(J.Bacteriol.，181444-453，1999)可以從除釀酒酵母之外的真菌物種中制備膜級(jí)分。
另外，可以通過(guò)在大腸桿菌，昆蟲和哺乳動(dòng)物細(xì)胞或其他非真菌細(xì)胞中表達(dá)來(lái)制備瘧原蟲GWT1蛋白。
當(dāng)使用哺乳動(dòng)物細(xì)胞時(shí)，將瘧原蟲GWT1基因與含有如CMV啟動(dòng)子的過(guò)表達(dá)載體連接，導(dǎo)入哺乳動(dòng)物細(xì)胞中。然后，按照Petaja-Repo等的方法(J.Biol.Chem.，2764416-23，2001)制備膜級(jí)分。
表達(dá)瘧原蟲GWT1基因的昆蟲細(xì)胞(如Sf9細(xì)胞)可以利用，例如桿狀病毒表達(dá)試劑盒如BAC-TO-BAC桿狀病毒表達(dá)系統(tǒng)(Invitrogen)來(lái)進(jìn)行制備。然后，按照Okamoto等的方法(J.Biol.Chem.，276742-751，2001)制備膜級(jí)分。
可通過(guò)下述步驟從大腸桿菌制備瘧原蟲GWT1蛋白例如將瘧原蟲GWT1基因與大腸桿菌表達(dá)載體如pGEX載體(Amersham Biosciences Corp.)連接，將此構(gòu)建體導(dǎo)入大腸桿菌如BL21中。
(2)結(jié)合分析法(a)標(biāo)記化合物的合成標(biāo)記化合物從已證實(shí)結(jié)合GWT1蛋白的化合物制備。與GWT1蛋白結(jié)合的任何化合物都是可以使用的。標(biāo)記化合物優(yōu)選從WO 02/04626中所述的化合物制備，更優(yōu)選從上述(1)至(5)的化合物制備。
任何標(biāo)記方法都是可以使用的?；衔飪?yōu)選是用放射性同位素，更優(yōu)選用3H標(biāo)記的化合物。采用放射性化合物作為起始物質(zhì)，用常規(guī)制備方法制得放射性標(biāo)記的化合物。另外，3H標(biāo)記可以通過(guò)3H交換反應(yīng)獲得。
(b)特異性結(jié)合的確定將標(biāo)記化合物加至制得的膜級(jí)分中，將該混合物置于冰上適當(dāng)長(zhǎng)的時(shí)間，如1至2小時(shí)，直到標(biāo)記化合物和膜級(jí)分間發(fā)生結(jié)合反應(yīng)。離心混合物，使膜級(jí)分沉淀，如15,000rpm離心3分鐘。沉淀物用結(jié)合緩沖液重懸浮，離心懸浮液。適當(dāng)?shù)刂貜?fù)(兩次)，除去未結(jié)合的標(biāo)記化合物。再用結(jié)合緩沖液懸浮沉淀物。將所得的懸浮液移至閃爍小管，加入閃爍劑。利用液體閃爍計(jì)數(shù)器測(cè)定放射性。
確認(rèn)標(biāo)記化合物與GWT1蛋白的特異性結(jié)合可以通過(guò)下述評(píng)估來(lái)進(jìn)行是否標(biāo)記化合物的結(jié)合會(huì)被加入的嚴(yán)重過(guò)量未標(biāo)記化合物(10倍或更多)所抑制，以及該化合物是否是可忽略地與由不表達(dá)GWT1蛋白的真菌細(xì)胞制得的膜級(jí)分結(jié)合。
(c)由試樣引起的標(biāo)記化合物的結(jié)合抑制將試樣和標(biāo)記化合物加至制得的膜級(jí)分中，將該混合物置于冰上適當(dāng)長(zhǎng)的時(shí)間，如1至2小時(shí)，期間與膜級(jí)分發(fā)生結(jié)合反應(yīng)。在本發(fā)明的篩選方法中使用的待測(cè)化合物包括簡(jiǎn)單的天然存在化合物，有機(jī)化合物，無(wú)機(jī)化合物，蛋白或肽，以及化合物文庫(kù)，基因文庫(kù)的表達(dá)產(chǎn)物，細(xì)胞提取物，細(xì)胞培養(yǎng)物上清液，來(lái)自發(fā)酵細(xì)菌的產(chǎn)物，海洋生物體提取物，植物提取物和類似物。
將混合物離心，例如15,000rpm離心3分鐘，使膜級(jí)分沉淀。在結(jié)合緩沖液中重懸浮沉淀物，離心該懸浮液。適當(dāng)?shù)刂貜?fù)此步驟(兩次)，從而除去所有未結(jié)合的標(biāo)記化合物。用結(jié)合緩沖液懸浮沉淀物。將懸浮液移至閃爍小管，加入閃爍劑。利用液體閃爍計(jì)數(shù)器測(cè)定放射性。
當(dāng)在試樣存在的條件下，標(biāo)記化合物與膜級(jí)分的結(jié)合被抑制，就判定該試樣具有結(jié)合瘧原蟲GWT1蛋白的活性。
用于篩選抑制瘧原蟲GWT1蛋白GlcN-PI?；D(zhuǎn)移酶活性的化合物的GlcN-PI?；D(zhuǎn)移酶分析系統(tǒng)酰基到GPI的轉(zhuǎn)移可以利用下述方法來(lái)進(jìn)行檢測(cè)Costello L.C和OrleanP.，J.Biol.Chem.(1992)2678599-8603；或Franzot S.P和Doering T.L.，Biochem.J.(1999)34025-32。該方法的具體實(shí)例在下面進(jìn)行了描述。下述實(shí)驗(yàn)條件優(yōu)選是對(duì)所用的各種瘧原蟲GWT1蛋白進(jìn)行了優(yōu)化的。
根據(jù)上述部分1所述的方法制備瘧原蟲GWT1蛋白。將包含瘧原蟲GWT1蛋白的膜級(jí)分加至緩沖液中，所述緩沖液含有合適的金屬離子(Mg2+，Mn2+)，ATP，輔酶A，優(yōu)選含有防止在其它反應(yīng)中消耗UDP-GlcNAc的抑制劑，如作為幾丁質(zhì)合成抑制劑的尼可霉素(nikkomycin)Z，或作為天冬酰胺-連接的糖基化抑制劑的衣霉素(tunicamycin)。然后，將試樣加到該混合物中，將所得的混合物在合適的溫度下孵育適當(dāng)長(zhǎng)的時(shí)間(如24℃孵育15分鐘)。
將已適當(dāng)標(biāo)記的，優(yōu)選放射性標(biāo)記的GlcN-(?；?PI前體(如UDP-GlcNAc，Acyl-輔酶A，優(yōu)選UDP-[14C]GlcNAc)加到混合物中。將所得的混合物孵育適當(dāng)長(zhǎng)的時(shí)間(如24℃孵育1小時(shí))。加入氯仿和甲醇(1∶2)的混合物，攪拌所得混合物以使反應(yīng)停止，提取脂質(zhì)。將提取的反應(yīng)產(chǎn)物溶解在合適的溶劑中，優(yōu)選丁醇。然后，利用如HPLC或薄層層析(TLC)，優(yōu)選TLC法分離反應(yīng)產(chǎn)生的GlcN-(?；?PI。當(dāng)使用TLC時(shí)，展開(kāi)劑(developing solvent)可以適當(dāng)?shù)剡x自，如CHCl3/CH3OH/H2O(65∶25∶4)，CHCl3/CH3OH/1M NH4OH(10∶10∶3)和CHCl3/吡啶/HCOOH(35∶30∶7)。優(yōu)選的展開(kāi)劑是CHCl3/CH3OH/H2O(65∶25∶4)。采用對(duì)所用標(biāo)記而言恰當(dāng)?shù)姆椒?，?duì)分離的GlcN-(?；?PI進(jìn)行定量。當(dāng)用放射性同位素作標(biāo)記時(shí)，可以基于其放射性對(duì)分離的GlcN-(?；?PI進(jìn)行定量。
當(dāng)在試樣存在的條件下，制得的GlcN-(酰基)PI量減少時(shí)，就判定該試樣具有抑制由瘧原蟲GWT1蛋白引起的?；D(zhuǎn)移的活性。
包括在細(xì)胞中表達(dá)瘧原蟲GWT1蛋白然后檢測(cè)細(xì)胞表面上的GPI-錨定蛋白的GPI-錨定蛋白檢測(cè)系統(tǒng)試樣抑制瘧原蟲GWT1蛋白活性的能力可以利用GPI-錨定蛋白檢測(cè)系統(tǒng)來(lái)進(jìn)行測(cè)定，該系統(tǒng)包括在細(xì)胞，優(yōu)選真菌細(xì)胞中表達(dá)GWT1蛋白，然后檢測(cè)細(xì)胞表面上的GPI-錨定蛋白。本發(fā)明的真菌是指屬于接合菌門(Zygomycota)，子囊菌門(Ascomycota)，擔(dān)子菌門(Basidiomycota)及半知菌綱(Deuteromycete)的真菌，且優(yōu)選的真菌是致病性真菌，毛霉屬(Mucor)，酵母屬(Saccharomyces)，念珠菌屬(Candida)，隱球菌屬(Cryptococcus)，絲孢酵母屬(Trichosporon)，鱗斑霉屬(Malassezia)，曲霉屬(Aspergillus)，發(fā)癬菌屬(Trichophyton)，小孢子菌屬(Microsporum)，孢子絲菌屬(Sporothrix)，芽生酵母屬(Blastmyces)，球孢子菌屬(Coccidioides)，副球孢子菌屬(Paracoccidioides)，青霉屬(Penicillinium)和鐮孢屬(Fusarium)，更優(yōu)選的真菌為白色念珠菌，光滑念珠菌(C.glabrata)，新生隱球菌和煙曲霉，甚至更優(yōu)選的真菌是酵母。所述酵母包括釀酒酵母和粟酒裂殖酵母。用于將包含編碼瘧原蟲GWT1蛋白的插入DNA的表達(dá)載體導(dǎo)入上述真菌細(xì)胞的方法是本領(lǐng)域技術(shù)人員所熟知的。
當(dāng)瘧原蟲GWT1蛋白在真菌細(xì)胞中表達(dá)時(shí)，轉(zhuǎn)運(yùn)到真菌細(xì)胞壁的GPI-錨定蛋白量可通過(guò)下述方法測(cè)定(1)利用報(bào)告酶的方法；(2)利用與真菌細(xì)胞壁表面糖蛋白反應(yīng)的抗體的方法；(3)利用該蛋白粘附動(dòng)物細(xì)胞的能力；或者(4)在光學(xué)顯微鏡或電子顯微鏡下觀察真菌細(xì)胞的方法。
(1)-(4)的方法已在WO 02/04626中公開(kāi)，所述方法在該發(fā)明的實(shí)施例中進(jìn)行了詳細(xì)描述。利用(1)-(4)的方法，并且優(yōu)選(1)-(4)的方法的組合可以判斷試樣是否抑制GPI-錨定蛋白到細(xì)胞壁上的轉(zhuǎn)運(yùn)過(guò)程或者GPI-錨定蛋白在真菌細(xì)胞表面的表達(dá)。
在下文中，將描述方法(1)-(4)。
(1) 利用報(bào)告酶進(jìn)行的方法GPI-錨定蛋白到細(xì)胞壁的轉(zhuǎn)運(yùn)過(guò)程可通過(guò)示蹤實(shí)驗(yàn)定量如用放射性同位素標(biāo)記GPI-錨定蛋白，獲得真菌細(xì)胞壁級(jí)分，并用抗GPI-錨定蛋白的抗體進(jìn)行免疫沉淀反應(yīng)。也可以用另一種方法更簡(jiǎn)便地定量在各種GPI-錨定蛋白中常見(jiàn)的、被認(rèn)為起轉(zhuǎn)運(yùn)信號(hào)作用的C-末端序列可與便于測(cè)量的酶(報(bào)告酶)表達(dá)為融合蛋白，獲得真菌細(xì)胞壁級(jí)分；然后采用報(bào)告系統(tǒng)測(cè)量每一級(jí)分的酶活性(Van Berkel MAA等，F(xiàn)EBSLetters，349135-138，1994)。在下文中，將要會(huì)對(duì)一種采用報(bào)告酶進(jìn)行的方法進(jìn)行描述，但是本發(fā)明并不僅限于此。
首先，構(gòu)建報(bào)告基因并將其引入真菌中。報(bào)告基因是這樣構(gòu)建的使在真菌中起作用的啟動(dòng)子序列，以與閱讀框匹配的方式連接分別編碼信號(hào)序列、報(bào)告酶和GPI-錨定蛋白C-末端序列的DNA。啟動(dòng)予序列的實(shí)例包括GAL10及ENO1。信號(hào)序列的實(shí)例包括如α-因子，轉(zhuǎn)化酶，溶菌酶。報(bào)告酶的實(shí)例是β-內(nèi)酰胺酶，溶菌酶，堿性磷酸酶和β-半乳糖苷酶?？梢允褂靡子跈z出的綠色螢光蛋白(GFP)，即便它不具有酶活性。GPI-錨定蛋白C-末端序列包括α-凝集素(agglutinin)C-末端序列，CWP2 C-末端序列等。此外，優(yōu)選將適宜的選擇標(biāo)記物如LEU2和URA3插到包含所構(gòu)建的報(bào)告基因的載體中。
將所構(gòu)建的報(bào)告基因通過(guò)適宜的方法插到真菌中，如乙酸鋰法(Gietz D等，Nucl.Acids Res.201425，1992)。并且在需要時(shí)，利用適于選擇標(biāo)記物的方法(如適于LEU2的Leu-培養(yǎng)基，以及適于URA3的Ura-培養(yǎng)基)培養(yǎng)真菌，然后挑選其中引入了DNA的真菌。
通過(guò)下列方法測(cè)定試樣對(duì)GPI-錨定蛋白到細(xì)胞壁的轉(zhuǎn)運(yùn)過(guò)程的影響在試樣存在時(shí)，于適宜的條件下(如30℃48小時(shí))培養(yǎng)引入了報(bào)告基因的真菌。培養(yǎng)之后，離心培養(yǎng)物上清液，并測(cè)量培養(yǎng)物上清液級(jí)分的報(bào)告酶活性。洗滌所得細(xì)胞級(jí)分，然后通過(guò)適當(dāng)?shù)姆椒ǚ蛛x細(xì)胞壁成分，例如用葡聚糖酶降解細(xì)胞壁葡聚糖，然后測(cè)量細(xì)胞壁級(jí)分和細(xì)胞質(zhì)級(jí)分的報(bào)告酶活性。該試驗(yàn)可以如下簡(jiǎn)單地進(jìn)行離心確定細(xì)胞級(jí)分中報(bào)告酶的量，然后在不洗滌細(xì)胞的情況下按比例計(jì)算來(lái)源于培養(yǎng)物上清液級(jí)分但殘留在細(xì)胞級(jí)分中的報(bào)告酶的量，從細(xì)胞級(jí)分的報(bào)告酶的量中減去此量。
如果試樣顯示出增加培養(yǎng)物上清液級(jí)分中報(bào)告酶活性的活性(活性/細(xì)胞)，或者降低細(xì)胞壁級(jí)分中報(bào)告酶活性的活性(活性/細(xì)胞)，那么就可以判定該試樣對(duì)GPI-錨定蛋白到細(xì)胞壁的轉(zhuǎn)運(yùn)過(guò)程有影響。
(2) 采用與真菌細(xì)胞壁表面糖蛋白反應(yīng)的抗體的方法對(duì)于試樣影響GPI-錨定蛋白在真菌表面層的表達(dá)的能力，可以通過(guò)利用與真菌細(xì)胞壁中的GPI-錨定蛋白反應(yīng)的抗體進(jìn)行定量來(lái)測(cè)定。
抗體可以如下制備例如，可以由GPI-錨定蛋白的氨基酸序列預(yù)測(cè)抗原決定簇，所述GPI-錨定蛋白如α-凝集素，Cwp2p，或Als1p(Chen MH等，J.Biol.Chem.，27026168-26177，1995；Van Der Vaat JM等，J.Bacteriol.，1773104-3110，1995；Hoyer LL等，Mol.Microbiol.，1539-54，1995)，合成該區(qū)的肽，將其與抗原性物質(zhì)如載體蛋白結(jié)合，然后通過(guò)免疫兔子等得到多克隆抗體，或者通過(guò)免疫小鼠等得到單克隆抗體。優(yōu)選是針對(duì)Als1p肽的兔多克隆抗體。
在可供選擇的方法中，可以通過(guò)用真菌，優(yōu)選過(guò)量表達(dá)GPI-錨定蛋白，如α-凝集素，Cwp2p及Als1p的真菌免疫小鼠等獲得抗GPI-錨定蛋白的單克隆抗體，(在某些情況下，可以進(jìn)一步用部分純化的GPI-錨定蛋白進(jìn)行免疫)，并利用ELISA，Western印跡分析等以此制備抗體為基礎(chǔ)選出產(chǎn)生的克隆。
通過(guò)下述方法可以檢測(cè)試樣對(duì)GPI-錨定蛋白到細(xì)胞壁的轉(zhuǎn)運(yùn)過(guò)程的影響，以及試樣對(duì)來(lái)源于細(xì)胞壁中GPI-錨定蛋白的蛋白量的影響。
在試樣存在時(shí)，于適宜的條件下如30℃48小時(shí)培養(yǎng)真菌。通過(guò)離心收集所培養(yǎng)的真菌并將細(xì)胞破碎，優(yōu)選采用玻璃珠。優(yōu)選用SDS離心提取洗滌過(guò)的破碎細(xì)胞，然后洗滌沉淀物。提取之后，用降解葡聚糖的酶，優(yōu)選葡聚糖酶處理破碎的細(xì)胞，其經(jīng)離心處理的上清液即為GPI-錨定蛋白樣品。
將抗-Als1p肽的抗體經(jīng)4℃過(guò)夜溫育包被在96-孔板上。用洗滌溶液，優(yōu)選含0.05％Tween 20的PBS(PBST)洗滌板之后，用封閉96-孔板非特異性吸附位點(diǎn)的試劑，優(yōu)選蛋白如BSA或明膠蛋白，更優(yōu)選BlockAce(Dainippon Pharmaceutical Co.，Ltd.)進(jìn)行封閉。用洗滌溶液，優(yōu)選PBST，再次洗滌之后，加入適當(dāng)稀釋的GPI-錨定蛋白樣品。在室溫進(jìn)行適當(dāng)長(zhǎng)時(shí)間如2小時(shí)的反應(yīng)。用洗滌溶液，優(yōu)選PBST，洗滌之后，使針對(duì)酶標(biāo)記的白色念珠菌的抗體，優(yōu)選HRP-標(biāo)記的抗-念珠菌抗體，在室溫反應(yīng)適當(dāng)長(zhǎng)的時(shí)間如2小時(shí)。標(biāo)記方法可以是酶標(biāo)記法或放射性同位素標(biāo)記法。用洗滌溶液，優(yōu)選PBST，洗滌之后，通過(guò)適于標(biāo)記類型即酶標(biāo)記的方法計(jì)算GPI-錨定蛋白樣品中Als1p的量，加入底物溶液，然后在終止反應(yīng)時(shí)測(cè)量在490nm處的吸光度。
(3) 利用粘附動(dòng)物細(xì)胞的能力進(jìn)行的方法試樣對(duì)GPI-錨定蛋白在真菌表面的表達(dá)的影響可以通過(guò)測(cè)量真菌細(xì)胞壁中GPI-錨定蛋白的活性，優(yōu)選通過(guò)測(cè)量真菌對(duì)動(dòng)物細(xì)胞等的粘附能力來(lái)進(jìn)行測(cè)定。除了Als1p，Hwp1p等參與粘附動(dòng)物細(xì)胞的活性之外，GPI-錨定蛋白活性還包括α-凝集素參與交配的活性，F(xiàn)lo1p參與酵母聚集的活性等。在下文中，將詳細(xì)描述利用真菌對(duì)動(dòng)物細(xì)胞的粘附活性進(jìn)行的方法，但是本發(fā)明并不僅限于此。
使用對(duì)細(xì)胞具有粘附能力的真菌，優(yōu)選的真菌為白色念珠菌。對(duì)于哺乳動(dòng)物細(xì)胞采用粘附真菌的細(xì)胞，優(yōu)選小腸上皮細(xì)胞。培養(yǎng)哺乳動(dòng)物細(xì)胞并通過(guò)適當(dāng)?shù)姆椒?，如乙醇固定法將其固定。將試樣和真菌培養(yǎng)適當(dāng)長(zhǎng)的時(shí)間，如30℃溫育48小時(shí)，然后接種并培養(yǎng)一定長(zhǎng)的時(shí)間，如在30℃溫育1小時(shí)。移出培養(yǎng)物上清液，用緩沖液洗滌，加樣至瓊脂培養(yǎng)基，如Sabouraud Dextrose Agar培養(yǎng)基(Becton Dickinson Company，Ltd.)。30℃過(guò)夜培養(yǎng)后，計(jì)數(shù)菌落的數(shù)目，并計(jì)算粘附率。
如果與未經(jīng)該化合物處理的真菌相比，觀察到試樣具有使細(xì)胞粘附而形成的菌落數(shù)目降低的活性，則可以判定該試樣已經(jīng)影響了GPI-錨定蛋白到細(xì)胞壁的轉(zhuǎn)運(yùn)過(guò)程。
(4) 利用光學(xué)顯微鏡或電子顯微鏡觀察真菌的方法可以通過(guò)用電子顯微鏡觀察真菌細(xì)胞壁的結(jié)構(gòu)來(lái)確定試樣對(duì)GPI-錨定蛋白在真菌表面的表達(dá)的影響。
在試樣存在的條件下，將真菌如白色念珠菌培養(yǎng)一定長(zhǎng)的時(shí)間，如30℃培養(yǎng)48小時(shí)，用透射電子顯微鏡觀察其超微形態(tài)結(jié)構(gòu)。在此，利用透射電子顯微鏡進(jìn)行的觀察可以通過(guò)例如根據(jù)Electron Microscope Chart Manual(Medical Publishing Center)的方法來(lái)進(jìn)行。真菌細(xì)胞最外層的絮狀纖維結(jié)構(gòu)具有高電子密度，并且可通過(guò)透射電子顯微鏡觀察到。此結(jié)構(gòu)不受其它現(xiàn)有抗真菌劑的影響，并認(rèn)為是包括GPI-錨定蛋白作為其構(gòu)成成分的表面糖蛋白層。與未經(jīng)處理的細(xì)胞相比，當(dāng)此結(jié)構(gòu)消失，只留下具有高電子密度的微層時(shí)，可以判定所述試樣已經(jīng)影響了GPI-錨定蛋白到細(xì)胞壁的轉(zhuǎn)運(yùn)過(guò)程。
當(dāng)在透射電子顯微鏡以及光學(xué)顯微鏡下的觀察都顯示真菌細(xì)胞大量溶脹且出芽(分裂)受到抑制時(shí)，可以判定試樣對(duì)細(xì)胞壁有影響。
本發(fā)明還提供了治療瘧疾的方法，該方法包括施用抑制瘧原蟲GWT1蛋白的活性的化合物的步驟。所述化合物包括WO 02/04626中所述化合物(如本文(1)-(5)中所述化合物)。
天然PfGWT1蛋白的核苷酸序列的特點(diǎn)是具有非常高的AT含量(80.41％)，因此其密碼子使用是有偏好的(biased)。此外，所述基因包含在23個(gè)分離位置含六個(gè)或更多個(gè)連續(xù)A殘基的序列段，且這些序列段作為假(pseudo)-poly(A)位點(diǎn)來(lái)制備截短的蛋白。由于如上所述的特性，該基因只能少量地在酵母中表達(dá)，而且很難利用PCR進(jìn)行擴(kuò)增或者在大腸桿菌中進(jìn)行復(fù)制。也很難確定其核苷酸序列。然而，利用該DNA的簡(jiǎn)并突變體(SEQID NO5)，其AT含量低于編碼PfGWT1蛋白的DNA，本發(fā)明人成功地使PfGWT1蛋白高效表達(dá)。本發(fā)明人還揭示簡(jiǎn)并突變體DNA的引入能拯救GWT1-缺陷型酵母的表型。此項(xiàng)發(fā)現(xiàn)說(shuō)明瘧原蟲的GPI合酶與真菌如酵母的GPI合酶是可以互換的。
編碼瘧原蟲GPI合酶的基因的AT含量例如對(duì)于惡性瘧原蟲的GPI8為79.35％，對(duì)于惡性瘧原蟲的GPI13為77.89％。其AT含量與PfGWT1的AT含量一樣高。由于惡性瘧原蟲基因組翻譯區(qū)內(nèi)的平均AT含量為76.3％，可以推斷大多數(shù)惡性瘧原蟲基因幾乎不能在其它物種中表達(dá)。本發(fā)明人成功地使比編碼PfGWT1蛋白的DNA具有更低AT含量的DNA簡(jiǎn)并突變體在酵母中表達(dá)。因此，通過(guò)利用該簡(jiǎn)并DNA突變體，可以在瘧原蟲之外的宿主中表達(dá)瘧原蟲GPI合酶。此外，GPI-缺陷型酵母和GWT1-缺陷型酵母已知表現(xiàn)出相似的表型，包括致死性等特性。這樣，可以采用上述簡(jiǎn)并突變體DNA拯救GPI合酶基因-缺陷型真菌的表型。
已導(dǎo)入了上述簡(jiǎn)并突變體DNA的GPI合酶基因-缺陷型真菌的表型由瘧原蟲GPI合酶的活性來(lái)決定。相應(yīng)地，可以利用GPI合酶基因-缺陷型真菌的表型作為指標(biāo)，經(jīng)篩選選出抑制瘧原蟲GPI合酶活性的化合物。這樣，事實(shí)上并不需要利用瘧原蟲就能選出靶向GPI生物合成路徑的的抗瘧藥物。
本發(fā)明提供了編碼具有拯救GPI合酶基因-缺陷型真菌表型的活性的蛋白的簡(jiǎn)并突變體DNA，其比編碼參與GPI生物合成的天然DNA具有更低的AT含量。所述DNA可以在本發(fā)明的篩選方法中使用。
如本文所用，術(shù)語(yǔ)“AT含量”是指GPI合酶基因編碼區(qū)完整核苷酸序列中的腺嘌呤和胸腺嘧啶含量。本發(fā)明簡(jiǎn)并突變體DNA中的AT含量范圍優(yōu)選從50％到70％，更優(yōu)選從53％到65％，更加優(yōu)選從55％到62％。
GPI合酶基因-缺陷型真菌的表型包括溫度敏感性(優(yōu)選高溫敏感性)和致死性。
在瘧原蟲中參與GPI生物合成的本發(fā)明蛋白包括GWT1，GPI1，GPI8，GPI3/PIG-A，GPI10/PIG-B，YJR013 W/PIG-M，GPI13/PIG-O，GAA1/GAA-1，DPM1，GPI2，GPI15，YDR437W，GPI12，MCD4，GPI11，GPI7，GPI17，GPI16，CDC91，DPM2，DPM3和SL15。上述蛋白中，已知GPI1和GPI8存在于瘧原蟲中，GPI3/PIG-A，GPI10/PIG-B，YJR013W/PIG-M，GPI13/PIG-O，GAA1/GAA-1和DPM1也認(rèn)為存在于瘧原蟲中(Delorenzi等，Infect.Immun.704510-4522，2002)。惡性瘧原蟲GWT1，GPI1，GPI8，GPI3/PIG-A，GPI10/PIG-B，YJR013W/PIG-M，GPI13/PIG-O，GAA1/GAA-1和DPM1的核苷酸序列分別示于SEQ ID NO1和SEQ ID NOs6-21中的偶數(shù)序列鑒定號(hào)中。各相應(yīng)氨基酸序列示于SEQ ID NO2和SEQ ID NOs6-21中的奇數(shù)序列鑒定編號(hào)中。此外，間日瘧原蟲GWT1的核苷酸序列示于SEQ ID NO3，相應(yīng)的氨基酸序列示于SEQ ID NO4。采用本領(lǐng)域技術(shù)人員熟知的方法，如采用雜交或PCR，利用包含SEQ ID NO1和3，以及偶數(shù)號(hào)的SEQ ID NOs6-21中任一核苷酸序列的DNA，可以對(duì)其它瘧原蟲的GWT1，GPI1，GPI8，GPI3/PIG-A，GPI10/PIG-B，YJR013W/PIG-M，GPI13/PIG-O，GAA1/GAA-1或DPM1進(jìn)行克隆。
另外，利用酵母或人GPI合酶基因可以對(duì)瘧原蟲的除GWT1，GPI1，GPI8，GPI3/PIG-A，GPI10/PIG-B，YJR013W/PIG-M，GPI13/PIG-O，GAA1/GAA-1和DPM1之外的GPI合酶基因進(jìn)行克隆。酵母(釀酒酵母)GPI2，GPI15，YDR437W，GPI12，MCD4，GPI11，GPI7，GPI17，GPI16和CDC91的核苷酸序列分別示于SEQ ID NOs22-41中的偶數(shù)序列鑒定號(hào)中；各相應(yīng)氨基酸序列示于SEQ ID NOs22-41中的奇數(shù)序列鑒定號(hào)中。此外，人DPM2，DPM3和SL1 5的核苷酸序列分別示于SEQ ID NOs42-47中的偶數(shù)序列鑒定號(hào)中；各相應(yīng)氨基酸序列示于SEQ ID NOs42-47中的奇數(shù)序列鑒定號(hào)中。
編碼參與瘧原蟲GPI生物合成的蛋白的簡(jiǎn)并突變體DNA的制備由兩步組成設(shè)計(jì)和合成，所述簡(jiǎn)并突變體DNA的AT含量比天然DNA低。在設(shè)計(jì)步驟中，首先對(duì)目的蛋白的氨基酸序列進(jìn)行反向翻譯，然后列出各氨基酸殘基的可能密碼子。反向翻譯可以利用商購(gòu)基因分析軟件(例如DNASIS-Pro；Hitachi Software Engineering Co.，Ltd)來(lái)實(shí)現(xiàn)。在列出的密碼子中，為各個(gè)氨基酸選出符合目的的密碼子(例如，AT含量較低的密碼子和在用于基因表達(dá)的宿主中使用的常規(guī)密碼子)。簡(jiǎn)并突變體DNA可以通過(guò)利用這些選出的密碼子重排目的蛋白的氨基酸序列來(lái)對(duì)其進(jìn)行設(shè)計(jì)。
如此設(shè)計(jì)的DNA可以通過(guò)本領(lǐng)域技術(shù)人員所熟知的方法來(lái)進(jìn)行合成。本發(fā)明的簡(jiǎn)并突變體DNA可以基于設(shè)計(jì)的核苷酸序列，通過(guò)如利用商購(gòu)DNA合成儀來(lái)進(jìn)行合成。
本發(fā)明還提供了插入了上述簡(jiǎn)并突變體DNA的載體，以及在可表達(dá)狀態(tài)包含該DNA或該載體的轉(zhuǎn)化體(優(yōu)選GPI合酶基因-缺陷型真菌)。載體和宿主可以是前面所述的載體和宿主。
如本文所用，表達(dá)“GPI合酶基因缺陷”是指該基因的功能產(chǎn)物未表達(dá)或表達(dá)水平減少。本發(fā)明的GPI合酶基因-缺陷型真菌可通過(guò)破壞GPI基因來(lái)加以制備。破壞可以通過(guò)在同源重組技術(shù)等的基礎(chǔ)上嵌入與該基因無(wú)關(guān)的DNA如選擇標(biāo)記來(lái)實(shí)現(xiàn)。更具體的說(shuō)，所述突變型真菌可通過(guò)將選擇標(biāo)記盒導(dǎo)入酵母，該選擇標(biāo)記盒含有利用引物擴(kuò)增的粟酒裂殖酵母his5基因或卡那霉素抗性基因(Longtine等，Yeast，14953-961，1998)，其均含有與部分該基因(50-70個(gè)核苷酸)同源的核苷酸序列。
本發(fā)明的GPI合酶基因-缺陷型真菌包括，如GWT1溫度敏感突變株gwt1-20，GPI7破壞菌株，GPI8突變株gpi8-1，以及GPI10溫度敏感突變株per13-1。
轉(zhuǎn)化了本發(fā)明簡(jiǎn)并突變體DNA的GPI合酶基因-缺陷型真菌可以通過(guò)將插入了該簡(jiǎn)并突變體DNA的載體導(dǎo)入真菌中來(lái)進(jìn)行制備。pRS316，YEp351等可用作釀酒酵母的載體，pcL，pALSK等可用作粟酒裂殖酵母的載體。
本發(fā)明還提供了篩選抗瘧藥物的方法，該方法包括利用上述GPI合酶基因-缺陷型真菌。
在該方法中，第一步包括將試樣與GPI合酶基因-缺陷型真菌接觸，其中所述真菌轉(zhuǎn)化了比參與瘧原蟲GPI生物合成的蛋白的編碼DNA具有更低AT含量的簡(jiǎn)并突變體DNA。所述“接觸”可通過(guò)將試樣加到上述真菌培養(yǎng)物中來(lái)實(shí)現(xiàn)。當(dāng)試樣是蛋白時(shí)，可以將包含該蛋白的編碼DNA的載體導(dǎo)入上述真菌中。
在本發(fā)明的方法中，下一步包括測(cè)量上述真菌的生長(zhǎng)程度。更具體地說(shuō)，在常規(guī)培養(yǎng)條件下接種真菌，具體地，將真菌接種至液體培養(yǎng)基，如酵母提取物聚蛋白胨葡萄糖培養(yǎng)基(YPD培養(yǎng)基)或瓊脂平板上，然后在25-37℃溫育4-72小時(shí)。這樣，就可以對(duì)已轉(zhuǎn)化本發(fā)明簡(jiǎn)并突變體DNA的GPI合酶基因-缺陷型真菌進(jìn)行生長(zhǎng)評(píng)估。生長(zhǎng)程度還可以以培養(yǎng)基液體的混濁度，菌落數(shù)量，或者瓊脂平板上形成的斑點(diǎn)大小或顏色作為指標(biāo)來(lái)進(jìn)行測(cè)定。在本發(fā)明的方法中，下一步包括選出抑制上述真菌生長(zhǎng)的化合物。
在另一可選的方法中，第一步包括將試樣與GPI合酶基因-缺陷型真菌接觸，其中該真菌已導(dǎo)入了上述簡(jiǎn)并突變體DNA。下一步包括測(cè)定轉(zhuǎn)運(yùn)到酵母細(xì)胞壁上的GPI-錨定蛋白量。該檢測(cè)方法包括(1)利用報(bào)告酶的方法；(2)利用與真菌細(xì)胞壁表面糖蛋白反應(yīng)的抗體的方法；(3)利用粘附動(dòng)物細(xì)胞的能力的方法；和(4)利用光學(xué)顯微鏡或電子顯微鏡觀察真菌的方法。在本發(fā)明的方法中，下一步包括選出降低轉(zhuǎn)運(yùn)到細(xì)胞壁的GPI-錨定蛋白量的樣品。
本發(fā)明提供了利用參與GPI生物合成的蛋白篩選抗瘧藥物的方法，該蛋白是利用本發(fā)明的簡(jiǎn)并突變體DNA制得的。所述方法包括例如結(jié)合分析系統(tǒng)，在該系統(tǒng)中進(jìn)行篩選是為了選出結(jié)合參與GPI生物合成的蛋白的化合物，該化合物與結(jié)合所述蛋白的標(biāo)記化合物競(jìng)爭(zhēng)。具體地說(shuō)，將本發(fā)明的簡(jiǎn)并突變體DNA導(dǎo)入GPI合酶基因-缺陷型真菌中，在該真菌中表達(dá)該DNA編碼的蛋白，獲得所表達(dá)的蛋白。然后，將制得的蛋白與試樣和能結(jié)合該蛋白的標(biāo)記化合物接觸。在下一步中，檢測(cè)結(jié)合該蛋白的標(biāo)記化合物，選出降低結(jié)合該蛋白的標(biāo)記化合物量的試樣。
本發(fā)明還提供了GlcN-PI?；D(zhuǎn)移酶分析系統(tǒng)。該系統(tǒng)包括使用GWT1蛋白，該蛋白是利用編碼具有補(bǔ)償GWT1-缺陷型酵母表型的活性的蛋白的DNA制得的，其中所述DNA是編碼瘧原蟲GWT1蛋白的DNA簡(jiǎn)并突變體，它比天然DNA具有更低的AT含量。具體地，將簡(jiǎn)并突變體DNA導(dǎo)入GWT1-缺陷型真菌，在該真菌中表達(dá)該簡(jiǎn)并突變體DNA編碼的蛋白，獲得所表達(dá)的蛋白。然后，將此蛋白與試樣接觸，檢測(cè)GlcN-(?；?PI，選出降低GlcN-(?；?PI量的試樣。
本文引用的所有專利，專利申請(qǐng)和公開(kāi)物其全部引入以供參考。
附圖的簡(jiǎn)單說(shuō)明

圖1顯示四分體分析結(jié)果的照片。在導(dǎo)入了opfGWT1-過(guò)表達(dá)質(zhì)粒后，gwt1遭破壞的菌株存活。將衍生自單個(gè)雙倍體細(xì)胞的四個(gè)孢子垂直點(diǎn)樣。
如果一個(gè)拷貝的GWT1基因被破壞，則僅有一半的孢子生長(zhǎng)。在這種情況下，[菌落-形成的斑點(diǎn)]∶[未顯示生長(zhǎng)的斑點(diǎn)]的比率為2∶2。在用箭頭標(biāo)記的縱列中，gwt1破壞體(disruptant)的致死表型由于導(dǎo)入了opfGWT1而得到補(bǔ)償，因此4個(gè)斑點(diǎn)全部生長(zhǎng)，均形成菌落。
圖2的圖表顯示化合物對(duì)于表達(dá)opfGWT1基因的酵母生長(zhǎng)的抑制活性。在GWT1基因遭破壞的酵母中表達(dá)酵母GWT1基因或opfGWT1基因。
具有抑制酵母GWT1-依賴性生長(zhǎng)活性的化合物也顯示了對(duì)于酵母的opfGWT1-依賴性生長(zhǎng)的抑制活性，其中opfGWT1在該酵母中表達(dá)。
圖3是顯示抗瘧活性的圖表。用惡性瘧原蟲感染人紅細(xì)胞。將GWT1-抑制性化合物加至這些紅細(xì)胞中，并確定對(duì)瘧原蟲感染的抑制。
表現(xiàn)抗真菌活性的5種化合物均抑制了紅細(xì)胞的瘧原蟲感染。
實(shí)施本發(fā)明的最佳方式下面，將利用實(shí)施例詳細(xì)地描述本發(fā)明，但是本發(fā)明并不限于此。
惡性瘧原蟲GWT1(PfGWT1)(1)惡性瘧原蟲GWT1(PfGWT1)的核苷酸序列(SEQ ID NO1)已公開(kāi)于惡性瘧原蟲基因組數(shù)據(jù)庫(kù)(PlasmoDB database，http://plasmodb.org/)中。用純化自惡性瘧原蟲(3D7菌株)的基因組DNA作為模板，通過(guò)PCR克隆PfGWT1基因。將PfGWT1基因的5和3’兩部分分別制備，并在XbaI(TCTAGA)限制酶位點(diǎn)對(duì)兩部分進(jìn)行組裝。這樣，就制得全長(zhǎng)PfGWT1基因。另外，在編碼區(qū)外的限制酶位點(diǎn)也包含在其中，從而可以插入到表達(dá)載體內(nèi)。
(2)用惡性瘧原蟲基因組DNA作為模板，以及引物pf152F(SEQ ID NO48)和pf136R(SEQ ID NO49)，通過(guò)PCR擴(kuò)增PfGWT1基因的5’部分。用pf137F(SEQ ID NO50)和pf151R(SEQ ID NO51)作為引物，通過(guò)上述相同的方法，擴(kuò)增PfGWT1基因的3’部分。將擴(kuò)增出的DNA片斷亞克隆到pT7-Blue載體(Novagen)中，對(duì)插入子的核苷酸序列進(jìn)行測(cè)序，以確定其與SEQ ID NO1的同源性。含有PfGWT1基因5’部分的克隆命名為PF15-5克隆。含有PfGWT1基因3’部分的克隆命名為PF20-9克隆。
(3)利用PCR將限制酶的切割位點(diǎn)添加到編碼區(qū)的外側(cè)，從而可以使PfGWT1基因插到表達(dá)載體內(nèi)。用PF15-5作為模板，以及引物pf154FE(SEQID NO52)和pf157R(SEQ ID NO53)，通過(guò)PCR將EcoRI切割位點(diǎn)加至5’部分。將擴(kuò)增的DNA片斷亞克隆到pT7-Blue載體(Novagen)中，制得pT7-plasmN2克隆。同樣地，用PF20-9作為模板，以及引物pf168BK(SEQID NO54)和pf155RK(SEQ ID NO55)，通過(guò)PCR擴(kuò)增3’部分。對(duì)擴(kuò)增的DNA片斷進(jìn)行亞克隆，制得pT7-plasmBK5克隆。
(4)用下述方法制備全長(zhǎng)PfGWT1基因。用限制性內(nèi)切酶EcoRI和KpnI消化酵母表達(dá)載體YEp352GAPII。將來(lái)源于pT7-plasmN2的EcoRI-XbaI片斷(約1500bp)，和來(lái)源于pT7-plasmBK5的XbaI-KpnI片斷(約1100bp)插入載體的切割位點(diǎn)。這樣，就構(gòu)建成了包含全長(zhǎng)PfGWT1的表達(dá)載體YEp352GAPII-PfGWT1。
ATGACAATGTGGGGAAGTCAACGGg(SEQ ID NO48)[pf136R]TGTGTGGTTACCGTTCTTTGAATACATAGA(SEQ ID NO49)[pf137F]ATAGAAAATGATTTATGGTACAGCTCAAA(SEQ ID NO50)[pf151R]AGACCAAATTAATTATGCCTTTACATGTAC(SEQ ID NO51)[pf154FE]agaattcaccATGAGCA ACATGA ATATACTTGCGTATCTT (SEQ TDNO52)[pf157R]GAAATTCCAATGTATTCCATATTCACTTAT(SEQ ID NO53)[pf168BK]AAGATCTAATACATTAAAACATTTTAGATTAATGAATATGTG(SEQ ID NO54)[pf155RK]aggtaccGTACACTCCACTCTATGATGATCATTC (SEQ ID NO55)[實(shí)施例2]全合成的PfGWT1基因惡性瘧原蟲DNA中的腺嘌呤和胸腺嘧啶(AT)比例非常高(80％或更高)，因此通常無(wú)法使用常規(guī)的生物技術(shù)(PCR，以大腸桿菌為基礎(chǔ)的基因工程，重組蛋白的表達(dá)系統(tǒng)等)(Sato和Horii；Protein，Nucleic acid，and Enzyme Vol.48，149-155，2003)。同樣地，PfGWT1 DNA的AT含量是80.41％，其包含多個(gè)連續(xù)的A或T序列段。因此，可以推斷該基因?qū)⒑茈y在酵母中復(fù)制并以蛋白形式表達(dá)。實(shí)際上，當(dāng)將連有酵母過(guò)表達(dá)載體的天然PfGWT1導(dǎo)入GWT1遭破壞的酵母時(shí)，PfGWT1根本不能拯救GWT1破壞體的致死表型。為了降低AT含量，用不改變?cè)景被犴樞虻耐x密碼子替換密碼子。
在惡性瘧原蟲GWT1(PfGWT1)核苷酸序列(SEQ ID NO1)的基礎(chǔ)上進(jìn)行密碼子取代，所述核苷酸序列已在惡性瘧原蟲基因組數(shù)據(jù)庫(kù)(PlasmoDBdatabase，http://plasmodb.org/)中公開(kāi)。將所得核苷酸序列命名為“優(yōu)化的PfGWT1(opfGWT1)”(SEQ ID NO5)。
將上述序列設(shè)計(jì)為編碼區(qū)外包含額外的序列；即EcoRI切割位點(diǎn)序列(GAATTC，5’末端)，Kozak序列(ACC，3’末端)，和KpnI切割位點(diǎn)序列(GGTACC，3’末端)。所得序列的合成委托美國(guó)的Blue Heron Inc。用這些額外的限制酶位點(diǎn)將完全合成的opfGWT1與YEp352GAPII載體連接，從而構(gòu)建opfGWT1的過(guò)表達(dá)質(zhì)粒。將該構(gòu)建體引入僅僅單個(gè)GWT1基因拷貝被破壞的二倍體細(xì)胞(WDG2)中。在含孢子形成培養(yǎng)基的平板上培養(yǎng)獲得的轉(zhuǎn)化體，形成用于四分體分析的孢子。
新設(shè)計(jì)的密碼子-修飾opfGWT1的AT含量減少至61.55％。四分體分析結(jié)果示于圖1。在導(dǎo)入opfGWT1過(guò)表達(dá)質(zhì)粒后，gwt1遭破壞的菌株存活。上述發(fā)現(xiàn)表明在通過(guò)密碼子修飾而使AT含量降低時(shí)，PfGWT1基因可以在酵母細(xì)胞中表達(dá)。
利用opfGWT1-表達(dá)型酵母進(jìn)行抗瘧活性分析利用opfGWT1-表達(dá)型酵母構(gòu)建抗瘧活性化合物的篩選系統(tǒng)。
通過(guò)將釀酒酵母GWT1終止子，釀酒酵母GAPDH啟動(dòng)子以及多克隆位點(diǎn)插到單拷貝載體pRS316的SacI-KpnI位點(diǎn)來(lái)構(gòu)建表達(dá)盒。分別將釀酒酵母GWT1和opfGWT1插到多克隆位點(diǎn)，制備pGAP-ScGWT1和pGAP-opfGWT1質(zhì)粒。將這些質(zhì)粒導(dǎo)入GWT1破壞體。用YPAD連續(xù)的兩倍稀釋化合物(1)，使其最高的終濃度為50μg/ml。將稀釋化合物的50μl等分試樣加到96-孔板的每個(gè)孔中。將含有各種質(zhì)粒的酵母細(xì)胞過(guò)夜培養(yǎng)物稀釋1000倍，然后將稀釋物的50μl等分試樣加到各孔中。將平板在30℃溫育2天，然后在660nm測(cè)定培養(yǎng)物混濁度(圖2和表1)[表1]

雖然GWT1破壞體不能存活，但菌株在導(dǎo)入每種質(zhì)粒后能存活(如0μg/ml的化合物濃度所示)?；衔?1)抑制ScGWT1-表達(dá)型酵母的生長(zhǎng)，化合物(1)是GWT1-特異性抑制劑。以25μg/ml使用該化合物，約有85％的生長(zhǎng)受抑制。當(dāng)以50μg/ml使用該化合物時(shí)，酵母完全不能存活。化合物(1)也抑制opfGWT1-表達(dá)型酵母的生長(zhǎng)。以25μg/ml使用該化合物，約有50％的生長(zhǎng)受抑制。當(dāng)以50μg/ml使用該化合物時(shí)，酵母完全不能存活。由于opfGWT1-表達(dá)型酵母的生長(zhǎng)是由導(dǎo)入的opfGWT1的活性來(lái)決定的，因此生長(zhǎng)抑制可歸因于化合物(1)對(duì)opfGWT1功能的抑制作用。此發(fā)現(xiàn)說(shuō)明利用該分析系統(tǒng)，通過(guò)篩選化合物可以鑒定出具有惡性瘧原蟲GWT1-特異性抑制活性GWT1的化合物。
抗瘧活性利用紅細(xì)胞培養(yǎng)系統(tǒng)，對(duì)抑制酵母GWT1的典型化合物(1)-(5)進(jìn)行抗瘧活性分析。

化合物(1)1-(4-丁基芐基)異喹啉

化合物(2)4-[4-(1-異喹啉基甲基)苯基]-3-丁炔-1-醇

化合物(3)5-丁基-2-(1-異喹啉基甲基)苯酚
化合物(4)2-(4-溴-2-氟芐基)-3-甲氧基吡啶 化合物(5)N-[2-(4-丁基芐基)-3-吡啶基]-N-甲胺具體地說(shuō)，將待測(cè)化合物溶解在100％DMSO中，用培養(yǎng)基稀釋，然后將稀釋物的80μl等分試樣加到96-孔培養(yǎng)板的每個(gè)孔中。惡性瘧原蟲FCR3菌株在37℃用含10％人血清的RPMI1640培養(yǎng)基預(yù)培養(yǎng)，然后在每個(gè)孔中加入20μl的培養(yǎng)細(xì)胞(含1 0％紅細(xì)胞)。此時(shí)，0.47％的紅細(xì)胞被感染。在5％O2，5％CO2，和90％N2的37℃條件培養(yǎng)48小時(shí)后，將瘧原蟲用吉姆薩(Giemsa)染色法染色。測(cè)定原蟲-感染的紅細(xì)胞數(shù)量，以估計(jì)感染率(圖3)。結(jié)果，顯示化合物(3)具有很強(qiáng)的抗瘧活性。其它4種化合物也顯示了抗瘧活性。化合物(4)表現(xiàn)的活性最低。這樣，抑制酵母GWT1的化合物包括具有抑制惡性瘧原蟲GWT1活性的化合物，這說(shuō)明抗瘧藥物可以在這些化合物的基礎(chǔ)上進(jìn)行合成。
工業(yè)實(shí)用性本發(fā)明成功地制得能表達(dá)瘧原蟲GWT1的真菌。采用該真菌，而不采用瘧原蟲，能篩選出靶向GPI生物合成途徑的抗瘧藥物。
到目前為止，還從未進(jìn)行過(guò)在真菌細(xì)胞中表達(dá)瘧原蟲基因以及篩選抑制該基因功能的物質(zhì)的嘗試。本發(fā)明的方法并不需要利用瘧原蟲，因此本方法證明了在后基因組時(shí)代，采用比較基因組進(jìn)行用于藥物發(fā)現(xiàn)的全新篩選方法的可能性。
序列表<110>衛(wèi)材株式會(huì)社(Eisai Co.，Ltd.)<120> 篩選在瘧原蟲中抑制GPI生物合成的化合物的方法<130> E1-A0210P<150> US60/428,589<151> 2002-11-22<160> 55<170> PatentIn version 3.1<210> 1<211> 2625<212> DNA<213>惡性瘧原蟲(Plasmodium falciparum))<220>
<221>CDS<222> (1)..(2625)<223>
<400> 1atg agc aac atg aat ata ctt gcg tat ctt ttg ata tgt ccc ttt aac48Met Ser Asn Met Asn Ile Leu Ala Tyr Leu Leu Ile Cys Pro Phe Asn1 5 10 15tta ata tat ata ttt gac ctt cct tca tat ata cct gag tta aat aaa96Leu Ile Tyr Ile Phe Asp Leu Pro Ser Tyr Ile Pro Glu Leu Asn Lys20 25 30aag ctg gag aat gac gag gtg ttt ata tat gga aaa gaa ata aga aag144Lys Leu Glu Asn Asp Glu Val Phe Ile Tyr Gly Lys Glu Ile Arg Lys35 40 45aat gaa tct gca tat tct tta cat tat gaa aaa tat tta tat gaa tta192Asn Glu Ser Ala Tyr Ser Leu His Tyr Glu Lys Tyr Leu Tyr Glu Leu50 55 60tca aga aga tat tat gag ata ata tta aaa tat aat aag gag ctc ggg240Ser Arg Arg Tyr Tyr Glu Ile Ile Leu Lys Tyr Asn Lys Glu Leu Gly65 70 75 80gtt aat cag gaa aaa gaa tat aat tta ata ata agt aga gag ata gat288Val Asn Gln Glu Lys Glu Tyr Asn Leu Ile Ile Ser Arg Glu Ile Asp85 90 95aaa aaa aaa aaa aaa caa aaa aat agt aca caa gga gaa tat aat aat336Lys Lys Lys Lys Lys Gln Lys Asn Set Thr Gln Gly Glu Tyr Asn Asn100 105 110gat gat gat aat aat tgg aaa tta ttc caa ata tat gag aag gaa gaa384Asp Asp Asp Asn Asn Trp Lys Leu Phe Gln Ile Tyr Glu Lys Glu Glu115 120 125ccc aga tca tac gaa tta ata cgt gtt gag att tac aaa aaa gat att432
Pro Arg Ser Tyr Glu Leu Ile Arg Val Glu Ile Tyr Lys Lys Asp Ile130 135 140ctt tta att tat aaa aat gaa aaa acc aaa tca tca ata aaa ttt ata480Leu Leu Ile Tyr Lys Asn Glu Lys Thr Lys Ser Ser Ile Lys Phe Ile145 150 155 160ata aag aaa aga aaa gat ata aaa aat tat ttc tca tta tgt tat caa528Ile Lys Lys Arg Lys Asp Ile Lys Asn Tyr Phe Ser Leu Cys Tyr Gln165 170175aat tgt ata aat aaa tta gat aaa aat gat tat aat att tta aaa agt576Asn Cys Ile Asn Lys Leu Asp Lys Asn Asp Tyr Asn Ile Leu Lys Ser180 185 190aca ata aat aat agt aaa gaa aat ata att aat agt gct tat ata tat624Thr Ile Asn Asn Ser Lys Glu Asn Ile Ile Asn Ser Ala Tyr Ile Tyr195 200 205atg tat ata ata ttt ttc ttt tta tgt ata tat gta gaa aaa aat tta672Met Tyr Ile Ile Phe Phe Phe Leu Cys Ile Tyr Val Glu Lys Asn Leu210 215 220ttt tta tac ttt cct ata tta tta caa aag tat gaa ata cta aca aca720Phe Leu Tyr Phe Pro Ile Leu Leu Gln Lys Tyr Glu Ile Leu Thr Thr225 230 235240tta ttt att tta ttt att cct cta att ctt ttt gtt ttt ttt tat ttt768Leu Phe Ile Leu Phe Ile Pro Leu Ile Leu Phe Val Phe Phe Tyr Phe245 250 255tat ttt act ata atc aag ttg ata tgt tct tgt cta gtt tta tat gta816Tyr Phe Thr Ile Ile Lys Leu Ile Cys Ser Cys Leu Val Leu Tyr Val260 265 270aca ttt caa tta att tat tat act caa ggt atg cct ata tat atg gaa864Thr Phe Gln Leu Ile Tyr Tyr Thr Gln Gly Met Pro Ile Tyr Met Glu275 280 285cat agc ata ttg aaa cat aaa gaa aaa gaa gaa att tgt gat gaa aaa912His Ser Ile Leu Lys His Lys Glu Lys Glu Glu Ile Cys Asp Glu Lys290 295 300gaa gaa att tgt gat gaa aaa gaa gaa att tgt gat gaa aaa gaa gaa960Glu Glu Ile Cys Asp Glu Lys Glu Glu Ile Cys Asp Glu Lys Glu Glu305 310 315320att tgt gat gaa aaa gaa gaa att tgt gat gaa aaa gaa gaa att tgt1008Ile Cys Asp Glu Lys Glu Glu Ile Cys Asp Glu Lys Glu Glu Ile Cys325 330 335gat gaa aaa gaa gaa att ctt gat aaa aaa aaa aaa att cat gaa aaa1056Asp Glu Lys Glu Glu Ile Leu Asp Lys Lys Lys Lys Ile His Glu Lys340 345 350aaa aaa aaa att cat gat aaa aaa gaa gaa att gat gaa aaa aaa aaa1104Lys Lys Lys Ile His Asp Lys Ly1s Glu Glu Ile Asp Glu Lys Lys Lys355 360 365
aaa att cat gat aaa aaa gac gaa agt cat gat aaa aat gaa gac ata1152Lys Ile His Asp Lys Lys Asp Glu Ser His Asp Lys Asn Glu Asp Ile370 375 380aca tat cct gtt caa tat aat ata gaa aat gat tta tgg tac agc tca1200Thr Tyr Pro Val Gln Tyr Asn Ile Glu Asn Asp Leu Trp Tyr Ser Ser385 390 395400aaa aat gta gat att aaa atg tat tca tct tca aat aaa gga gaa gaa1248Lys Asn Val Asp Ile Lys Met Tyr Ser Ser Ser Asn Lys Gly Glu Glu405 410 415tat att ata cag aat aca tta aaa cat ttt aga tta atg aat atg tgt1296Tyr Ile Ile Gln Asn Thr Leu Lys His Phe Arg Leu Met Asn Met Cys420 425 430atg aca tat ata tgt atc ttt gct gtt gat ttt tat ttc ttt cca aat1344Met Thr Tyr Ile Cys Ile Phe Ala Val Asp Phe Tyr Phe Phe Pro Asn435 440 445cat ttt tgt aaa tcc tat tat tat gga aat aca cta atg gat ata ggg1392His Phe Cys Lys Ser Tyr Tyr Tyr Gly Asn Thr Leu Met Asp Ile Gly450 455 460ata ggt gct tct ata agc tcc agt gca tat tct caa gaa ata aaa aag1440Ile Gly Ala Ser Ile Ser Ser Ser Ala Tyr Ser Gln Glu Ile Lys Lys465 470 475 480ttt aca tat ata aaa gag aag aaa aga ata att gaa tta aaa cat ata1488Phe Thr Tyr Ile Lys Glu Lys Lys Arg Ile Ile Glu Leu Lys His Ile485 490495gtt tta ttt att tta gga ata tct aga ttt att gga ata tat ctt ttt1536Val Leu Phe Ile Leu Gly Ile Ser Arg Phe Ile Gly Ile Tyr Leu Phe500 505 510aat tat aat tat aat ata agt gaa tat gga ata cat tgg aat ttc ttt1584Asn Tyr Asn Tyr Asn Ile Ser Glu Tyr Gly Ile His Trp Asn Phe Phe515 520525tta aca tta tgt act aca ttt ctt ata tct aat ata tgt ttt att tta1632Leu Thr Leu Cys Thr Thr Phe Leu Ile Ser Asn Ile Cys Phe Ile Leu530 535 540tta aaa agg ata cgt tat att ttt ctt ttt agt ata atc tct ata att1680Leu Lys Arg Ile Arg Tyr Ile Phe Leu Phe Ser Ile Ile Ser Ile Ile545 550 555 560ctt ttt gaa ata gcc ata tac tat ttt gac tta cat aat tat ata tta1728Leu Phe Glu Ile Ala Ile Tyr Tyr Phe Asp Leu His Asn Tyr Ile Leu565 570 575tta aaa aat gat aga tta aat ttc ttt tca tca aac aaa gaa ggc tta1776Leu Lys Asn Asp Arg Leu Asn Phe Phe Ser Ser Asn Lys Glu Gly Leu580 585 590ttt aat att ata ggt tct gtt aat ttg tat tta ttt tct ttc tca tta1824
Phe Asn Ile Ile Gly Ser Val Asn Leu Tyr Leu Phe Ser Phe Ser Leu595 600 605ttt aaa tat tta aca aaa caa agg aca tac ata aca acc tcg aac att1872Phe Lys Tyr Leu Thr Lys Gln Arg Thr Tyr Ile Thr Thr Ser Asn Ile610 615 620cca aaa aat aaa aag gat atg aat aat tct atg tat tca aag aac ggt1920Pro Lys Asn Lys Lys Asp Met Asn Asn Ser Met Tyr Ser Lys Asn Gly625 630 635 640aac cac aca aat agc aat ata aat aat agg aat cat aaa att gta att1968Asn His Thr Asn Ser Asn Ile Asn Asn Arg Asn His Lys Ile Val Ile645 650655cgg aat aat cat ata aat aaa tat gaa caa gat aac aca aat aag tat2016Arg Asn Asn His Ile Asn Lys Tyr Glu Gln Asp Asn Thr Asn Lys Tyr660 665 670att aat aaa caa ata aat aat aat aag aat aaa ctt gat gaa gaa gaa2064Ile Asn Lys Gln Ile Asn Asn Asn Lys Asn Lys Leu Asp Glu Glu Glu675 680 685aaa tta aaa aaa tta aaa aaa tta aaa aac aaa aaa aaa aat tta aaa2112Lys Leu Lys Lys Leu Lys Lys Leu Lys Asn Lys Lys Lys Asn Leu Lys690 695 700aaa aaa att aaa tat tat ttg tta tac ctt caa tat ata ata aat ata2160Lys Lys Ile Lys Tyr Tyr Leu Leu Tyr Leu Gln Tyr Ile Ile Asn Ile705 710 715 720tat aaa gaa gaa tat tat act att tat tat aat ata aaa tta att ata2208Tyr Lys Glu Glu Tyr Tyr Thr Ile Tyr Tyr Asn Ile Lys Leu Ile Ile725 730735tca tct ttt att ttt tat tta tta cat ata att ctt aat tta tat aaa2256Ser Ser Phe Ile Phe Tyr Leu Leu His Ile Ile Leu Asn Leu Tyr Lys740 745 750aat tat agt gtg cgt ata tta tgt aat gca aat tat att ttt tta ata2304Asn Tyr Ser Val Arg Ile Leu Cys Asn Ala Asn Tyr Ile Phe Leu Ile755 760 765aca tca ttg ggt ttg ttt tct tgt gcc ttg agt ttt tcc tta gaa gat2352Thr Ser Leu Gly Leu Phe Ser Cys Ala Leu Ser Phe Ser Leu Glu Asp770 775 780ata tta tta aga tat aaa aaa tat aaa ata aat att gat ata aca gta2400Ile Leu Leu Arg Tyr Lys Lys Tyr Lys Ile Asn Ile Asp Ile Thr Val785 790 795 800ttg gat aag ata aat aaa aat aca ttg ata gtt ttt ctt ttc tcg aat2448Leu Asp Lys Ile Asn Lys Asn Thr Leu Ile Val Phe Leu Phe Ser Asn805 810 815ata ctt gtg ggc atg ttc aat att cta ttt caa act tta tta tta cct2496Ile Leu Val Gly Met Phe Asn Ile Leu Phe Gln Thr Leu Leu Leu Pro820 825830
ctt ata ttt gta ata cct ata ttg gta ttt tat tct ttc tta ata ctg2544Leu Ile Phe Val Ile Pro Ile Leu Val Phe Tyr Ser Phe Leu Ile Leu835 840 845ctt ttt aca aaa tgc tta cca cct tcc ata cgc cat cca aaa aaa aaa2592Leu Phe Thr Lys Cys Leu Pro Pro Ser Ile Arg His Pro Lys Lys Lys850855 860aca cat cat gag gaa aag caa aaa aaa gaa tga2625Thr His His Glu Glu Lys Gln Lys Lys Glu865 870<210> 2<211> 874<212>PRT<213>惡性瘧原蟲(Plasmodium falciparum)<400> 2Met Ser Asn Met Asn Ile Leu Ala Tyr Leu Leu Ile Cys Pro Phe Asn1 5 10 15Leu Ile Tyr Ile Phe Asp Leu Pro Ser Tyr Ile Pro Glu Leu Asn Lys20 25 30Lys Leu Glu Asn Asp Glu Val Phe Ile Tyr Gly Lys Glu Ile Arg Lys35 40 45Asn Glu Ser Ala Tyr Ser Leu His Tyr Glu Lys Tyr Leu Tyr Glu Leu50 55 60Ser Arg Arg Tyr Tyr Glu Ile Ile Leu Lys Tyr Asn Lys Glu Leu Gly65 70 75 80Val Asn Gln Glu Lys Glu Tyr Asn Leu Ile Ile Ser Arg Glu Ile Asp85 90 95Lys Lys Lys Lys Lys Gln Lys Asn Ser Thr Gln Gly Glu Tyr Asn Asn100 105 110Asp Asp Asp Asn Asn Trp Lys Leu Phe Gln Ile Tyr Glu Lys Glu Glu115 120 125Pro Arg Ser Tyr Glu Leu Ile Arg Val Glu Ile Tyr Lys Lys Asp Ile130 l35 140Leu Leu Ile Tyr Lys Asn Glu Lys Thr Lys Ser Ser Ile Lys Phe Ile145 150 155 160Ile Lys Lys Arg Lys Asp Ile Lys Asn Tyr Phe Ser Leu Cys Tyr Glnl65 170 175Asn Cys Ile Asn Lys Leu Asp Lys Asn Asp Tyr Asn Ile Leu Lys Ser180 185 190Thr Ile Asn Asn Ser Lys Glu Asn Ile Ile Asn Ser Ala Tyr Ile Tyr195 200 205Met Tyr Ile Ile Phe Phe Phe Leu Cys Ile Tyr Val Glu Lys Asn Leu210 215 220
Phe Leu Tyr Phe Pro Ile Leu Leu Gln Lys Tyr Glu Ile Leu Thr Thr225 230 235240Leu Phe Ile Leu Phe Ile Pro Leu Ile Leu Phe Val Phe Phe Tyr Phe245 250 255Tyr Phe Thr Ile Ile Lys Leu Ile Cys Ser Cys Leu Val Leu Tyr Val260265 270Thr Phe Gln Leu Ile Tyr Tyr Thr Gln Gly Met Pro Ile Tyr Met Glu275 280 285His Ser Ile Leu Lys His Lys Glu Lys Glu Glu Ile Cys Asp Glu Lys290 295 300Glu Glu Ile Cys Asp Glu Lys Glu Glu Ile Cys Asp Glu Lys Glu Glu305 310 315320Ile Cys Asp Glu Lys Glu Glu Ile Cys Asp Glu Lys Glu Glu Ile Cys325 330 335Asp Glu Lys Glu Glu Ile Leu Asp Lys Lys Lys Lys Ile His Glu Lys340 345 350Lys Lys Lys Ile His Asp Lys Lys Glu Glu Ile Asp Glu Lys Lys Lys355 360 365Lys Ile His Asp Lys Lys Asp Glu Ser His Asp Lys Asn Glu Asp Ile370 375 380Thr Tyr Pro Val Gln Tyr Asn Ile Glu Asn Asp Leu Trp Tyr Ser Ser385 390 395400Lys Asn Val Asp Ile Lys Met Tyr Ser Ser Ser Asn Lys Gly Glu Glu405 410 415Tyr Ile Ile Gln Asn Thr Leu Lys His Phe Arg Leu Met Asn Met Cys420 425 430Met Thr Tyr Ile Cys Ile Phe Ala Val Asp Phe Tyr Phe Phe Pro Asn435 440 445His Phe Cys Lys Ser Tyr Tyr Tyr Gly Asn Thr Leu Met Asp Ile Gly450 455 460Ile Gly Ala Ser Ile Ser Ser Ser Ala Tyr Ser Gln Glu Ile Lys Lys465 470 475 480Phe Thr Tyr Ile Lys Glu Lys Lys Arg Ile Ile Glu Leu Lys His Ile485 490 495Val Leu Phe Ile Leu Gly Ile Ser Arg Phe Ile Gly Ile Tyr Leu Phe500 505 510Asn Tyr Asn Tyr Asn Ile Ser Glu Tyr Gly Ile His Trp Asn Phe Phe515 520 525Leu Thr Leu Cys Thr Thr Phe Leu Ile Ser Asn Ile Cys Phe Ile Leu
530 535 540Leu Lys Arg Ile Arg Tyr Ile Phe Leu Phe Ser Ile Ile Ser Ile Ile545 550 555 560Leu Phe Glu Ile Ala Ile Tyr Tyr Phe Asp Leu His Asn Tyr Ile Leu565 570 575Leu Lys Asn Asp Arg Leu Asn Phe Phe Ser Ser Asn Lys Glu Gly Leu580 585 590Phe Asn Ile Ile Gly Ser Val Asn Leu Tyr Leu Phe Ser Phe Ser Leu595 600 605Phe Lys Tyr Leu Thr Lys Gln Arg Thr Tyr Ile Thr Thr Ser Asn Ile610 615 620Pro Lys Asn Lys Lys Asp Met Asn Asn Ser Met Tyr Ser Lys Asn Gly625 630 635 640Asn His Thr Asn Ser Asn Ile Asn Asn Arg Asn His Lys Ile Val Ile645 650 655Arg Asn Asn His Ile Asn Lys Tyr Glu Gln Asp Asn Thr Asn Lys Tyr660 665670Ile Asn Lys Gln Ile Asn Asn Asn Lys Asn Lys Leu Asp Glu Glu Glu675 680 685Lys Leu Lys Lys Leu Lys Lys Leu Lys Asn Lys Lys Lys Asn Leu Lys690 695 700Lys Lys Ile Lys Tyr Tyr Leu Leu Tyr Leu Gln Tyr Ile Ile Asn Ile705 710 715 720Tyr Lys Glu Glu Tyr Tyr Thr Ile Tyr Tyr Asn Ile Lys Leu Ile Ile725 730 735Ser Ser Phe Ile Phe Tyr Leu Leu His Ile Ile Leu Asn Leu Tyr Lys740 745 750Asn Tyr Ser Val Arg Ile Leu Cys Asn Ala Asn Tyr Ile Phe Leu Ile755 760 765Thr Ser Leu Gly Leu Phe Ser Cys Ala Leu Ser Phe Ser Leu Glu Asp770 775780Ile Leu Leu Arg Tyr Lys Lys Tyr Lys Ile Asn Ile Asp Ile Thr Val785 790 795 800Leu Asp Lys Ile Asn Lys Asn Thr Leu Ile Val Phe Leu Phe Ser Asn805 810 815Ile Leu Val Gly Met Phe Asn Ile Leu Phe Gln Thr Leu Leu Leu Pro820 825 830Leu Ile Phe Val Ile Pro Ile Leu Val Phe Tyr Ser Phe Leu Ile Leu835 840 845Leu Phe Thr Lys Cys Leu Pro Pro Ser Ile Arg His Pro Lys Lys Lys
850 855 860Thr His His Glu Glu Lys Gln Lys Lys Glu865 870<210> 3<211> 1956<212>DNA<213> 間日瘧原蟲(Plasmodium vivax))<220>
<221>CDS<222> (1)..(19 56)<223>
<400> 3atg gcg cat ttg aac ctc ctc gtc tac ctc atc atg tgc ccc ttc aat48Met Ala His Leu Asn Leu Leu Val Tyr Leu Ile Met Cys Pro Phe Asn1 5 10 15gtg agg cac atg ctg gat gcg ccc agc ttc cea ttc cgg tta gga agc96Val Arg His Met Leu Asp Ala Pro Ser Phe Pro Phe Arg Leu Gly Ser20 25 30aaa gca gca agt ggt gaa acc ttc acg tat gga gcg act gca aga gag144Lys Ala Ala Ser Gly Glu Thr Phe Thr Tyr Gly Ala Thr Ala Arg Glu35 40 45aac ctg ggg agt tac tct ccc gca cat gac gag cta tac atg tta gag192Asn Leu Gly Ser Tyr Ser Pro Ala His Asp Glu Leu Tyr Met Leu Glu50 55 60tta gcc aaa atg tac tat aaa att gtg tta aea tat aag aag gat gtt240Leu Ala Lys Met Tyr Tyr Lys Ile Val Leu Thr Tyr Lys Lys Asp Val65 70 75 80agg aaa gga cag gag gag agt tac aac ttg gtg gta ggc tcc ttt ggg288Arg Lys Gly Gln Glu Glu Ser Tyr Asn Leu Val Val Gly Ser Phe Gly85 90 95aag gaa gcc aaa ggg gag gtc tcc ctc caa aga gtt ctc atc aca aat336Lys Glu Ala Lys Gly Glu Val Ser Leu Gln Arg Val Leu Ile Thr Asn100 105 110gat gcc gtg tac ctg tcg tac cag gat gtg caa aac gaa cgt ggg atc384Asp Ala Val Tyr Leu Ser Tyr Gln Asp Val Gln Asn Glu Arg Gly Ile115 120 125caa gtt aag ata aaa agg ggg gaa ata tct tcc tat tta gac ctc cta432Gln Val Lys Ile Lys Arg Gly Glu Ile Ser Ser Tyr Leu Asp Leu Leu130 135 140tcg tgg gat tct tgt ttg tat aag ctt aac tca gac gat tat aat tta480Ser Trp Asp Ser Cys Leu Tyr Lys Leu Asn Ser Asp Asp Tyr Asn Leu145 150 155 160atg aag agc gca tcg gat cat agc aag cca atg gtg gtc agc aca tac528Met Lys Ser Ala Ser Asp His Ser Lys Pro Met Val Val Ser Thr Tyr165 170 175
cac ata tac atg ctg ctg ctg gtg ttt tct ctt tgc act tac gtg gag576His Ile Tyr Met Leu Leu Leu Val Phe Ser Leu Cys Thr Tyr Val Glu180 185 190aag agc ctc ctg ctt gaa ttc cct gcg ttg aaa aag tgc caa gta ttt624Lys Ser Leu Leu Leu Glu Phe Pro Ala Leu Lys Lys Cys Gln Val Phe195 200 205cta acc cta tgt ttg gtg tac tgc ccg ata atc agt tac ctt ttt ttt672Leu Thr Leu Cys Leu Val Tyr Cys Pro Ile Ile Ser Tyr Leu Phe Phe210 215220ttt tac tcc cat gtg agc cta ctt ggg gtg tta ctc gtc tat gtg ttt720Phe Tyr Ser His Val Ser Leu Leu Gly Val Leu Leu Val Tyr Val Phe225 230 235 240ttt tgc ggg ctc ttc agg ggc gtc tct tgc aga agg ggg ggg cag cac768Phe Cys Gly Leu Phe Arg Gly Val Ser Cys Arg Arg Gly Gly Gln His245 250 255atg ggg gag caa acg ggc caa cac acg ggc gat tgg cac acc atc cgc816Met Gly Glu Gln Thr Gly Gln His Thr Gly Asp Trp His Thr Ile Arg260 265 270ggc aac cca caa ggt gat gat acg caa gag gag aga cgc aag tgt ttg864Gly Asn Pro Gln Gly Asp Asp Thr Gln Glu Glu Arg Arg Lys Cys Leu275 280 285gtc cat atg agg cta gcc aac ctg tgc atc acc tac ata tgc ata ttc912Val His Met Arg Leu Ala Asn Leu Cys Ile Thr Tyr Ile Cys Ile Phe290 295 300gct gtg gac ttt tat ttt ttc cca agg caa ttt tcc aag tct ttt ttt960Ala Val Asp Phe Tyr Phe Phe Pro Arg Gln Phe Ser Lys Ser Phe Phe305 310 315 320ttt ggt aac act ttg atg gat tta ggg gtg ggg ggg tgc atc aca tcg1008Phe Gly Asn Thr Leu Met Asp Leu Gly Val Gly Gly Cys Ile Thr Ser325 330 335agc gcg tat tct cta aac agt aaa aag ctc cat tct gcg aac cgc aag1056Ser Ala Tyr Ser Leu Asn Ser Lys Lys Leu His Ser Ala Asn Arg Lys340 345 350gga cac cta atc gat tgg aag cat ttc att tta ttt ttc ctt gga ata1104Gly His Leu Ile Asp Trp Lys His Phe Ile Leu Phe Phe Leu Gly Ile355 360 365gct aga tac att gca gtg aag ctt ttc aat tat aat tac agc tta act1152Ala Arg Tyr Ile Ala Val Lys Leu Phe Asn Tyr Asn Tyr Ser Leu Thr370 375 380gag tat ggg atg cac tgg aat ttt ttt ctt act ctc ttt ttt act ctc1200Glu Tyr Gly Met His Trp Asn Phe Phe Leu Thr Leu Phe Phe Thr Leu385 390 395400cta act tgt aac gcc cta ctc tgc ttg ata aga ggg gtt aaa cgc acc1248Leu Thr Cys Asn Ala Leu Leu Cys Leu Ile Arg Gly Val Lys Arg Thr
405410415ttt cac ctg agc tgc gtc ctc atc tgt ttg tat gaa att ata att tgg1296Phe His Leu Ser Cys Val Leu Ile Cys Leu Tyr Glu Ile Ile Ile Trp420 425430cgc ctg gac att acg agt tat tta gtg gtt gac gag gca gaa cgg agc1344Arg Leu Asp Ile Thr Ser Tyr Leu Val Val Asp Glu Ala Glu Arg Ser435 440 445ggc ttt ttt tcg cag aac aga gag ggc ctt atg aac gtc atc ggg tcc1392Gly Phe Phe Ser Gln Asn Arg Glu Gly Leu Met Asn Val Ile Gly Ser450 455 460gtc aat ttg tac ctc ttt tcg ttt tcg cta tgg aat ggc tat gtg ttt1440Val Asn Leu Tyr Leu Phe Ser Phe Ser Leu Trp Asn Gly Tyr Val Phe465 470 475480ccg gat gag ggg cag cag tgg gag cga gga aag gcg gcg cga aga ccg1488Pro Asp Glu Gly Gln Gln Trp Glu Arg Gly Lys Ala Ala Arg Arg Pro485 490 495gat gag gcg gcg cga acg ccg ggg gag gga cat ggc cag cgc tcc cct1536Asp Glu Ala Ala Arg Thr Pro Gly Glu Gly His Gly Gln Arg Ser Pro500 505 510gtc cgc ctc acc ctg aag ttg ctt gcc ctg tcc ctc ctc ttc cac ctg1584Val Arg Leu Thr Leu Lys Leu Leu Ala Leu Ser Leu Leu Phe His Leu515 520 525ctg cac ctg ctg ttg aat tac tac cga aat tac agt gtg cgc atc ctt1632Leu His Leu Leu Leu Asn Tyr Tyr Arg Asn Tyr Ser Val Arg Ile Leu530 535 540tgc aac gcg aac tac ata tgt gtt gtc tcc tcc gtg agt ctc ttc gcg1680Cys Asn Ala Asn Tyr Ile Cys Val Val Ser Ser Val Ser Leu Phe Ala545 550 555 560gct gcc ctg agc tac ctc gta gag aag gta ctc ctc cgc gag aag acc1728Ala Ala Leu Ser Tyr Leu Val Glu Lys Val Leu Leu Arg Glu Lys Thr565 570 575acc acc atc cca gtt ttg caa caa atg aac cgg cac tcc ctg gca gtg1776Thr Thr Ile Pro Val Leu Gln Gln Met Asn Arg His Ser Leu Ala Val580 585 590ttc ctc ttt tgc aac gta aca atg ggc act ttc aac ctc ctc ttt cag1824Phe Leu Phe Cys Asn Val Thr Met Gly Thr Phe Asn Leu Leu Phe Gln595 600 605tct ctc ttg ttt ccc cta ttt ttt gcg tgc ctc gtt ttg gcg gcg tac1872Ser Leu Leu Phe Pro Leu Phe Phe Ala Cys Leu Val Leu Ala Ala Tyr610 615 620tcc tat ggc atg ttg cgc ttc gcc tcc ctg ttg ccc ggc ccc gcg cag1920Ser Tyr Gly Met Leu Arg Phe Ala Ser Leu Leu Pro Gly Pro Ala Gln625 630 635 640
ggg gag aag gga gag aag cgg gag aag cag caa taa1936Gly Glu Lys Gly Glu Lys Arg Glu Lys Gln Gin645 650<210>4<211>651<212>PRT<213>間日瘧原蟲(Plasmodium vivax)<400>4Met Ala His Leu Asn Leu Leu Val Tyr Leu Ile Met Cys Pro Phe Asn1 5 101 5Val Arg His Met Leu Asp Ala Pro Ser Phe Pro Phe Arg Leu Gly Ser20 25 30Lys Ala Ala Ser Gly Glu Thr Phe Thr Tyr Gly Ala Thr Ala Arg Glu35 40 45Asn Leu Gly Ser Tyr Ser Pro Ala His Asp Glu Leu Tyr Met Leu Glu50 55 60Leu Ala Lys Met Tyr Tyr Lys Ile Val Leu Thr Tyr Lys Lys Asp Val65 70 75 80Arg Lys Gly Gln Glu Glu Ser Tyr Asn Leu Val Val Gly Ser Phe Gly85 90 95Lys Glu Ala Lys Gly Glu Val Ser Leu Gln Arg Val Leu Ile Thr Asn100 105 110Asp Ala Val Tyr Leu Ser Tyr Gln Asp Val Gln Asn Glu Arg Gly Ile115 120 125Gln Val Lys Ile Lys Arg Gly Glu Ile Ser Ser Tyr Leu Asp Leu Leu130 135 140Ser Trp Asp Ser Cys Leu Tyr Lys Leu Asn Ser Asp Asp Tyr Asn Leu145 150 l55 160Met Lys Ser Ala Ser Asp His Ser Lys Pro Met Val Val Ser Thr Tyr165 170 175His Ile Tyr Met Leu Leu Leu Val Phe Ser Leu Cys Thr Tyr Val Glu180 185 190Lys Ser Leu Leu Leu Glu Phe Pro Ala Leu Lys Lys Cys Gln Val Phe195 200 205Leu Thr Leu Cys Leu Val Tyr Cys Pro Ile Ile Ser Tyr Leu Phe Phe210 215 220Phe Tyr Ser His Val Ser Leu Leu Gly Val Leu Leu Val Tyr Val Phe225 230 235 240Phe Cys Gly Leu Phe Arg Gly Val Ser Cys Arg Arg Gly Gly Gln His245 250 255
Met Gly Glu Gln Thr Gly Gln His Thr Gly Asp Trp His Thr Ile Arg260 265 270Gly Asn Pro Gln Gly Asp Asp Thr Gln Glu Glu Arg Arg Lys Cys Leu275 280 285Val His Met Arg Leu Ala Asn Leu Cys Ile Thr Tyr Ile Cys Ile Phe290 295 300Ala Val Asp Phe Tyr Phe Phe Pro Arg Gln Phe Ser Lys Ser Phe Phe305 310 315 320Phe Gly Asn Thr Leu Met Asp Leu Gly Val Gly Gly Cys Ile Thr Ser325 330 335Ser Ala Tyr Ser Leu Asn Ser Lys Lys Leu His Ser Ala Asn Arg Lys340 345 350Gly His Leu Ile Asp Trp Lys His Phe Ile Leu Phe Phe Leu Gly Ile355 360 365Ala Arg Tyr Ile Ala Val Lys Leu Phe Asn Tyr Asn Tyr Ser Leu Thr370 375 380Glu Tyr Gly Met His Trp Asn Phe Phe Leu Thr Leu Phe Phe Thr Leu385 390 395 400Leu Thr Cys Asn Ala Leu Leu Cys Leu Ile Arg Gly Val Lys Arg Thr405 410 415Phe His Leu Ser Cys Val Leu Ile Cys Leu Tyr Glu Ile Ile Ile Trp420 425 430Arg Leu Asp Ile Thr Ser Tyr Leu Val Val Asp Glu Ala Glu Arg Ser435 440 445Gly Phe Phe Ser Gln Asn Arg Glu Gly Leu Met Asn Val Ile Gly Ser450 455 460Val Asn Leu Tyr Leu Phe Ser Phe Ser Leu Trp Asn Gly Tyr Val Phe465 470 475 480Pro Asp Glu Gly Gln Gln Trp Glu Arg Gly Lys Ala Ala Arg Arg Pro485 490 495Asp Glu Ala Ala Arg Thr Pro Gly Glu Gly His Gly Gln Arg Ser Pro500 505 510Val Arg Leu Thr Leu Lys Leu Leu Ala Leu Ser Leu Leu Phe His Leu515 520 525Leu His Leu Leu Leu Asn Tyr Tyr Arg Asn Tyr Ser Val Arg Ile Leu530 535 540Cys Asn Ala Asn Tyr Ile Cys Val Val Ser Ser Val Ser Leu Phe Ala545 550 555 560Ala Ala Leu Ser Tyr Leu Val Glu Lys Val Leu Leu Arg Glu Lys Thr565 570 575
Thr Thr Ile Pro Val Leu Gln Gln Met Asn Arg His Ser Leu Ala Val580 585 590Phe Leu Phe Cys Asn Val Thr Met Gly Thr Phe Asn Leu Leu Phe Gln595 600 605Ser Leu Leu Phe Pro Leu Phe Phe Ala Cys Leu Val Leu Ala Ala Tyr610 615 620Ser Tyr Gly Met Leu Arg Phe Ala Ser Leu Leu Pro Gly Pro Ala Gln625 630 635 640Gly Glu Lys Gly Glu Lys Arg Glu Lys Gln Gln645 650<210>5<211>262 5<212>DNA<213>人工的<220>
<223>人工合成的序列<400> 5atgagtaaca tgaacatcct ggcctacctg ctgatctgtc cattcaacct gatctacatc60ttcgacctgc ctagctacat ccctgagcta aacaagaagc tggagaacga cgaagtcttc 120atctacggta aggagatccg taagaacgaa tccgcatact ctctacacta cgagaagtac 180ctatacgaat tgtcacgaag atactacgag atcatcctga agtacaacaa ggagttggga 240gtcaaccaag agaaggaata caacctgatt atctccagag agatcgataa gaagaagaag 300aagcagaaga atagtaccca gggtgaatac aataacgacg atgataacaa ttggaagttg 360ttccagattt acgagaagga agaacctagg agctatgaat tgatcagggt agagatatac 420aagaaggaca ttctgttgat ctacaagaat gagaagacga agtcctctat caagttcatt 480atcaagaagc gtaaggatat caagaattac ttctccttgt gttaccagaa ctgtatcaat 540aagctggaca agaatgatta caacatcttg aagtctacca tcaacaattc caaggaaaac 600attatcaact ctgcatacat ttacatgtac attatcttct tcttcctgtg catatacgtc 660gagaagaacc tgttcttgta cttcccaata ttgcttcaga agtatgagat tctcactacg 720ttgttcatcc tcttcatccc attgatccta ttcgtattct tctatttcta cttcacgatt 780atcaagctga tttgctcatg cttagtccta tacgtgactt tccagttgat ctactatacg 840cagggaatgc ctatttacat ggaacatagt attctcaagc acaaggagaa ggaagagatt 900tgcgatgaga aggaggaaat ctgtgatgaa aaggaagaga tttgcgatga gaaggaagag 960atttgcgatg agaaggaaga gatttgtgat gagaaggaag aaatctgcga tgagaaagaa 1020
gaaatccttg ataagaagaa gaagatccac gagaagaaga agaagatcca tgataagaaa 1080gaggaaatcg atgagaagaa gaagaagatt catgacaaaa aggacgaaag tcatgataag 1140aacgaggaca ttacgtatcc agtccagtac aatatcgaga atgacctatg gtattcatcc 1200aagaacgtgg acatcaagat gtattcatcc agcaacaagg gtgaagaata cattatccag 1260aacacgttga aacatttccg attgatgaac atgtgtatga cgtacatttg tatcttcgct 1320gttgacttct acttcttccc taaccatttc tgcaagtcct actattacgg aaatacgttg 1380atggacattg gaatcggtgc atccatttct tccagtgcat actcgcagga gatcaagaag 1440ttcacgtaca ttaaggagaa gaaacgaatt atcgagttga aacatatcgt gttattcatt 1500ctgggaatta gcagatttat cggtatctac ctattcaact ataactacaa catttctgag 1560tatggaatcc attggaactt ctttcttacg ctatgtacaa cctttctgat tagcaacatt 1620tgtttcatcc tcctcaagag gattcgttat atcttcctat tcagtattat ctcgatcatc 1680ttattcgaaa tcgctattta ctacttcgat ctacataatt acatcctcct gaagaatgac 1740cgtttaaact tcttctcttc taacaaggag ggtctattca acattatcgg ttccgtgaac 1800ctttatttgt tcagtttctc actcttcaag taccttacga agcagcgtac gtatatcacg 1860acgtccaata tccctaagaa caagaaagat atgaataact caatgtattc gaagaatgga 1920aaccatacta attcgaatat caacaaccgt aaccataaga tcg tcatccg taataaccac 1980attaacaagt atgagcagga caacacgaac aagtacatca acaagcaaat caataacaac 2040aagaacaaat tggatgagga agagaagctg aagaaactca agaagcttaa gaataagaag 2100aagaacctca agaagaaaat caagtactat ctattgtacc ttcagtatat catcaacatc 2160tacaaggagg aatactatac gatctactat aacatcaagc taatcatttc gtcgtttata 2220ttctacctcc ttcacataat ccttaatctg tacaagaact actctgtgcg tattctgtgt 2280aatgcaaact acatcttcct gattacgagt ctgggtctgt tctcctgtgc attatccttc 2340tccctggagg acatcctact ccgttataag aaatacaaga tcaatatcga tattactgtg 2400ctagataaga ttaacaagaa caccttaatc gtgttcctgt ttagtaatat cctagtggga 2460atgttcaaca tcctgttcca aactttgctc ctaccactga tattcgttat ccctattcta 2520gtattctaca gcttcttgat cctgctgttc accaagtgtt tgccaccttc tatccgacat 2580cctaagaaga agacgcatca cgaagagaag cagaagaaag agtga 2625<210>6<211>2010<212>DNA<213>惡性瘧原蟲(Plasmodium falciparum)
<400>6atgtgcacta caaaaaatga agacaataat aaagtgaatt atctgtatct catttattat60aataatatac ctatatttaa aaaaaaaagt ggaaatgaac aaaacataag tgcgctattt 120attttatatg atgtaaaaaa gtatgtgtat aatatggttc atgatcacgt aaatacatta 180gtcctagaag cttttagaag agaagacata ataaaaaaaa taaaagtaaa agaaaaacaa 240aataataatg ataaaaataa agaaagtaat attgaaaaag ataaaaatga acaaaccaaa 300tttacagata tatatgatac aaatagtaag agtgacaaag atatacaaaa gaataatatg 360aacgatggtg atagtaataa taaaaatagt agtttattta ttgatccttt tgaaagtgat 420tcatatgaaa agaataattt tagtaatgaa aaatgtgctt tccaaaatgt cgataaatca 480aaaaaagata aagaacatat atattctgaa aatattactc ctagtagtag taataataat 540aatgataata ataaagaaaa tgattgtgat aaggaacaat tagataaata taataaagat 600aaagaaaata aattaaaatt aaatgataag gatgaatata tttctttcaa ttttattgaa 660gataaattaa ccgaatcatt tcatatgaat caaataattc atttaattaa taaaaaatgt 720gtatttacca aatgcctaga aaattataaa aatagatatt ttgtactgaa aaaagaagag 780attttaaaaa aaaaaaaaaa gcaaaaaaaa atgtctatat tttcatatat tgtatcaatc 840atattatttt ttacatatat catatcactt ataaatagtt gtttatatta tataatatgt 900acaccaaaat tgttcagtga atatatattt tcaaaaaaat gtgatggata tctgcagaat 960tcggcttacc ctaaatttat atttccttct gaatggcata atatattccg cagcttcatg 1020aaaaataaac aaaacccatc tgaatattat aaatatagag aaatcttatt aattcgtatt 1080atcaatttaa taatcgatat tttcttggga tttctgatat ttttactact ttattttaac 1140gtaataaacc tacattatat atcagagaag gctcaaatat tttatggaac cagtacttta 1200acttctatct tgggtacctt gttacagaat ccattaggat ttaaattaaa taataatttt 1260acgtctttca ttggtagcat ccttgtttcg atattagaca aatgggattt atttacaaat 1320accatccctg tgaataatag tacagttttg aattttgttg gatatacatc actgctgggt 1380ttttcttttt ttttgtcttt tgttattgat tatttgagat tcgtaacagc acatgttacc 1440attatttatt tatttttgaa aaaaatatgt accctttttc ataaaaatat gtattcctta 1500tacctattat tcaatgggaa aaaatggaac atactaaaac taagagttga tacaaattac 1560tactcaaatg aagaagtact tttaggaacc atattattta ccattttaat ttttttgtac 1620cctaccattt ttgttcttgt tttggttttt ggtcttatat atcttataat aaatagaatt 1680
atataccttt tgtgtgttat ggagaaaata attttgtaca cccctttcta tattttcttt 1740attcaaccga attgtaataa atatattagt aagggtttca agtttacaaa atatgaagtt 1800ggggaacatg aactgttaaa aaggtatccg actaacagct acttgttgct agaaaagatt 1860cattttttat tttttgataa aataaaattg tttataaata ttttccttta ttttaaaaac 1920ctcgagtcaa tgtcatcata ttcatatata tttatttctt tttgtttttg tcttttaaaa 1980aaaaaatata tatatatata tataatataa2010<210>7<211>669<212>PRT<213>惡性瘧原蟲(Plasmodium falciparum)<400>7Met Cys Thr Thr Lys Asn Glu Asp Asn Asn Lys Val Asn Tyr Leu Tyr1 5 10 15Leu Ile Tyr Tyr Asn Asn Ile Pro Ile Phe Lys Lys Lys Ser Gly Asn20 25 30Glu Gln Asn Ile Ser Ala Leu Phe Ile Leu Tyr Asp Val Lys Lys Tyr35 40 45Val Tyr Asn Met Val His Asp His Val Asn Thr Leu Val Leu Glu Ala50 55 60Phe Arg Arg Glu Asp Ile Ile Lys Lys Ile Lys Val Lys Glu Lys Gln65 70 75 80Asn Asn Asn Asp Lys Asn Lys Glu Ser Asn Ile Glu Lys Asp Lys Asn85 90 95Glu Gln Thr Lys Phe Thr Asp Ile Tyr Asp Thr Asn Set Lys Ser Asp100 105 110Lys Asp Ile Gln Lys Asn Asn Met Asn Asp Gly Asp Ser Asn Asn Lys115 120 125Asn Ser Ser Leu Phe Ile Asp Pro Phe Glu Ser Asp Ser Tyr Glu Lys130 135 140Asn Asn Phe Ser Asn Glu Lys Cys Ala Phe Gln Asn Val Asp Lys Ser145 150 155 160Lys Lys Asp Lys Glu His Ile Tyr Ser Glu Asn Ile Thr Pro Ser Ser165 170 175Ser Asn Asn Asn Asn Asp Asn Asn Lys Glu Asn Asp Cys Asp Lys Glu180 185 190Gln Leu Asp Lys Tyr Asn Lys Asp Lys Glu Asn Lys Leu Lys Leu Asn195 200 205
Asp Lys Asp Glu Tyr Ile Ser Phe Asn Phe Ile Glu Asp Lys Leu Thr210 215 220Glu Ser Phe His Met Asn Gln Ile Ile His Leu Ile Asn Lys Lys Cys225 230 235 240Val Phe Thr Lys Cys Leu Glu Asn Tyr Lys Asn Arg Tyr Phe Val Leu245 250 255Lys Lys Glu Glu Ile Leu Lys Lys Lys Lys Lys Gln Lys Lys Met Ser260 265 270Ile Phe Ser Tyr Ile Val Ser Ile Ile Leu Phe Phe Thr Tyr Ile Ile275 280 285Ser Leu Ile Asn Ser Cys Leu Tyr Tyr Ile Ile Cys Thr Pro Lys Leu290 295 300Phe Ser Glu Tyr Ile Phe Ser Lys Lys Cys Asp Gly Tyr Leu Gln Asn305 310 315 320Ser Ala Tyr Pro Lys Phe Ile Phe Pro Ser Glu Trp His Asn Ile Phe325 330 335Arg Ser Phe Met Lys Asn Lys Gln Asn Pro Ser Glu Tyr Tyr Lys Tyr340 345 350Arg Glu Ile Leu Leu Ile Arg Ile Ile Asn Leu Ile Ile Asp Ile Phe355 360 365Leu Gly Phe Leu Ile Phe Leu Leu Leu Tyr Phe Asn Val Ile Asn Leu370 375 380His Tyr Ile Ser Glu Lys Ala Gln Ile Phe Tyr Gly Thr Ser Thr Leu385 390 395 400Thr Ser Ile Leu Gly Thr Leu Leu Gln Asn Pro Leu Gly Phe Lys Leu405 410 415Asn Asn Asn Phe Thr Ser Phe Ile Gly Ser Ile Leu Val Ser Ile Leu420 425 430Asp Lys Trp Asp Leu Phe Thr Asn Thr Ile Pro Val Asn Asn Ser Thr435 440 445Val Leu Asn Phe Val Gly Tyr Thr Ser Leu Leu Gly Phe Ser Phe Phe450 455 460Leu Ser Phe Val Ile Asp Tyr Leu Arg Phe Val Thr Ala His Val Thr465 470 475 480Ile Ile Tyr Leu Phe Leu Lys Lys Ile Cys Thr Leu Phe His Lys Asn485 490 495Met Tyr Ser Leu Tyr Leu Leu Phe Asn Gly Lys Lys Trp Asn Ile Leu500 505 510Lys Leu Arg Val Asp Thr Asn Tyr Tyr Ser Asn Glu Glu Val Leu Leu
515 520 525Gly Thr Ile Leu Phe Thr Ile Leu Ile Phe Leu Tyr Pro Thr Ile Phe530 535 540Val Leu Val Leu Val Phe Gly Leu Ile Tyr Leu Ile Ile Asn Arg Ile545 550 555 560Ile Tyr Leu Leu Cys Val Met Glu Lys Ile Ile Leu Tyr Thr Pro Phe565 570 575Tyr Ile Phe Phe Ile Gln Pro Asn Cys Asn Lys Tyr Ile Ser Lys Gly580 585 590Phe Lys Phe Thr Lys Tyr Glu Val Gly Glu His Glu Leu Leu Lys Arg595 600 605Tyr Pro Thr Asn Ser Tyr Leu Leu Leu Glu Lys Ile His Phe Leu Phe610 615 620Phe Asp Lys Ile Lys Leu Phe Ile Asn Ile Phe Leu Tyr Phe Lys Asn625 630 635 640Leu Glu Ser Met Ser Ser Tyr Ser Tyr Ile Phe Ile Ser Phe Cys Phe645 650 655Cys Leu Leu Lys Lys Lys Tyr Ile Tyr Ile Tyr Ile Ile660 665<210> 8<211>1482<212>DNA<213>惡性瘧原蟲(Plasmodium falciparum)<400>8atggggataa aaattataat ttatatattt tttctatcgt gggcgaaatg ggtgtgtggg 60tctgttaatt ttaccgggtt tgataacaaa aatatgatag gtaagcacgt ggaactagaa120gggaggtata aaaaggaata tatagatagg ttttttttag aagaattaag aaaacacaat180tatatgaata acaatgttat attattaagt acatcaagac attattttaa ttatagacat240accactaatt tattgattgc atataaatat cttaaatatt tcggtgatac tatggataag300aatattttat taatgattcc atttgatcaa gcttgtgatt gtaggaatat aagagaaggt360caaatatttc gagaatatga attatttcct agtagtcata ataaagaaac aaaaatagaa420aatataaatt tatatgagaa tttaaatatt gattataaaa ataataatgt acgtgatgaa480caaattagaa gagtacttag acatagatat gatgctttta cacctaaaaa aaatagatta540tataataatg gaaataatga gaaaaattta tttctttata tgaccggaca tggtggtgtg600aattttttaa aaattcaaga atttaatatt attagttctt ctgaatttaa tatatatata660
caagaattac ttatcaaaaa tttttataaa tatatattcg taattattga tacgtgtcaa 720ggatatagtt tttatgatga tattctaaat tttgtatata aaaaaaaaat taataatatc 780ttctttttat catcttctaa aagaaatgaa aatagttata gtttattttc cagtagttat 840ttaagcgttt caacggtcga cagatttaca taccattttt ttaattatct tcaacaaata 900cataaaatat atgaaaaaga acgatctaaa aatataaaag ccttttcatt atataacatt 960ttaaattatt taaaaacaca acatattatg tcagaaccta ctacaaataa ttctaaattt 1020aattcgtcca tttttttaca tgataaaaat attcttttct tcaattctaa tttgttaatt 1080atacataaag atgatgtttc tataatatat caagataaac aaacacacaa tcacaaatat 1140atatgtttgg ataatctatc taaatgtggt catataaaaa ataatgtaca taaaaaaatg 1200caaactctat atgaacaaac gttatattat aataataatc aacagaattt cttttctaat 1260catatgtcta attttacaga ttattttttt acacatgata tatataatat atataatata 1320tataatgtat ataatatata taatgtatat aatatatata atgtatatga tatatataat 1380gtatattctt ttcttatatt attgctctct ttatttttta ttatgtgttc tcttcttaca 1440tattatattg ttttttttac agaaaaggct aaaatgacat aa 1482<210>9<211>493<212>PRT<213>惡性瘧原蟲(Plasmodium falciparum)<400>9Met Gly Ile Lys Ile Ile Ile Tyr Ile Phe Phe Leu Ser Trp Ala Lys15 10 15Trp Val Cys Gly Ser Val Asn Phe Thr Gly Phe Asp Asn Lys Asn Met20 25 30Ile Gly Lys His Val Glu Leu Glu Gly Arg Tyr Lys Lys Glu Tyr Ile35 40 45Asp Arg Phe Phe Leu Glu Glu Leu Arg Lys His Asn Tyr Met Asn Asn50 55 60Asn Val Ile Leu Leu Ser Thr Ser Arg His Tyr Phe Asn Tyr Arg His65 70 75 80Thr Thr Asn Leu Leu Ile Ala Tyr Lys Tyr Leu Lys Tyr Phe Gly Asp85 90 95Thr Met Asp Lys Asn Ile Leu Leu Met Ile Pro Phe Asp Gln Ala Cys100 105 110Asp Cys Arg Asn Ile Arg Glu Gly Gln Ile Phe Arg Glu Tyr Glu Leu115 120 125
Phe Pro Ser Ser His Asn Lys Glu Thr Lys Ile Glu Asn Ile Asn Leu130 135 140Tyr Glu Asn Leu Asn Ile Asp Tyr Lys Asn Asn Asn Val Arg Asp Glu145 150 155 160Gln Ile Arg Arg Val Leu Arg His Arg Tyr Asp Ala Phe Thr Pro Lys165 170 175Lys Asn Arg Leu Tyr Asn Asn Gly Asn Asn Glu Lys Asn Leu Phe Leu180 185 190Tyr Met Thr Gly His Gly Gly Val Asn Phe Leu Lys Ile Gln Glu Phe195 200 205Asn Ile Ile Ser Ser Ser Glu Phe Asn Ile Tyr Ile Gln Glu Leu Leu210 215 220Ile Lys Asn Phe Tyr Lys Tyr Ile Phe Val Ile Ile Asp Thr Cys Gln225 230 235 240Gly Tyr Ser Phe Tyr Asp Asp Ile Leu Asn Phe Val Tyr Lys Lys Lys245 250 255Ile Asn Asn Ile Phe Phe Leu Ser Ser Ser Lys Arg Asn Glu Asn Ser260 265 270Tyr Ser Leu Phe Ser Ser Ser Tyr Leu Ser Val Ser Thr Val Asp Arg275 280 285Phe Thr Tyr His Phe Phe Asn Tyr Leu Gln Gln Ile His Lys Ile Tyr290 295 300Glu Lys Glu Pro Ser Lys Asn Ile Lys Ala Phe Ser Leu Tyr Asn Ile305 310 315 320Leu Asn Tyr Leu Lys Thr Gln His Ile Met Ser Glu Pro Thr Thr Asn325 330 335Asn Ser Lys Phe Asn Ser Ser Ile Phe Leu His Asp Lys Asn Ile Leu340 345 350Phe Phe Asn Ser Asn Leu Leu Ile Ile His Lys Asp Asp Val Ser Ile355 360 365Ile Tyr Gln Asp Lys Gln Thr His Asn His Lys Tyr Ile Cys Leu Asp370 375 380Asn Leu Ser Lys Cys Gly His Ile Lys Asn Asn Val His Lys Lys Met385 390 395 400Gln Thr Leu Tyr Glu Gln Thr Leu Tyr Tyr Asn Asn Asn Gln Gln Asn405 410 415Phe Phe Ser Asn His Met Ser Asn Phe Thr Asp Tyr Phe Phe Thr His420 425 430Asp Ile Tyr Asn Ile Tyr Asn Ile Tyr Asn Val Tyr Asn Ile Tyr Asn
435 440 445Val Tyr Asn Ile Tyr Asn Val Tyr Asp Ile Tyr Asn Val Tyr Ser Phe450 455 460Leu Ile Leu Leu Leu Ser Leu Phe Phe Ile Met Cys Ser Leu Leu Thr465 470 475 480Tyr Tyr Ile Val Phe Phe Thr Glu Lys Ala Lys Met Thr485 490<210>10<211>1494<212>DNA<213>惡性瘧原蟲(Pla smodium falciparum)<400>10atggaaagtg cagttagcga atgcaatata tacaaaaagg aaaaagataa aaatataatt60tacaaacaag aaagaaaatg ttgtatttgc atggtgagtg atttttttta tccaaatttg120ggaggaatag aaactcacat ttttgaattg tctaagaatt tgataaaaaa gggtttcaag180gttatagttg taacaaattt taataataat aggcatggta taagatggat gggtaatggt240attaaggttt attatttgcc cttccagcct tttttagatg tagtgagttt tccaaatatt300atagggactt taccactatg tagaaatata ttatataggg aaaaagtcga catagtacat360ggtcaccagg ctacgtcagc attagctcat caattcattc ttcatgccaa aaccttagga420ataaaaacta tttatacaga tcactcatta tacagttttt cagacaaagg atgcatacat480gtaaacaaat tattgaaata ttgtataaac gatgttgatc attctatatg tgttteccat540acgaatagag aaaatttagt tttgagaaca gaaagtaatc catataaaac gtcagttata600ggaaatgccc ttgatactac aaaatttgtt ccttgtatta gtaaaagacc aaagtttcca660agaataaata ttattgttat aagtaggtta acatatagaa aaggtataga tttgatagtt720aaggtaatac cattagtatg tcaaaaatat ccattcataa aatttattat agggggagaa780ggtcctaaaa gattgttatt agaagaaatg agagaaaagt atcacttaca taattctgtt840gtattattag gaaaagtaaa acaagaaaat gtaaaaaata ttttacaaac tggtcatata900ttcttaaata catctttaac agaagctttt tgtatagcca taattgaagc agctagttgt960ggtttgcttg tcatatctac ggatgtaggt ggaatatctg aagttttacc acacgatatg1020atgattttag ctaaaccaaa tcatatcgaa ttatgtaaag cagtcgataa agcattaaaa1080attgtacaaa aggtggactc aaatttattc cacgaaaggg taaacatgag tttattaaca1140tatgtaaata tatatatata tatatatata tatatatata taatatatat gaataatttt1200
atttataatc taaccaaaat gtactcttgg gaaaaagtag cggaaaagac ggtaaaatca 1260cataatacat atattatgaa tattgaaaag gtgtatatga acgtattaaa ttatgcaaat 1320ccaagcttgt ttaatagaat aagaaaaata tacgaaatta atacacctat tcatgtttcc 1380tttttttttt ttttagacaa aatattgaag aggtcgttag ctttcctcat ttttatgatg 1440acgaaaataa aaatgaaaaa taagagtttt aaaatgtcga tatataccac ataa1494<210>11<211>497<212>PRT<213>惡性瘧原蟲(Plasmodium falciparum)<400>11Met Glu Ser Ala Val Ser Glu Cys Asn Ile Tyr Lys Lys Glu Lys Asp1 5 10 15Lys Asn Ile Ile Tyr Lys Gln Glu Arg Lys Cys Cys Ile Cys Met Val20 25 30Ser Asp Phe Phe Tyr Pro Asn Leu Gly Gly Ile Glu Thr His Ile Phe35 40 45Glu Leu Ser Lys Asn Leu Ile Lys Lys Gly Phe Lys Val Ile Val Val50 55 60Thr Asn Phe Asn Asn Asn Arg His Gly Ile Arg Trp Met Gly Asn Gly65 70 75 80Ile Lys Val Tyr Tyr Leu Pro Phe Gln Pro Phe Leu Asp Val Val Ser85 90 95Phe Pro Asn Ile Ile Gly Thr Leu Pro Leu Cys Arg Asn Ile Leu Tyr100 105 110Arg Glu Lys Val Asp Ile Val His Gly His Gln Ala Thr Ser Ala Leu115 120 125Ala His Gln Phe Ile Leu His Ala Lys Thr Leu Gly Ile Lys Thr Ile130 135 140Tyr Thr Asp His Ser Leu Tyr Ser Phe Ser Asp Lys Gly Cys Ile His145 150 155 160Val Asn Lys Leu Leu Lys Tyr Cys Ile Asn Asp Val Asp His Ser Ile165 170 175Cys Val Ser His Thr A sn Arg Glu Asn Leu yal Leu Arg Thr Glu Ser180 185 190Asn Pro Tyr Lys Thr Ser Val Ile Gly Asn Ala Leu Asp Thr Thr Lys195 200 205Phe Val Pro Cys Ile Ser Lys Arg Pro Lys Phe Pro Arg Ile Asn Ile210 215 220
Ile Val Ile Ser Arg Leu Thr Tyr Arg Lys Gly Ile Asp Leu Ile Val225 230 235 240Lys Val Ile Pro Leu Val Cys Gln Lys Tyr Pro Phe Ile Lys Phe Ile245 250 255Ile Gly Gly Glu Gly Pro Lys Arg Leu Leu Leu Glu Glu Met Arg Glu260 265 270Lys Tyr His Leu His Asn Ser Val Val Leu Leu Gly Lys Val Lys Gln275 280 285Glu Asn Val Lys Asn Ile Leu Gln Thr Gly His Ile Phe Leu Asn Thr290 295 300Ser Leu Thr Glu Ala Phe Cys Ile Ala Ile Ile Glu Ala Ala Ser Cys305 310 315 320Gly Leu Leu Val Ile Ser Thr Asp Val Gly Gly Ile Ser Glu Val Leu325 330 335Pro His Asp Met Met Ile Leu Ala Lys Pro Asn His Ile Glu Leu Cys340 345 350Lys Ala Val Asp Lys Ala Leu Lys Ile Val Gln Lys Val Asp Ser Asn355 360 365Leu Phe His Glu Arg Val Asn Met Ser Leu Leu Thr Tyr Val Asn Ile370 375 380Tyr Ile Tyr Ile Tyr Ile Tyr Ile Tyr Ile Ile Tyr Met Asn Asn Phe385 390 395 400Ile Tyr Asn Leu Thr Lys Met Tyr Ser Trp Glu Lys Val Ala Glu Lys405 410 415Thr Val Lys Ser His Asn Thr Tyr Ile Met Asn Ile Glu Lys Val Tyr420 425 430Met Asn Val Leu Asn Tyr Ala Asn Pro Ser Leu Phe Asn Arg Ile Arg435 440 445Lys Ile Tyr Glu Ile Asn Thr Pro Ile His Val Ser Phe Phe Phe Phe450 455 460Leu Asp Lys Ile Leu Lys Arg Ser Leu Ala Phe Leu Ile Phe Met Met465 470 475 480Thr Lys Ile Lys Met Lys Asn Lys Ser Phe Lys Met Ser Ile Tyr Thr485 490 495Thr<210>12<211>2361<212>DNA<213>惡性瘧原蟲(Plasmodium falciparum)
<400>12atgatttaca atgacatttt aacattatgt gcaaataaat taagaggact aattgattca60aagatattta tttggttgtt aatatttttt agaatattta attgtttatt tgttgtaaca120tcattttatc ctgatgaata ttttcaatct gtagaaattg ctcatttttg ggcttatgga180tatggacata tgtcatggga atgggaacct tgtgtagcat taagatcagt gataactcct240tttatttatt atgtattatt tttattttta aaacttatta acatagatca tcctgtttgt300gtattataca ttcctaaatt atgtcatggt atatgtgctg ctttgtgtga tttaggtatt360tataaattat tgatatattg gtatgttgaa ttgtataatg acgcatggat aaatgaagat420aatataaaac gtaatgagaa agatgaaaat aatggaaata acaacaacaa taataataat480aacaataata ataacaataa taataataat tattattatc ataataatat attatacaac540acaaatgaca ttatttctac aattctatgt tgtcattttt tttgttggtt ttatttttat600tccatatgta ggacatcctc gcattccttt gaatgcttgt ttaatatatg gggtgtatat660tttttatcac aaaattatta ccccttgaaa aatcaatcaa acaaaataga aaagatagac720ttattattac agaacgatgt aatcatacaa aaaggaaaga aacatttaaa tgaatggaca780aatttaaaag aaagaaggaa tgatcatcat tttgatacat acgaaaataa ttttatatat840cataaaggaa cacaaaattg taaacaatat gataaaaata tggttgatca aaatgtttgt900ggtcaaaata tggttgatca cataattcag aacaggaaca atttgtgtag gacacatttt960tattcctcca aatttaacaa aatacaagag gcaaaaaatt tattatttag tttattcttt1020agctccttgt ctgttatatt tcgtccaaac gctttagtat tttggttatc tttatatata1080ctatatatta taaagaatat atttgaaaaa caaaataaaa taaattataa agaaatattc1140aaaataggta ttacgtacac tttttttttc ttaactatta ttattataat tgattcttat1200tattttgggc acattacatt tcccttttgg aatttttttg tttataattt tttaagtgga1260aacaataaat attttggagg gcattctttt tttttttact ttgtatgtgt tataccttct1320atatatttaa ctttaacacc ttttttgttt tatggttatt acattatata taataatata1380ttgaataagg tgaagtataa gacaattaat atatatatgt atatattgaa acgaattgat1440tggatagtat atttagttac acacttagaa atcctatctt tatctttcag taagcataag1500gaacataaaa ttgttatagg atatataccg tttcttacaa tttttgttgg atatgcatta1560tatataataa aattacatta taaaaaatat aatggcaaaa atggaaagaa tatatataat1620aataataaaa tccaatatgg taatataacc ataaagggaa gaaataaata tattttttta1680
atttcatctt ctctttttac aaatataagt tttttacttc aatttttatg tattcttttt 1740ttctgcctta tacataacag atcacctgaa catgtagcct cttattttag aaacttagaa 1800acgaaagatg atcaaaatat ttatatattt ataacaaatt gttatgatat acctttatat 1860tcgcatatac atagaaaatt caatatagga tttttagact gttctcctta tgacacgagt 1920aatgatgaag ctaccaaaaa ttggagaaaa cgtaratatg aagataaatt taaggaacaa 1980ttttttaata tttttcaaga aaaaaaaaat aataatcatc atataaatag tacatatgga 2040gatacaatta ctccatatat aataccagat aaatcatttt attggtttgg tcatcatcat 2100tttaataaaa aaaataattt tcaatatatt tatcaaaaca ttaatttgtc atgtttaaat 2160tatagatttc atataccttt acaaggacaa ttacctactt atatagttac aacaactatt 2220gaacttacac atttacaatt atttctgagt acatataatt ataaacttga aacaaagcct 2280tttttttctt attttatgat aagtgaagca aaaggaatag ttccagtata tcattacata 2340ttcaagaggg ttccttccta a2361<210>13<211>786<212>PRT<213>惡性瘧原蟲(Plasmodium falciparum)<400>13Met Ile Tyr Asn Asp Ile Leu Thr Leu Cys Ala Asn Lys Leu Arg Gly1 5 10 15Leu Ile Asp Ser Lys Ile Phe Ile Trp Leu Leu Ile Phe Phe Arg Ile20 25 30Phe Asn Cys Leu Phe Val Val Thr Ser Phe Tyr Pro Asp Glu Tyr Phe35 40 45Gln Ser Val Glu Ile Ala His Phe Trp Ala Tyr Gly Tyr Gly His Met50 55 60Ser Trp Glu Trp Glu Pro Cys Val Ala Leu Arg Ser Val Ile Thr Pro65 70 75 80Phe Ile Tyr Tyr Val Leu Phe Leu Phe Leu Lys Leu Ile Asn Ile Asp85 90 95His Pro Val Cys Val Leu Tyr Ile Pro Lys Leu Cys His Gly Ile Cys100 105 110Ala Ala Leu Cys Asp Leu Gly Ile Tyr Lys Leu Leu Ile Tyr Trp Tyr115 120 125Val Glu Leu Tyr Asn Asp Ala Trp Ile Asn Glu Asp Asn Ile Lys Arg130 135 140
Asn Glu Lys Asp Glu Asn Asn Gly Asn Asn Asn Asn Asn Asn Asn Asn145 150 155 160Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Tyr Tyr Tyr His Asn Asn165 170 175Ile Leu Tyr Asn Thr Asn Asp Ile Ile Ser Thr Ile Leu Cys Cys His180 l85 190Phe Phe Cys Trp Phe Tyr Phe Tyr Ser Ile Cys Arg Thr Ser Ser His195 200 205Ser Phe Glu Cys Leu Phe Asn Ile Trp Gly Val Tyr Phe Leu Ser Gln210 215 220Asn Tyr Tyr Pro Leu Lys Asn Gln Ser Asn Lys Ile Glu Lys Ile Asp225 230 235 240Leu Leu Leu Gln Asn Asp Val Ile Ile Gln Lys Gly Lys Lys His Leu245 250 255Asn Glu Trp Thr Asn Leu Lys Glu Arg Arg Asn Asp His His Phe Asp260 265 270Thr Tyr Glu Asn Asn Phe Ile Tyr His Lys Gly Thr Gln Asn Cys Lys275 280 285Gln Tyr Asp Lys Asn Met Val Asp Gln Asn Val Cys Gly Gln Asn Met290 295 300Val Asp His Ile Ile Gln Asn Arg Asn Asn Leu Cys Arg Thr His Phe305 310 315 320Tyr Ser Ser Lys Phe Asn Lys Ile Gln Glu Ala Lys Asn Leu Leu Phe325 330 335Ser Leu Phe Phe Ser Ser Leu Ser Val Ile Phe Arg Pro Asn Ala Leu340 345 350Val Phe Trp Leu Ser Leu Tyr Ile Leu Tyr Ile Ile Lys Asn Ile Phe355 360 365Glu Lys Gln Asn Lys Ile Asn Tyr Lys Glu Ile Phe Lys Ile Gly Ile370 375 380Thr Tyr Thr Phe Phe Phe Leu Thr Ile Ile Ile Ile Ile Asp Ser Tyr385 390 395 400Tyr Phe Gly His Ile Thr Phe Pro Phe Trp Asn Phe Phe Val Tyr Asn405 410 415Phe Leu Ser Gly Asn Asn Lys Tyr Phe Gly Gly His Ser Phe Phe Phe420 425 430Tyr Phe Val Cys Val Ile Pro Ser Ile Tyr Leu Thr Leu Thr Pro Phe435 440 445Leu Phe Tyr Gly Tyr Tyr Ile Ile Tyr Asn Asn Ile Leu Asn Lys Val450 455 460
Lys Tyr Lys Thr Ile Asn Ile Tyr Met Tyr Ile Leu Lys Arg Ile Asp465 470 475 480Trp Ile yal Tyr Leu Val Thr Hi s Leu Glu Ile Leu Ser Leu Ser Phe485 490 495Ser Lys His Lys Glu His Lys Ile Val Ile Gly Tyr Ile Pro Phe Leu500 505 510Thr Ile Phe Val Gly Tyr Ala Leu Tyr Ile Ile Lys Leu His Tyr Lys515 520 525Lys Tyr Asn Gly Lys Asn Gly Lys Asn Ile Tyr Asn Asn Asn Lys Ile530 535 540Gln Tyr Gly Asn Ile Thr Ile Lys Gly Arg Asn Lys Tyr Ile Phe Leu545 550 555 560Ile Ser Ser Ser Leu Phe Thr Asn Ile Ser Phe Leu Leu Gln Phe Leu565 570 575Cys Ile Leu Phe Phe Cys Leu Ile His Asn Arg Ser Pro Glu His Val580 585 590Ala Ser Tyr Phe Arg Asn Leu Glu Thr Lys Asp Asp Gln Asn Ile Tyr595 600 605Ile Phe Ile Thr Asn Cys Tyr Asp Ile Pro Leu Tyr Ser His Ile His610 615 620Arg Lys Phe Asn Ile Gly Phe Leu Asp Cys Ser Pro Tyr Asp Thr Ser625 630 635 640Asn Asp Glu Ala Thr Lys Asn Trp Arg Lys Arg Ile Tyr Glu Asp Lys645 650 655Phe Lys Glu Gln Phe Phe Asn Ile Phe Gln Glu Lys Lys Asn Asn Asn660 665 670His His Ile Asn Ser Thr Tyr Gly Asp Thr Ile Thr Pro Tyr Ile Ile675 680 685Pro Asp Lys Ser Phe Tyr Trp Phe Gly His His His Phe Asn Lys Lys690 695 700Asn Asn Phe Gln Tyr Ile Tyr Gln Asn Ile Asn Leu Ser Cys Leu Asn705 710 715 720Tyr Arg Phe His Ile Pro Leu Gln Gly Gln Leu Pro Thr Tyr Ile Val725 730 735Thr Thr Thr Ile Glu Leu Thr His Leu Gln Leu Phe Leu Ser Thr Tyr740 745 750Asn Tyr Lys Leu Glu Thr Lys Pro Phe Phe Ser Tyr Phe Met Ile Ser755760 765Glu Ala Lys Gly Ile Val Pro Val Tyr His Tyr Ile Phe Lys Arg Val
770 775 780Pro Ser785<210>14<211>1233<212>DNA<213>惡性瘧原蟲(Plasmodium falciparum)<400>14atggggcata cacataaaga atataaaaac aatgagaaga gtgcaatatt ttttgagtgg60ttgatttttt ttgtgggtat aataattcgt ataattattt attattatgg aaggtggcaa120gataaaaact ttaatgttaa gtttacagat gtagattatt atgttttttc tgatgctgca180aaatatgtac ttatgaacaa atcaccatat gaaagatata catatagata tacaccttta240ttagcatata taatgatacc aaattttttt gttcattttt cttttgggaa aatattattt300tcatttatcg atattcttgt tactattctt ataaatcaaa ttataaaaat caaatatact360aattgtaaaa attatatatt ttatacttgt ttatggtttt taaatccatt agtcataatt420atatcccttc gaggtaatgc agatgttatc ccatgcttct taataatagt aacaatcttt480tgtatatata aaaaacatat ctttttgtct tcgatttttt atggactagc tgtgaacttt540aaaatatata caattattta tgcactacca ttcatgttat atttaaataa aaattattta600cttggggaaa atatttttca attaaatgaa aaaaaaaaaa aaaaaaaaaa tgacttccta660ttaaacacat ttttttatat ttttcgtatt atatctaatt tttttgtgga attatttaaa720ttaaattatg aacagttttt atttgccata tgtagttcct cggtatttct aattttaaac780tgtgtattct atatcatata tggatatgaa tttttgtatg agtcttttat atatcatatt840attagacgtg atcataggca taacttttcc cttttttttt accttatgta tttaagtatt900gagaagaatt caaagattat tcccttaata acctttgtac cacaaataat tttagtagcc960ttatttggat ttaaatatgc aagaacgaat ttggaattat ccatgttttt acaaactatt1020tcttttattg cattgaataa agtgtgcaca tctcagtatt tcatttggtg tattccattt1080ttaccaatta tactttgtgc cataacctta agcaagagaa atatgtttct tataatatcc1140tccattttat tttttattgt ggcaaaagtg ggctcaaaaa gatttctttt attatataaa1200tatgtctttt catatattat ttttttttca tga 1233<210>15<211>410<212>PRT
<213>惡性瘧原蟲(Plasmodium falciparum)<400>15Met Gly His Thr His Lys Glu Tyr Lys Asn Asn Glu Lys Ser Ala Ile1 5 10 15Phe Phe Glu Trp Leu Ile Phe Phe Val Gly Ile Ile Ile Arg Ile Ile20 25 30Ile Tyr Tyr Tyr Gly Arg Trp Gln Asp Lys Asn Phe Asn Val Lys Phe35 40 45Thr Asp Val Asp Tyr Tyr Val Phe Ser Asp Ala Ala Lys Tyr Val Leu50 55 60Met Asn Lys Ser Pro Tyr Glu Arg Tyr Thr Tyr Arg Tyr Thr Pro Leu65 70 75 80Leu Ala Tyr Ile Met Ile Pro Asn Phe Phe Val His Phe Ser Phe Gly85 90 95Lys Ile Leu Phe Ser Phe Ile Asp Ile Leu Val Thr Ile Leu Ile Asn100 105 110Gln Ile Ile Lys Ile Lys Tyr Thr Asn Cys Lys Asn Tyr Ile Phe Tyr115 120 125Thr Cys Leu Trp Phe Leu Asn Pro Leu Val Ile Ile Ile Ser Leu Arg130 135 140Gly Asn Ala Asp Val Ile Pro Cys Phe Leu Ile Ile Val Thr Ile Phe145 150 155 160Cys Ile Tyr Lys Lys His Ile Phe Leu Ser Ser Ile Phe Tyr Gly Leu165 170 175Ala Val Asn Phe Lys Ile Tyr Thr Ile Ile Tyr Ala Leu Pro Phe Met180 185 190Leu Tyr Leu Asn Lys Asn Tyr Leu Leu Gly Glu Asn Ile Phe Gln Leu195 200 205Asn Glu Lys Lys Lys Lys Lys Lys Asn Asp Phe Leu Leu Asn Thr Phe210 215 220Phe Tyr Ile Phe Arg Ile Ile Ser Asn Phe Phe Val Glu Leu Phe Lys225 230 235 240Leu Asn Tyr Glu Gln Phe Leu Phe Ala Ile Cys Ser Ser Ser Val Phe245 250 255Leu Ile Leu Asn Cys Val Phe Tyr Ile Ile Tyr Gly Tyr Glu Phe Leu260 265 270Tyr Glu Ser Phe Ile Tyr His Ile Ile Arg Arg Asp His Arg His Asn275 280 285Phe Ser Leu Phe Phe Tyr Leu Met Tyr Leu Ser Ile Glu Lys Asn Ser290 295 300
Lys Ile Ile Pro Leu Ile Thr Phe Val Pro Gln Ile Ile Leu Val Ala305 310 315 320Leu Phe Gly Phe Lys Tyr Ala Arg Thr Asn Leu Glu Leu Ser Met Phe325 330 335Leu Gln Thr Ile Ser Phe Ile Ala Leu Asn Lys Val Cys Thr Ser Gln340 345 350Tyr Phe Ile Trp Cys Ile Pro Phe Leu Pro Ile Ile Leu Cys Ala Ile355 360 365Thr Leu Ser Lys Arg Asn Met Phe Leu Ile Ile Ser Ser Ile Leu Phe370 375 380Phe Ile Val Ala Lys Val Gly Ser Lys Arg Phe Leu Leu Leu Tyr Lys385 390 395 400Tyr Val Phe Ser Tyr Ile Ile Phe Phe Ser405 410<210>16<211>3852<212>DNA<213>惡性瘧原蟲(Plasmodium falciparum)<400>16atgaaaatta aaaataatat taaaaaaaaa aatgataacc tacatcattt cataagtaat 60catacgctaa tattttctat tatcctactt ataaatctat tgattttttt ctcatttatc 120aatggctatt tttatgcaag acaaaaactt gaagaaaaat cagaaaacct tgagcttttt 180agtaggaaag tatttggaga tgaatacgta gaaagtttga aaaagaaaaa aaatactttt 240tcgattatta atgcaccata tgataaagtt gttatacttt taatagattc cttaagattt 300gattttaccc tatacgatac taattatgaa aaggaattca taggaaaaga aaaaaataca 360gatatctaca ataatatatc tagcgaaaaa aaaaatatat caaatgatgg ggaaaaaaaa 420aactcattat tttttttaaa taatatgata aatgtacatc acattttaca aaatgaaaaa 480aacaacactt tattattccg gtttgatgca gatgccccta caataacaac ttcaaggata 540aaatctatat ttatgggtac cataccaaat tatatggaag taaatgaaaa ctttagtcct 600acgactagtg ttgaagataa tttttttgaa cagcttcatt taaataataa aaaagtaatt 660gctatcggtg ataataccat tactcatttg atgaaacatt tttctaaaga attagtttat 720gagagcttta atgtttttga tttttattca ttagatattg ctgcaaagaa acatttttat 780gaagaatacg aatcaaatga ttgggatatt atgtatatac atatgttggg agttgatcat 840attggacata taaaaacacc caactcaaaa atcatgggag atgccttaaa agattttgat 900
acattcatat atgatattat aaataaaata aaattagata atcttaaaaa tattagcaca960gaagaaaaag aaaaaatgaa aataataaaa aaaaaaatat ataaaactta tcgaaacggt1020catcacatac aaaatgaaaa tgatcatatt attgataata tacaaaatga aaatgatcat1080attattgata atatacaaaa tgaaaatgat catactattg ataatataca aagtgaaaat1140gatcatacta ttgataatat acaaaatgaa aatgatcata ctattgaaga tatacaaatt1200gataatgatc atactattga agatattgaa aatcaaagtg aacaaaaaaa tgatgataaa1260aaaacactgt tcatattttt tggtgatcat ggacagctag atacaggaga tcacggagga1320tacagcttgg acgaaaccca tagtgctctt tttgcatatt cacccttaaa ctttatatct1380ttagataatg acatcattca aaataatttt gttttatatg ataaagataa attaaaaaaa1440aatgtgaata cactgaatga agaaaataat aataatgaga atatagataa ttataaaaaa1500tatcattcat atttaaaaga tagaaataaa aaatattctt accattataa tgttaaatat1560acaaaacaag taaatttaat gagtacctta tccttattaa ttggatcaac attaccttat1620ggaaatatag gaaatattat tatggatttc attcccaatg catatataaa aaataataat1680aaaaaaaaaa ataataataa taataataat aataataatt cttcattacc taatgaacag1740acaaatttat attatgatct tttaaattta cattatattg ctgaattaaa ttatgctaac1800ttatggcaat tgaatagata cctgaatgaa tatgaaaaaa agtataatat aataaaaaat1860gaagattatc attttattaa atcctcatgg catattatac aaaaagacaa aaaagaattg1920ttttttcaac caaataaaaa atttattaaa aatgacattt tattaaaaaa agaaaaagaa1980tcatatatag aattcataaa tgaaatgaca actctaatgg atatcacaca aaaatatttt2040tactatatat tcaacataaa agaaaaatat ttcttaattt tatctattgt tttaaatata2100ttcttattat tatttttaaa acatttttat tattattcta aattaaatta ttaccataaa2160ttaattaaag taacctttaa tgattttaat aaaaatattt atttattact ttgtatatgt2220gcattacttt tatatttttt tatcttctgt ttatcaatta aagaatataa agatatattc2280cgaatatttt ctcatgctaa aatcattttc ataagcaata atatagatat gataatacct2340agtataaaaa attaccatat gagcgctaaa cgtaacatga atattacgaa taatgacacc2400taccatacat cacataagga tcggaaatca ttcacaaata aagaagagaa acaaaataat2460acacttatga atatattcta taatattatt tatttcttaa gaataattcg aaaaaaaatt2520gttcagtata taatctttat acatttgaca atatacaatc taactatagg taatattatc2580
tttattctat ttaaattatt tccgaaaatt ataactaatt catttcaaat attaagaagt 2640aattatttcc ttttgtttgt tattatatgg agctgttgtg aaatgtcctt taattatata 2700gataaagaaa gatattatat tcattacatt ttaattgtat atgttatatt tgggatgctt 2760aaatggaagt atcaccgtgt ctttaatata ctaaaagcct tcattttgtt ggtgcttcta 2820ataattaatg ctttgtatag ccatacaccc gaatattttg accatggtaa agaaaaaata 2880tatttaaaag aatctgtact aaaatcagtt tttccaatat catcttatat tcttagctta 2940atattaataa atagtggtat taataattta ctgaaaaaac gaataaaaat aataataacc 3000caaatatgga cattacaata tatcctcgtt tttttgttct taaataatat ataccataga 3060tatattcaat ttataacacc cccaagtatt tatttcttaa ccatttcaac cttcatattc 3120atattcaata caaatttagg tgtattattt ctcttttata tgacatttct ttttttctac 3180tttattctta taagttccaa ttgttcagaa aatatgatcc aaatgaatga cataacatcg 3240acatggataa atgaaaatat acataataga aatgatccta taattacaaa aggaaatttg 3300gagaataaag aaaagtgtac ttcatgtaat acatctataa aagaaaaatt ttattataaa 3360ttaatgatag aaaaattcaa attattaaat acatgctctg tattaaatga agaagaaata 3420atgggaatac aaatttataa attaattaga gatatttctt atttttatat aaatgaaaca 3480gatttttata ttttatcatg tgtactttta atatattctt tttttataac gggacataaa 3540tttattttaa ataacttacc actagtttca ggttatgttg gattgtataa atatgtgtgg 3600ccaataagtc agttttatat ttttaatcat attttttttc cattcttctt ttcgttattt 3660tttatcattt atatttataa cataagaagg ataaaaatta taaattcttt taagcaattt 3720gatttgtatt atttttatgt ttatccacta atgaactttt ccttcaaggc ctcctttttg 3780ttttgttgca agtttattat atctgtatgg gtttcatatt acttgaatct gcacataatg 3840gtaaaaatat aa 3852<210>17<211>1283<212>PRT<213>惡性瘧原蟲(Plasmodium falciparum)<400>17Met Lys Ile Lys Asn Asn Ile Lys Lys Lys Asn Asp Asn Leu His His1 5 10 15Phe Ile Ser Asn His Thr Leu Ile Phe Ser Ile Ile Leu Leu Ile Asn20 25 30Leu Leu Ile Phe Phe Ser Phe Ile Asn Gly Tyr Phe Tyr Ala Arg Gln
35 40 45Lys Leu Glu Glu Lys Ser Glu Asn Leu Glu Leu Phe Ser Arg Lys Val50 55 60Phe Gly Asp Glu Tyr Val Glu Ser Leu Lys Lys Lys Lys Asn Thr Phe65 70 75 80Ser Ile Ile Asn Ala Pro Tyr Asp Lys Val Val Ile Leu Leu Ile Asp85 90 95Ser Leu Arg Phe Asp Phe Thr Leu Tyr Asp Thr Asn Tyr Glu Lys Glu100 105 110Phe Ile Gly Lys Glu Lys Asn Thr Asp Ile Tyr Asn Asn Ile Ser Ser115 120 125Glu Lys Lys Asn Ile Ser Asn Asp Gly Glu Lys Lys Asn Ser Leu Phe130 135 140Phe Leu Asn Asn Met Ile Asn Val His His Ile Leu Gln Asn Glu Lysl45 150 155 160Asn Asn Thr Leu Leu Phe Arg Phe Asp Ala Asp Ala Pro Thr Ile Thrl65 170 175Thr Ser Arg Ile Lys Ser Ile Phe Met Gly Thr Ile Pro Asn Tyr Met180 185 190Glu Val Asn Glu Asn Phe Ser Pro Thr Thr Ser Val Glu Asp Asn Phe195 200 205Phe Glu Gln Leu His Leu Asn Asn Lys Lys Val Ile Ala Ile Gly Asp210 215 220Asn Thr Ile Thr His Leu Met Lys His Phe Ser Lys Glu Leu Val Tyr225 230 235 240Glu Ser Phe Asn Val Phe Asp Phe Tyr Ser Leu Asp Ile Ala Ala Lys245 250 255Lys His Phe Tyr Glu Glu Tyr Glu Ser Asn Asp Trp Asp Ile Met Tyr260 265 270Ile His Met Leu Gly Val Asp His Ile Gly His Ile Lys Thr Pro Asn275 280 285Ser Lys Ile Met Gly Asp Ala Leu Lys Asp Phe Asp Thr Phe Ile Tyr290 295 300Asp Ile Ile Asn Lys Ile Lys Leu Asp Asn Leu Lys Asn Ile Ser Thr305 310 315 320Glu Glu Lys Glu Lys Met Lys Ile Ile Lys Lys Lys Ile Tyr Lys Thr325 330 335Tyr Arg Asn Gly His His Ile Gln Asn Glu Asn Asp His Ile Ile Asp340 345 350
Asn Ile Gln Asn Glu Asn Asp His Ile Ile Asp Asn Ile Gln Asn Glu355 360 365Asn Asp His Thr Ile Asp Asn Ile Gln Ser Glu Asn Asp His Thr Ile370 375 380Asp Asn Ile Gln Asn Glu Asn Asp His Thr Ile Glu Asp Ile Gln Ile385 390 395 400Asp Asn Asp His Thr Ile Glu Asp Ile Glu Asn Gln Ser Glu Gln Lys405 410 415Asn Asp Asp Lys Lys Thr Leu Phe Ile Phe Phe Gly Asp His Gly Gln420 425 430Leu Asp Thr Gly Asp His Gly Gly Tyr Ser Leu Asp Glu Thr His Ser435 440 445Ala Leu Phe Ala Tyr Ser Pro Leu Asn Phe Ile Ser Leu Asp Asn Asp450 455 460Ile Ile Gln Asn Asn Phe Val Leu Tyr Asp Lys Asp Lys Leu Lys Lys465 470 475 480Asn Val Asn Thr Leu Asn Glu Glu Asn Asn Asn Asn Glu Asn Ile Asp485 490 495Asn Tyr Lys Lys Tyr His Ser Tyr Leu Lys Asp Arg Asn Lys Lys Tyr500 505 510Ser Tyr His Tyr Asn Val Lys Tyr Thr Lys Gln Val Asn Leu Met Ser515 520 525Thr Leu Ser Leu Leu Ile Gly Ser Thr Leu Pro Tyr Gly Asn Ile Gly530 535 540Asn Ile Ile Met Asp Phe Ile Pro Asn Ala Tyr Ile Lys Asn Asn Asn545 550 555 560Lys Lys Lys Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Ser Ser Leu565 570 575Pro Asn Glu Gln Thr A sn Leu Tyr Tyr Asp Leu Leu Asn Leu His Tyr580 585 590Ile Ala Glu Leu Asn Tyr Ala Asn Leu Trp Gln Leu Asn Arg Tyr Leu595 600 605Asn Glu Tyr Glu Lys Lys Tyr Asn Ile Ile Lys Asn Glu Asp Tyr His610 615 620Phe Ile Lys Ser Ser Trp His Ile Ile Gln Lys Asp Lys Lys Glu Leu625 630 635 640Phe Phe Gln Pro Asn Lys Lys Phe Ile Lys Asn Asp Ile Leu Leu Lys645 650 655Lys Glu Lys Glu Ser Tyr Ile Glu Phe Ile Asn Glu Met Thr Thr Leu660 665 670
Met Asp Ile Thr Gln Lys Tyr Phe Tyr Tyr Ile Phe Asn Ile Lys Glu675 680 685Lys Tyr Phe Leu Ile Leu Ser Ile Val Leu Asn Ile Phe Leu Leu Leu690 695 700Phe Leu Lys His Phe Tyr Tyr Tyr Ser Lys Leu Asn Tyr Tyr His Lys705 710 715 720Leu Ile Lys Val Thr Phe Asn Asp Phe Asn Lys Asn Ile Tyr Leu Leu725 730 735Leu Cys Ile Cys Ala Leu Leu Leu Tyr Phe Phe Ile Phe Cys Leu Ser740 745 750Ile Lys Glu Tyr Lys Asp Ile Phe Arg Ile Phe Ser His Ala Lys Ile755 760 765Ile Phe Ile Ser Asn Asn Ile Asp Met Ile Ile Pro Ser Ile Lys Asn770 775 780Tyr His Met Ser Ala Lys Arg Asn Met Asn Ile Thr Asn Asn Asp Thr785 790 795 800Tyr His Thr Ser His Lys Asp Arg Lys Ser Phe Thr Asn Lys Glu Glu805 810 815Lys Gln Asn Asn Thr Leu Met Asn Ile Phe Tyr Asn Ile Ile Tyr Phe820 825 830Leu Arg Ile Ile Arg Lys Lys Ile Val Gln Tyr Ile Ile Phe Ile His835 840 845Leu Thr Ile Tyr Asn Leu Thr Ile Gly Asn Ile Ile Phe Ile Leu Phe850 855 860Lys Leu Phe Pro Lys Ile Ile Thr Asn Ser Phe Gln Ile Leu Arg Ser865 870 875 880Asn Tyr Phe Leu Leu Phe Val Ile Ile Trp Ser Cys Cys Glu Met Ser885 890 895Phe Asn Tyr Ile Asp Lys Glu Arg Tyr Tyr Ile His Tyr Ile Leu Ile900 905 910Val Tyr Val Ile Phe Gly Met Leu Lys Trp Lys Tyr His Arg Val Phe915 920 925Asn Ile Leu Lys Ala Phe Ile Leu Leu Val Leu Leu Ile Ile Asn Ala930 935 940Leu Tyr Ser His Thr Pro Glu Tyr Phe Asp His Gly Lys Glu Lys Ile945 950 955 960Tyr Leu Lys Glu Ser Val Leu Lys Ser Val Phe Pro Ile Ser Ser Tyr965 970 975
Ile Leu Ser Leu Ile Leu Ile Asn Ser Gly Ile Asn Asn Leu Leu Lys980 985 990Lys Arg Ile Lys Ile Ile Ile Thr Gln Ile Trp Thr Leu Gln Tyr Ile995 10001005Leu Val Phe Leu Phe Leu Asn Asn Ile Tyr His Arg Tyr Ile Gln10101015 1020Phe Ile Thr Pro Pro Ser Ile Tyr Phe Leu Thr Ile Ser Thr Phe102510301035Ile Phe Ile Phe Asn Thr Asn Leu Gly Val Leu Phe Leu Phe Tyr104010451050Met Thr Phe Leu Phe Phe Tyr Phe Ile Leu Ile Ser Ser Asn Cys105510601065Ser Glu Asn Met Ile Gln Met Asn Asp Ile Thr Ser Thr Trp Ile107010751080Asn Glu Asn Ile His Asn Arg Asn Asp Pro Ile Ile Thr Lys Gly108510901095Asn Leu Glu Asn Lys Glu Lys Cys Thr Ser Cys Asn Thr Ser Ile110011051110Lys Glu Lys Phe Tyr Tyr Lys Leu Met Ile Glu Lys Phe Lys Leu111511201125Leu Asn Thr Cys Ser Val Leu Asn Glu Glu Glu Ile Met Gly Ile113011351140Gln Ile Tyr Lys Leu Ile Arg Asp Ile Ser Tyr Phe Tyr Ile Asn114511501155Glu Thr Asp Phe Tyr Ile Leu Ser Cys Val Leu Leu Ile Tyr Ser116011651170Phe Phe Ile Thr Gly His Lys Phe Ile Leu Asn Asn Leu Pro Leu117511801185Val Ser Gly Tyr Val Gly Leu Tyr Lys Tyr Val Trp Pro Ile Ser119011951200Gln Phe Tyr Ile Phe Asn His Ile Phe Phe Pro Phe Phe Phe Ser120512101215Leu Phe Phe Ile Ile Tyr Ile Tyr Asn Ile Arg Arg Ile Lys Ile122012251230Ile Asn Ser Phe Lys Gln Phe Asp Leu Tyr Tyr Phe Tyr Val Tyr123512401245Pro Leu Met Asn Phe Ser Phe Lys Ala Ser Phe Leu Phe Cys Cys125012551260Lys Phe Ile Ile Ser Val Trp Val Ser Tyr Tyr Leu Asn Leu His126512701275
Ile Met Val Lys Ile1280<210>18<211>2433<212>DNA<213>惡性瘧原蟲(Plasmodium falciparum)<400>18atgggatttt cagaaaatcc caaattctgc ctgctcataa ataagcttat caagaaatct60aaagttatag gcgttttctt atcaattatt ggtgtgattt tctttttggt gtttaataag 120tttaataaga atgctgaatt agacgctagg acatttaccc aatttgttgg taattcagta 180ttgaacaaaa agaatgagaa gttttataat gatacaaatt cttatttcat gaactataca 240tatgaaggga aagaagatat aataaagttg atatatgatt atattagaaa aaatatatta 300gtaaatgtag agaatgagat ggttaaaata aaattaacgg atagaattga acagaatata 360ttgataagta atgtaggatg taaatattgt aataatatgg aaagtttagt tgtggtaata 420aattttgatt ttaaagaaag gaaatatttt catagcgtaa ttatcggttt aacgttaatg 480gaacattttt ctaaatgtaa ctatatgagt aaggatgtga cttttttatt taccaataaa 540gaattattat attctttagg tgttcaagaa tttatacaaa aatattttta taataatact 600aatagaattg gaaaaaaaat tattagatct tctactatta ttgaatttga ttctatttat 660ccttcttata ttaaaattaa ttatgaagga ttaaatggta tgttacctaa tcaagactta 720atattattat taacaaatga acttcatttt tattctattc ctattaaaat ggagcttact 780catggttcca tatttgatat ggccctagaa aagaattatg aaaatggtca catatacttt 840ctgaggtaca aaaaaaaata tgaatatata cgtgatgata atgatgaaat caagaacatt 900cccgcattta ctgcaacggg gggtagcaaa gtacccataa gaaataaaat gattaatttg 960ttcaacttaa ccaaagcatt acaaagttat ttaagaagtc agagtaatac acatgaaggc 1020ttttgtcatt cttcaaattt ttattttttt aatacattca gaaggcatat accaataagt 1080atatattgtt atagtgttta tttaatatgc gcatatagca taatgaaatt atttaaatca 1140acgatattta gaagctacat aaatttttta acaggtttct acacttattt gattacaata 1200ttaattattt ccctacctat atatttaatt tcaacaaata aaaaatttta tgagttgttg 1260aattttgaag aaaattatat tccttcatgt tatgaatggc atcctgataa ttttgataaa 1320tatataaaaa ttgcaaatat atggtggaat gttttatttt tctcaatttt tggtgcattt 1380ttttttaatt tatttatttc ttttttagtt aataaaaaaa gaaaagttat accaaaaaaa 1440
aatgatcaaa atgaatcatt tgatggctat aaaaaggtag agaaggtaga acgaatttta 1500atattagaaa aaatcaaaga attgcaaaat gaaataatga aacgaaaagg tattacaaat 1560aatcataata atattaaaaa ttataatatt tatacaaatg aaaatatata caataataat 1620ataaataata taaataataa taataatatt tatgaaaact tatatgataa tggagaagta 1680aaaaaaaaca ttctggttaa accaaaaatt ataaatagcg atgatgaaga ttttcttctt 1740gaaaaaaaaa attccgaatt cattaaaaaa atagaaaaac aaatagaaat attagaagaa 1800aaattggaat ttttaagtaa tgatgaaaat gtaaaatata ttttttataa taattctata 1860gcaccattta atacaatgat gatatatatg aatatttttt atttcatatt agtagcgcta 1920ttaagttcgt tatataattg gtcctattct gtattattta gcctgctatt tgtaattcct 1980atatcaattt tacataattt aaaaacaaaa ccagtcagaa tttttaagaa gattattctt 2040tcacttttta ttctttgtat gtttatttat atgtatccta atgataatca tctttggaat 2100ataagacaga agctaactaa tttatttagg aataatatat caaaatgttg taaatattta 2160gacaagcaca aaatattaca aagcaaatat ttcccagaaa gtttgcaatt tatttgttcg 2220aataggttat ttgattcatt ttactcaaat aaatattttt tggataatct aaatattaaa 2280tttagttatg tcttggatat tcaaaatgga ttcttattaa ctttatacaa tttagcaaga 2340aatcattttt gtattggtac agctacctat cctttaatat gctttacctt attcccaata 2400gtattttata tagttttttt atttttttgt taa 2433<210>19<211>810<212>PRT<213>惡性瘧原蟲(Plasmodium falciparum)<400>19Met Gly Phe Ser Glu Asn Pro Lys Phe Cys Leu Leu Ile Asn Lys Leu1 510 15Ile Lys Lys Ser Lys Val Ile Gly Val Phe Leu Ser Ile Ile Gly Val20 25 30Ile Phe Phe Leu Val Phe Asn Lys Phe Asn Lys Asn Ala Glu Leu Asp35 40 45Ala Arg Thr Phe Thr Gln Phe Val Gly Asn Set Val Leu Asn Lys Lys50 55 60Asn Glu Lys Phe Tyr Asn Asp Thr Asn Ser Tyr Phe Met Asn Tyr Thr65 70 75 80Tyr Glu Gly Lys Glu Asp Ile Ile Lys Leu Ile Tyr Asp Tyr Ile Arg85 90 95
Lys Asn Ile Leu Val Asn Val Glu Asn Glu Met Val Lys Ile Lys Leu100 105 110Thr Asp Arg Ile Glu Gln Asn Ile Leu Ile Ser Asn Val Gly Cys Lys115 120125Tyr Cys Asn Asn Met Glu Ser Leu Val Val Val Ile Asn Phe Asp Phe130 135 140Lys Glu Arg Lys Tyr Phe His Ser Val Ile Ile Gly Leu Thr Leu Met145 150 155 160Glu His Phe Ser Lys Cys Asn Tyr Met Ser Lys Asp Val Thr Phe Leu165 170 175Phe Thr Asn Lys Glu Leu Leu Tyr Ser Leu Gly Val Gln Glu Phe Ile180 185 190Gln Lys Tyr Phe Tyr Asn Asn Thr Asn Arg Ile Gly Lys Lys Ile Ile195 200 205Arg Ser Ser Thr Ile Ile Glu Phe Asp Ser Ile Tyr Pro Ser Tyr Ile210 215 220Lys Ile Asn Tyr Glu Gly Leu Asn Gly Met Leu Pro Asn Gln Asp Leu225 230 235 240Ile Leu Leu Leu Thr Asn Glu Leu His Phe Tyr Ser Ile Pro Ile Lys245 250 255Met Glu Leu Thr His Gly Ser Ile Phe Asp Met Ala Leu Glu Lys Asn260 265 270Tyr Glu Asn Gly His Ile Tyr Phe Leu Arg Tyr Lys Lys Lys Tyr Glu275 280 285Tyr Ile Arg Asp Asp Asn Asp Glu Ile Lys Asn Ile Pro Ala Phe Thr290 295 300Ala Thr Gly Gly Ser Lys Val Pro Ile Arg Asn Lys Met Ile Asn Leu305 310 315 320Phe Asn Leu Thr Lys Ala Leu Gln Ser Tyr Leu Arg Ser Gln Ser Asn325 330 335Thr His Glu Gly Phe Cys His Ser Ser Asn Phe Tyr Phe Phe Asn Thr340 345 350Phe Arg Arg His Ile Pro Ile Ser Ile Tyr Cys Tyr Ser Val Tyr Leu355 360 365Ile Cys Ala Tyr Ser Ile Met Lys Leu Phe Lys Ser Thr Ile Phe Arg370 375 380Ser Tyr Ile Asn Phe Leu Thr Gly Phe Tyr Thr Tyr Leu Ile Thr Ile385 390 395 400Leu Ile Ile Ser Leu Pro Ile Tyr Leu Ile Ser Thr Asn Lys Lys Phe
405 410 415Tyr Glu Leu Leu Asn Phe Glu Glu Asn Tyr Ile Pro Ser Cys Tyr Glu420 425 430Trp His Pro Asp Asn Phe Asp Lys Tyr Ile Lys Ile Ala Asn Ile Trp435 440 445Trp Asn Val Leu Phe Phe Ser Ile Phe Gly Ala Phe Phe Phe Asn Leu450 455 460Phe Ile Ser Phe Leu Val Asn Lys Lys Arg Lys Val Ile Pro Lys Lys465 470 475 480Asn Asp Gln Asn Glu Ser Phe Asp Gly Tyr Lys Lys Val Glu Lys yal485 490 495Glu Arg Ile Leu Ile Leu Glu Lys Ile Lys Glu Leu Gln Asn Glu Ile500 505 510Met Lys Arg Lys Gly Ile Thr Asn Asn His Asn Asn Ile Lys Asn Tyr515 520 525Asn Ile Tyr Thr Asn Glu Asn Ile Tyr Asn Asn Asn Ile Asn Asn Ile530 535 540Asn Asn Asn Asn Asn Ile Tyr Glu Asn Leu Tyr Asp Asn Gly Glu Val545 550 555 560Lys Lys Asn Ile Leu Val Lys Pro Lys Ile Ile Asn Ser Asp Asp Glu565 570 575Asp Phe Leu Leu Glu Lys Lys Asn Ser Glu Phe Ile Lys Lys Ile Glu580 585 590Lys Gln Ile Glu Ile Leu Glu Glu Lys Leu Glu Phe Leu Ser Asn Asp595 600 605Glu Asn Val Lys Tyr Ile Phe Tyr Asn Asn Ser Ile Ala Pro Phe Asn610 615 620Thr Met Met Ile Tyr Met Asn Ile Phe Tyr Phe Ile Leu Val Ala Leu625 630 635 640Leu Ser Ser Leu Tyr Asn Trp Ser Tyr Ser Val Leu Phe Ser Leu Leu645 650 655Phe Val Ile Pro Ile Ser Ile Leu His Asn Leu Lys Thr Lys Pro Val660 665 670Arg Ile Phe Lys Lys Ile Ile Leu Ser Leu Phe Ile Leu Cys Met Phe675 680 685Ile Tyr Met Tyr Pro Asn Asp Asn His Leu Trp Asn Ile Arg Gln Lys690 695 700Leu Thr Asn Leu Phe Arg Asn Asn Ile Ser Lys Cys Cys Lys Tyr Leu705 710 715 720
Asp Lys His Lys Ile Leu Gln Ser Lys Tyr Phe Pro G1u Ser Leu Gln725 730 735Phe Ile Cys Ser Asn Arg Leu Phe Asp Ser Phe Tyr Ser Asn Lys Tyr740 745 750Phe Leu Asp Asn Leu Asn Ile Lys Phe Ser Tyr Val Leu Asp Ile Gln755 760 765Asn Gly Phe Leu Leu Thr Leu Tyr Asn Leu Ala Arg Asn His Phe Cys770 775 780Ile Gly Thr Ala Thr Tyr Pro Leu Ile Cys Phe Thr Leu Phe Pro Ile785 790 795 800Val Phe Tyr Ile Val Phe Leu Phe Phe Cys805 810<210>20<211>780<212>DNA<213>惡性瘧原蟲(Plasmodium falciparum)<400>20atggttattc gatttttcct gtttgtcatt acactcttag gcttatgcat aaatatggtg 60tgttgtaatt ttaaatattc gattatatta cctacttaca atgaaaaaga aaacttacca 120tatcttattt atatgataat tgatgaatta aataaacatg aaattaaatt tgaaataatt 180gtaatagatg ataatagtca agatggtact gcagatgtgt acaaaaagtt acaaaacatt 240tttaaggatg aagaattatt attaatacaa agaaaaggaa aattagggtt aggttctgca 300tatatggaag gtttaaaaaa tgtaacagga gattttgtta taataatgga tgctgattta 360tcacatcatc ctaaatatat ttataacttt attaaaaaac aaagagaaaa aaattgtgac 420attgttacag gcacaagata taagaaccaa ggtggaatat caggatggtc atttaataga 480attataataa gtagagtagc aaatttttta gctcaatttc tattattcat taatctatca 540gatttaaccg ggtcttttag attatataaa actaatgtac tgaaggaact tatgcaatct 600attaataata caggttatgt ttttcaaatg gaagttcttg taagagcata taaaatggga 660aaatctatag aagaagttgg ttacgttttt gttgatagat tatttggaaa atcaaaactg 720gaaactacag atattttaca atacttatca ggtcttttca agttattctg gtcaatataa 780<210>21<211>259<212>PRT<213>惡性瘧原蟲(Plasmodium falciparum)
<400>21Met Val Ile Arg Phe Phe Leu Phe Val Ile Thr Leu Leu Gly Leu Cys1 5 10 15Ile Asn Met Val Cys Cys Asn Phe Lys Tyr Ser Ile Ile Leu Pro Thr20 25 30Tyr Asn Glu Lys Glu Asn Leu Pro Tyr Leu Ile Tyr Met Ile Ile Asp35 40 45Glu Leu Asn Lys His Glu Ile Lys Phe Glu Ile Ile Val Ile Asp Asp50 55 60Asn Ser Gln Asp Gly Thr Ala Asp Val Tyr Lys Lys Leu Gln Asn Ile65 70 75 80Phe Lys Asp Glu Glu Leu Leu Leu Ile Gln Arg Lys Gly Lys Leu Gly85 90 95Leu Gly Ser Ala Tyr Met Glu Gly Leu Lys Asn Val Thr Gly Asp Phe100 105 110Val Ile Ile Met Asp Ala Asp Leu Ser His His Pro Lys Tyr Ile Tyr115120 125Asn Phe Ile Lys Lys Gln Arg Glu Lys Asn Cys Asp Ile Val Thr Gly130 135 140Thr Arg Tyr Lys Asn Gln Gly Gly Ile Ser Gly Trp Ser Phe Asn Arg145 150 155 160Ile Ile Ile Ser Arg Val Ala Asn Phe Leu Ala Gln Phe Leu Leu Phe165 170 175Ile Asn Leu Ser Asp Leu Thr Gly Ser Phe Arg Leu Tyr Lys Thr Asn180 185 190Val Leu Lys Glu Leu Met Gln Ser Ile Asn Asn Thr Gly Tyr Val Phe195 200 205Gln Met Glu Val Leu Val Arg Ala Tyr Lys Met Gly Lys Ser Ile Glu210 215 220Glu Val Gly Tyr Val Phe Val Asp Arg Leu Phe Gly Lys Ser Lys Leu225 230 235 240Glu Thr Thr Asp Ile Leu Gln Tyr Leu Ser Gly Leu Phe Lys Leu Phe245 250 255Trp Ser Ile<210> 22<211> 843<212>DNA<213>釀酒酵母(Saccharomyces cerevisiae)<400>22atgacaagat ctccctggaa gcgcctacta tggttgaaac aggagtaccc agataattat60
acagatccaa gttttattga gttgagagca agacaaaagg ctgagagtaa ccagaagtct120gatagaaaat tatcagaagc tgctcgcgct caaattaggt tggattttat aagtttctac180caaaccatat tgaacacttc tttcatttac atcactttta catatattta ctattatggc240ttcgatccta ttccgccaac tattttcctt tcatttataa cattgattat atcaaggacg300aaagtcgacc ctctattgtc ctcattcatg gacgtaaagt cttcgctgat tatcacattt360gcaatgttga ctctctctcc agtcctcaaa tctctttcta aaacaactgc atctgattcc420atatggacat tgtctttttg gctgacccta tggtacattt tcgttatttc gtcaacaaag480tccaaagata aaccctctaa cctttccacc aatatacttg tcgcccttgt tgctgtccta540tcatcgaggc tttcgaccac aatcgacgta ttctgttttc ttttaatttg tattcagttg600aatatcattc tacccactta tttatcggtg acgaataagg tagtaccaat aatttcaaat660attattgtat actcattttt gaatgttgct ctaggttgga tttatatgct gttgattttc720tttgcttcag tattttatat tactgtttta cctaagtggt tcatctactg gaaaatcaat780tatcataaac gggataacga tctactaagt acatgggatg caagaacacc aatattggat840tag 843<210>23<211>280<212>PRT<213>釀酒酵母(Saccharomyces cerevisiae)<400>23Met Thr Arg Ser Pro Trp Lys Arg Leu Leu Trp Leu Lys Gln Glu Tyr1 510 15Pro Asp Asn Tyr Thr Asp Pro Ser Phe Ile Glu Leu Arg Ala Arg Gln20 25 30Lys Ala Glu Ser Asn Gln Lys Ser Asp Arg Lys Leu Ser Glu Ala Ala35 40 45Arg Ala Gln Ile Arg Leu Asp Phe Ile Ser Phe Tyr Gln Thr Ile Leu50 55 60Asn Thr Ser Phe Ile Tyr Ile Thr Phe Thr Tyr Ile Tyr Tyr Tyr Gly65 70 75 80Phe Asp Pro Ile Pro Pro Thr Ile Phe Leu Ser Phe Ile Thr Leu Ile85 90 95Ile Ser Arg Thr Lys Val Asp Pro Leu Leu Ser Ser Phe Met Asp Val100 105 110Lys Ser Ser Leu Ile Ile Thr Phe Ala Met Leu Thr Leu Ser Pro Val115 120 125
Leu Lys Ser Leu Ser Lys Thr Thr A1a Ser Asp Ser Ile Trp Thr Leu130 135 140Ser Phe Trp Leu Thr Leu Trp Tyr Ile Phe Val Ile Ser Set Thr Lys145 150 155 160Ser Lys Asp Lys Pro Ser Asn Leu Ser Thr Asn Ile Leu Val Ala Leu165 170 175Val Ala Val Leu Ser Ser Arg Leu Ser Thr Thr Ile Asp Val Phe Cys180 185 190Phe Leu Leu Ile Cys Ile Gln Leu Asn Ile Ile Leu Pro Thr Tyr Leu195 200 205Ser Val Thr Asn Lys Val Val Pro Ile Ile Ser Asn Ile Ile Val Tyr210 215 220Ser Phe Leu Asn Val Ala Leu Gly Trp Ile Tyr Met Leu Leu Ile Phe225 230 235 240Phe Ala Ser Val Phe Tyr Ile Thr Val Leu Pro Lys Trp Phe Ile Tyr245 250 255Trp Lys Ile Asn Tyr His Lys Arg Asp Asn Asp Leu Leu Ser Thr Trp260 265 270Asp Ala Arg Thr Pro Ile Leu Asp275 280<210>24<211>764<212>DNA<213>釀酒酵母(Saccharomyces cerevisiae)<400>24atgattagta aagagtatga atttggtaag actagtatac tgaatagaaa gaagtataca 60ttagttatcg atgaagacaa gaatggcaat tttataagat ttaccgtttt acctgtatct 120aaccgaaagt tcaaaaaagt caagcaaaat gggagggtag agattaacat gggcatacga 180tatcaccaaa ttgtacttat tttactactg aatattttgt tctatgtaat ttgcctaaga 240teaagatttc tcgaacatat taatagaact tttgaagtga caatcgcgcg aagtttccag 300atcttaatta taatgggatt gtttgcctta ggtacaatta tacttgtgag gggacctagt 360gtggaaactg taacaatttt caaagaaagt ggactacagc tgtccagagt gaagggtatg 420gttatatttc ctcaacaatg gaatcggaag ttctttgaac aagtagagtt tatatcgaat 480gaaagaatta ttgatgtagt gatcaatgaa ggattctgtc ggggatttcg agtgatattc 540tatcttgcag caattgtacg taaatcgtct acgcttaagc tattatttcc agtatgtatt 600
caagcgaatt taagattgtt tcttacttca acgatctgat tagaaatact aacacaacaa660tgcagtcaaa tttgcccagt atcgatgacc aacgtctaat atacaacata tctagaaaat 720atctcagtaa gcaagaaaaa cccctgagca gaccaaaaga ttga 764<210>25<211>229<212>PRT<213>釀酒酵母(Saccharomyces cerevisiae)<400>25Met Ile Ser Lys Glu Tyr Glu Phe Gly Lys Thr Ser Ile Leu Asn Arg1 5 10 15Lys Lys Tyr Thr Leu Val Ile Asp Glu Asp Lys Asn Gly Asn Phe Ile20 25 30Arg Phe Thr Val Leu Pro Val Ser Asn Arg Lys Phe Lys Lys Val Lys35 40 45Gln Asn Gly Arg Val Glu Ile Asn Met Gly Ile Gln Tyr His Gln Ile50 55 60Val Leu Ile Leu Leu Leu Asn Ile Leu Phe Tyr Val Ile Cys Leu Arg65 70 75 80Ser Arg Phe Leu Glu His Ile Asn Arg Thr Phe Glu Val Thr Ile Ala85 90 95Arg Ser Phe Gln Ile Leu Ile Ile Met Gly Leu Phe Ala Leu Gly Thr100 105 110Ile Ile Leu Val Arg Gly Pro Ser Val Glu Thr Val Thr Ile Phe Lys115 120 125Glu Ser Gly Leu Gln Leu Ser Arg Val Lys Gly Met Val Ile Phe Pro130 135 140Gln Gln Trp Asn Arg Lys Phe Phe Glu Gln Val Glu Phe Ile Ser Asn145 150 155 160Glu Arg Ile Ile Asp Val Val Ile Asn Glu Gly Phe Cys Arg Gly Phe165 170 175Arg Val Ile Phe Tyr Leu Ala Ala Ile Val Arg Lys Ser Ser Thr Leu180 185 190Lys Leu Leu Phe Pro Ser Asn Leu Pro Ser Ile Asp Asp Gln Arg Leu195 200 205Ile Tyr Asn Ile Ser Arg Lys Tyr Leu Ser Lys Gln Glu Lys Pro Leu210 215 220Ser Arg Pro Lys Asp225
<210>26<211>423<212>DNA<213>釀酒酵母(Saccharomyces cerevisiae)<400>26atgtatacaa aagagtacta ctggttttca caatatatga taataacaag cactttggtg 60ctcaccataa tatggtccat cttaccatca tcgctgggtg aggctgcacc aaagcagttt120atcaacacgc tattggacat cttcccacaa agaagatgga ttattacctt ggagagcata180atgctgatgg gcatgctatg cacatacatc ggccttctga tgtacaatga agatacatta240acaccgccgc tagattctct atctacagta acggatgccg gtggtcaact tgtaatagag300gacgacccgg acgtattcgt taagaaatgg gcctttaaag aaacaagtgg tatttacgat360ctgtctctga tggatgcctg ccaacttctc tacctatatg ataacgacca taccagcaca420tag 423<210>27<211>140<212>PRT<213>釀酒酵母(Saccharomyces cerevisiae)<400>27Met Tyr Thr Lys Glu Tyr Tyr Trp Phe Ser Gln Tyr Met Ile Ile Thr1 5 10 15Ser Thr Leu Val Leu Thr Ile Ile Trp Ser Ile Leu Pro Ser Ser Leu20 25 30Gly Glu Ala Ala Pro Lys Gln Phe Ile Asn Thr Leu Leu Asp Ile Phe35 40 45Pro Gln Arg Arg Trp Ile Ile Thr Leu Glu Ser Ile Met Leu Met Gly50 55 60Met Leu Cys Thr Tyr Ile Gly Leu Leu Met Tyr Asn Glu Asp Thr Leu65 70 75 80Thr Pro Pro Leu Asp Ser Leu Ser Thr Val Thr Asp Ala Gly Gly Gln85 90 95Leu Val Ile Glu Asp Asp Pro Asp Val Phe Val Lys Lys Trp Ala Phe100 105 110Lys Glu Thr Ser Gly Ile Tyr Asp Leu Ser Leu Met Asp Ala Cys Gln115 120 125Leu Leu Tyr Leu Tyr Asp Asn Asp His Thr Ser Thr130 135 140<210>28
<211>915<212>DNA<213>釀酒酵母(Saccharomyces cerevisiae)<400>28atgaagatgt tgaggcgtac aaaggtaaat ttttcaaaat tgctttacaa gattactaaa 60ctagcaatag tactgacgat cctatatatt tattttacgc ceaaaatcgt ctcccgaaac120aatgcatcat tgcagcatat ttttcctcat aaatatggcg attatgaaat caatttggtc180atagcgcacc ctgacgacga agttatgttt ttttccccea taatttctca actgaattcg240tactttccga gaaccgtccc atttaacata atctgcttat caaagggcaa cgccgaaggt300cttggcgaaa ccagggtaag agaattaaat gagtcggccg ctttattgct acacaatgaa360agagcagtct ccgtacaggt gatggatttc caggatggta tggacgaaat atgggatatt420gattctataa cttcttctct ttcacaaaag atagatataa agaatcataa cttgaaccag480attatcgtta cctttgattc atatggtgta tcaaatcata tcaaccacaa aagctgttat540gctgccgtta aaaagttggt ggatgattat gctcaaccta agaceaaaag aaatgaacaa600ccacctcatg tcactgcgct ttatttgaga agctacaaga acaacatcgt tttaaagtac660aactccttta tttgggaaat cctaaaaata ctttacgacc tgatttctcc attccgtaga720ataattcagg cgcttccgcc taacacagcc gccgaaaaag acaagctttc acttatgaat780acacatgcac aatacgtact agcgtttgcc actatgctaa atgctcacga atcccaagtt840gtgtggttta gatacggatg gtggatattt tccagatttg tcttcgttaa tgaatttgat900gtttatacat attag915<210>29<211>304<212>PRT<213>釀酒酵母(Saccharomyces cerevisiae)<400>29Met Lys Met Leu Arg Arg Thr Lys Val Asn Phe Ser Lys Leu Leu Tyr1 5 10 15Lys Ile Thr Lys Leu Ala Ile Val Leu Thr Ile Leu Tyr Ile Tyr Phe20 25 30Thr Pro Lys Ile Val Ser Arg Asn Asn Ala Ser Leu Gln His Ile Phe35 40 45Pro His Lys Tyr Gly Asp Tyr Glu Ile Asn Leu Val Ile Ala His Pro50 55 60Asp Asp Glu Val Met Phe Phe Ser Pro Ile Ile Ser Gln Leu Asn Ser65 70 75 80
Tyr Phe Pro Arg Thr Val Pro Phe Asn Ile Ile Cys Leu Ser Lys Gly85 90 95Asn Ala Glu Gly Leu Gly Glu Thr Arg Val Arg Glu Leu Asn Glu Ser100 105 110Ala Ala Leu Leu Leu His Asn Glu Arg Ala Val Ser Val Gln Val Met115 120 125Asp Phe Gln Asp Gly Met Asp Glu Ile Trp Asp Ile Asp Ser Ile Thr130 135 140Ser Ser Leu Ser Gln Lys Ile Asp Ile Lys Asn His Asn Leu Asn Gln145 150 155 160Ile Ile Val Thr Phe Asp Ser Tyr Gly Val Ser Asn His Ile Asn His165 170 175Lys Ser Cys Tyr Ala Ala Val Lys Lys Leu Val Asp Asp Tyr Ala Gln180 185 190Pro Lys Thr Lys Arg Asn Glu Gln Pro Pro His Val Thr Ala Leu Tyr195 200 205Leu Arg Ser Tyr Lys Asn Asn Ile Val Leu Lys Tyr Asn Ser Phe Ile210 215 220Trp Glu Ile Leu Lys Ile Leu Tyr Asp Leu Ile Ser Pro Phe Arg Arg225 230 235 240Ile Ile Gln Ala Leu Pro Pro Asn Thr Ala Ala Glu Lys Asp Lys Leu245 250 255Ser Leu Met Asn Thr His Ala Gln Tyr Val Leu Ala Phe Ala Thr Met260 265 270Leu Asn Ala His Glu Ser Gln Val Val Trp Phe Arg Tyr Gly Trp Trp275 280 285Ile Phe Ser Arg Phe Val Phe Val Asn Glu Phe Asp Val Tyr Thr Tyr290 295 300<210>30<211>2760<212>DNA<213>釀酒酵母(Saccharomyces cerevi siae)<400>30atgtggaaca aaaccagaac gacgcttctg gctgttggtg tcttatttca tttattttac 60ctatggtcta tttttgatat ctatttcatt tcaccgctcg ttcatggtat gagcccatat120caaagtactc caacccctcc tgcaaagaga ttgtttttga ttgtcggtga tggtttacgt180gcagatacca cttttgataa agtcactcat ccagtatccg gaaaaacaga atttctggca240
ccttttatta gatctttggt aatgaataat gccacctacg gtatatcaca taccagaatg300ccaactgaat cccgtcctgg tcatgttgct atgattgctg ggttttacga agatgttagt360gccgtcacaa aaggttggaa gtcaaaccct gtcaatttcg atagtttttt caaccaatct420actcatactt attcattcgg ttcacctgac attttaccta tgttcaaaga tggcgcttct480gacccaaata aagttgacac ttggatgtat gatcatactt tcgaggattt tacgcaatct540tccatcgagc tggatgcttt tgtctttaga cacttggatc aattattcca caattccaca600ctgaactcaa cattggatta tgaaattagg caagacggta atgtattctt tctacatcta660ctaggttgcg atactgccgg acattcttat agaccatatt ctgccgagta ttatgacaat720gtcaaatata ttgatgatca aatccctatc cttatagaca aagtcaacaa gttttttgcg780gacgacaaaa ccgcatttat ttttacagca gatcatggta tgagtgcatt tggatcacat840ggtgacggtc atcctaacaa cacaaggacc cctcttgttg cttggggtgc gggtttgaat900aaaccagtac ataatccttt tccggtatcc gacaactata ctgaaaattg ggagctttcg960agcattaaaa gaaatgatgt caagcaagca gatattgctt ctttaatgtc atacttgatt1020ggtgtgaact atcctaaaaa ttcagttggt gagttaccaa tagcatatat cgatggaaaa1080gaaagtgaca agcttgccgc attgtacaac aacgcaagaa gcattttaga gcagtactta1140gtcaagcaag atgaggtaat agactctcaa tttttttata aggaatactt caagtttgtt1200gaaaagtctc attcacatta cttagaagag atagaaacct taattcagcg tatatctgaa1260ggagaaaact atttggaaca agaagcaatc acccttacag aggaattaat gcagataaca1320ttggaaggtt tacattattt gacaacctat aattggagat tcattagaac tattgttaca1380tttgggtttg ttgggtggat ctttttttct tttataatat ttttgaaatc attcatatta1440gagaatgtaa ttgatgacca aaaagcgtca ccattaagcc atgcagtatt tggttccata1500ggaattttac taaattggat tttgttctac caacattctc cgttcaattt ttacatgtac1560cttcttttcc cattatactt ttggagctat atttttacaa atagatccgt actacgttca1620ggtatcaagg aattcttcaa aggtacctct ccttggaaaa gagttttaat aacaatctct1680attatatcag tttatgaggg aattgtatat ggatttttcc atagatggac gtttacgcta1740attacaaata tattggcgtt ttacccgttt atttgtgggg tgagagagct atccgtgaat1800atattgtgga tcataactag tgttctttta tctacattta ccttatttga cgctgttaaa1860attgaggact tgaaccagat acatctagca gggttattaa tcattctcag tgccttttat1920gctctttaca aaatacattc caggataaat tcctacacgc gtgctatatt tgccattcaa1980
atttccttgg tggctgccat gttggcggtt actcatcgtt cagttatctc tttacagcta2040agacaagggt taccaagaga gtcacaggtc gctggatgga taattttttt tgtatctctt2100tttgtaatgc caattttaca ttataggaag cccaacaatg attacaaagt gagattattg2160atcatttatt taaccttcgc accatccttt atcattttga ctatatcatt cgaatccctt2220ttctacttct tgttcactag ttacatggta caatggattg aaattgagaa caaaatcaaa2280gaaatgaaga cccaaaaaga tgaaaattgg ttacaagtgc taagagtttc agtaatcggg2340ttctttttac ttcaagtcgc attctttgga actggtaacg tcgcttcaat ctcttcattt2400tcattggagt ctgtttgtag attgttgcca atttttgatc ctttcctgat gggcgcatta2460ttgatgttga aattgataat tccctacggg ctattgtcca catgcctagg tatactgaat2520ttaaaactta acttcaagga ctacacaatc tcatcattaa ttatttccat gagtgatatt2580ctgtcgttga attttttcta ccttttaaga acggaggggt cgtggttgga tattggcata2640accatttcca actattgttt ggcgatccta tcatctttgt tcatgcttat tttggaagta2700ctcggtcatg tgttgctaaa aaatgtcatc atacaggata aaaccaaaaa aacacaatag2760<210>31<211>919<212>PRT<213>釀酒酵母(Saccharomyces cerevisiae)<400>31Met Trp Asn Lys Thr Arg Thr Thr Leu Leu Ala Val Gly Val Leu Phe1 5 10 15His Leu Phe Tyr Leu Trp Ser Ile Phe Asp Ile Tyr Phe Ile Ser Pro20 25 30Leu Val His Gly Met Ser Pro Tyr Gln Ser Thr Pro Thr Pro Pro Ala35 40 45Lys Arg Leu Phe Leu Ile Val Gly Asp Gly Leu Arg Ala Asp Thr Thr50 55 60Phe Asp Lys Val Thr His Pro Val Ser Gly Lys Thr Glu Phe Leu Ala65 70 75 80Pro Phe Ile Arg Ser Leu Val Met Asn Asn Ala Thr Tyr Gly Ile Ser85 90 95His Thr Arg Met pro Thr Glu Ser Arg Pro Gly His Val Ala Met Ile100 105 110Ala Gly Phe Tyr Glu Asp Val Ser Ala Val Thr Lys Gly Trp Lys Ser115 120 125Asn Pro Val Asn Phe Asp Ser Phe Phe Asn Gln Ser Thr His Thr Tyr
130 135 140Ser Phe Gly Ser Pro Asp Ile Leu Pro Met Phe Lys Asp Gly Ala Ser145 150 155 160Asp Pro Asn Lys Val Asp Thr Trp Met Tyr Asp His Thr Phe Glu Asp165 170 175Phe Thr Gln Ser Ser Ile Glu Leu Asp Ala Phe Val Phe Arg His Leu180 185 190Asp Gln Leu Phe His Asn Ser Thr Leu Asn Ser Thr Leu Asp Tyr Glu195 200 205Ile Arg Gln Asp Gly Asn Val Phe Phe Leu His Leu Leu Gly Cys Asp210 215 220Thr Ala Gly His Ser Tyr Arg Pro Tyr Ser Ala Glu Tyr Tyr Asp Asn225 230 235 240Val Lys Tyr Ile Asp Asp Gln Ile Pro Ile Leu Ile Asp Lys Val Asn245 250 255Lys Phe Phe Ala Asp Asp Lys Thr Ala Phe Ile Phe Thr Ala Asp His260 265 270Gly Met Ser Ala Phe Gly Ser His Gly Asp Gly His Pro Asn Asn Thr275 280 285Arg Thr Pro Leu Val Ala Trp Gly Ala Gly Leu Asn Lys Pro Val His290 295 300Asn Pro Phe Pro Val Ser Asp Asn Tyr Thr Glu Asn Trp Glu Leu Ser305 310 315 320Ser Ile Lys Arg Asn Asp Val Lys Gln Ala Asp Ile Ala Ser Leu Met325 330 335Ser Tyr Leu Ile Gly Val Asn Tyr Pro Lys Asn Ser Val Gly Glu Leu340 345 350Pro Ile Ala Tyr Ile Asp Gly Lys Glu Ser Asp Lys Leu Ala Ala Leu355 360 365Tyr Asn Asn Ala Arg Ser Ile Leu Glu Gln Tyr Leu Val Lys Gln Asp370 375 380Glu Val Ile Asp Ser Gln Phe Phe Tyr Lys Glu Tyr Phe Lys Phe Val385 390 395 400Glu Lys Ser His Ser His Tyr Leu Glu Glu Ile Glu Thr Leu Ile Gln405 410 415Arg Ile Ser Glu Gly Glu Asn Tyr Leu Glu Gln Glu Ala Ile Thr Leu420 425 430Thr Glu Glu Leu Met Gln Ile Thr Leu Glu Gly Leu His Tyr Leu Thr435 440 445
Thr Tyr Asn Trp Arg Phe Ile Arg Thr Ile Val Thr Phe Gly Phe Val450 455 460Gly Trp Ile Phe Phe Ser Phe Ile Ile Phe Leu Lys Ser Phe Ile Leu465 470 475 480Glu Asn Val Ile Asp Asp Gln Lys Ala Ser Pro Leu Ser His Ala Val485 490 495Phe Gly Ser Ile Gly Ile Leu Leu Asn Trp Ile Leu Phe Tyr Gln His500 505 510Ser Pro Phe Asn Phe Tyr Met Tyr Leu Leu Phe Pro Leu Tyr Phe Trp515 520 525Ser Tyr Ile Phe Thr Asn Arg Ser Val Leu Arg Ser Gly Ile Lys Glu530 535 540Phe Phe Lys Gly Thr Ser Pro Trp Lys Arg Val Leu Ile Thr Ile Ser545 550 555 560Ile Ile Ser Val Tyr Glu Gly Ile Val Tyr Gly Phe Phe His Arg Trp565 570 575Thr Phe Thr Leu Ile Thr Asn Ile Leu Ala Phe Tyr Pro Phe Ile Cys580 585 590Gly Val Arg Glu Leu Ser Val Asn Ile Leu TrpIle Ile Thr Ser Val595 600605Leu Leu Ser Thr Phe Thr Leu Phe Asp Ala Val Lys Ile Glu Asp Leu610 615 620Asn Gln Ile His Leu Ala Gly Leu Leu Ile Ile Leu Ser Ala Phe Tyr625 630 635 640Ala Leu Tyr Lys Ile His Ser Arg Ile Asn Ser Tyr Thr Arg Ala Ile645 650 655Phe Ala Ile Gln Ile Ser Leu Val Ala Ala Met Leu Ala Val Thr His660 665 670Arg Ser Val Ile Ser Leu Gln Leu Arg Gln Gly Leu Pro Arg Glu Ser675 680 685Gln Val Ala Gly Trp Ile Ile Phe Phe Val Ser Leu Phe Val Met Pro690 695 700Ile Leu His Tyr Arg Lys Pro Asn Asn Asp Tyr Lys Val Arg Leu Leu705 710 715 720Ile Ile Tyr Leu Thr Phe Ala Pro Ser Phe Ile Ile Leu Thr Ile Ser725 730 735Phe Glu Ser Leu Phe Tyr Phe Leu Phe Thr Ser Tyr Met Val Gln Trp740 745 750Ile Glu Ile Glu Asn Lys Ile Lys GIu Met Lys Thr Gln Lys Asp Glu
755 760 765Asn Trp Leu Gln Val Leu Arg Val Ser Val Ile Gly Phe Phe Leu Leu770 775 780Gln Val Ala Phe Phe Gly Thr Gly Asn Val Ala Ser Ile Ser Ser Phe785 790 795 800Ser Leu Glu Ser Val Cys Arg Leu Leu Pro Ile Phe Asp Pro Phe Leu805 810 815Met Gly Ala Leu Leu Met Leu Lys Leu Ile Ile Pro Tyr Gly Leu Leu820 825 830Ser Thr Cys Leu Gly Ile Leu Asn Leu Lys Leu Asn Phe Lys Asp Tyr835 840 845Thr Ile Ser Ser Leu Ile Ile Ser Met Ser Asp Ile Leu Ser Leu Asn850 855 860Phe Phe Tyr Leu Leu Arg Thr Glu Gly Ser Trp Leu Asp Ile Gly Ile865 870 875 880Thr Ile Ser Asn Tyr Cys Leu Ala Ile Leu Ser Ser Leu Phe Met Leu885 890 895Ile Leu Glu Val Leu Gly His Val Leu Leu Lys Asn Val Ile Ile Gln900 905 910Asp Lys Thr Lys Lys Thr Gln915<210>32<211>660<212>DNA<213>釀酒酵母(Saccharomyces cerevisiae)<400>32atgccagcta aaaaaaggac tagaaagaca gtgaaaaaaa ccgtatcatt ctccgatgac 60acaacattaa caacgcacca aaatcgtgag aaaaagaacg tagatcatga tcgtccacct120gtgtatgtga ggaaaacccc tctgatgaca tttccatacc atttagtagc actactttat180tactacgttt ttgtatcttc aaatttcaat acggtgaagt tgctaagttt tttgattcct240acacaagttg cttatttagt tttacaattc aataaatgca cagtttacgg taacaaaatc300attaagatca attactcatt gaccattatt tgtctaggtg ttacattttt gttgagcttt360cccacaatgt tattaactat attatttggt gcgccattaa tggacttatt gtgggaaacc420tggctgttgt cactgcattt tgcattttta gcataccctg cagtttattc tgtatttaat480tgtgatttca aagtgggatt atggaagaag tattttatct ttatcgttgt agggggttgg540attagttgtg ttgtcattcc tttggattgg gatagagatt ggcagaattg gccaattcct600
attgttgttg gaggttattt gggcgctttg gtgggctata ctatcggtgc ctatatataa660<210>33<211>219<212>PRT<213>釀酒酵母(Saccharomyces cerevisiae)<400>33Met Pro Ala Lys Lys Arg Thr Arg Lys Thr Val Lys Lys Thr Val Ser1 510 15Phe Ser Asp Asp Thr Thr Leu Thr Thr His Gln Asn Arg Glu Lys Lys20 25 30Asn Val Asp His Asp Arg Pro Pro Val Tyr Val Arg Lys Thr Pro Leu35 40 45Met Thr Phe Pro Tyr His Leu Val Ala Leu Leu Tyr Tyr Tyr Val Phe50 55 60Val Ser Ser Asn Phe Asn Thr Val Lys Leu Leu Ser Phe Leu Ile Pro65 70 75 80Thr Gln Val Ala Tyr Leu Val Leu Gln Phe Asn Lys Cys Thr Val Tyr85 90 95Gly Asn Lys Ile Ile Lys Ile Asn Tyr Ser Leu Thr Ile Ile Cys Leu100 105 110Gly Val Thr Phe Leu Leu Ser Phe Pro Thr Met Leu Leu Thr Ile Leu115 120 125Phe Gly Ala Pro Leu Met Asp Leu Leu Trp Glu Thr Trp Leu Leu Ser130 135 140Leu His Phe Ala Phe Leu Ala Tyr Pro Ala Val Tyr Ser Val Phe Asn145 150 155 160Cys Asp Phe Lys Val Gly Leu Trp Lys Lys Tyr Phe Ile Phe Ile Val165 170 175Val Gly Gly Trp Ile Ser Cys Val Val Ile Pro Leu Asp Trp Asp Arg180 185 190A sp Trp Gln Asn Trp Pro Ile Pro Ile Val Val Gly Gly Tyr Leu Gly195 200 2 05Ala Leu Val Gly Tyr Thr Ile Gly Ala Tyr Ile210 215<210> 34<211> 2493<212>DNA<213>釀酒酵母(Saccharomyces cerevisiae)
<400> 34atgaacttga agcagttcac gtgcctatca tgcgctcaat tactcgctat tctgctcttt60atctttgctt ttttccctag aaaaatcgtg ctgacaggta tatcaaagca agatccggat120caagaccgtg atctccagcg cgataggccc ttccagaaat tggtgtttgt gatcattgat180gctctcagat cagactttct ttttgattcg cagatttccc acttcaacaa cgtgcaccaa240tggctcaata cgggcgaagc atggggttac acgtcatttg ctaatccgcc taccgtgacg300ctgcctagac tcaaaagtat tactacggga tctacaccta gcttcattga cttgctgctg360aatgtagccc aggacataga ttccaacgat ctttcggagc acgattcctg gctgcagcag420ttcatccaac ataataacac gattcgtttc atgggcgatg acacctggct gaaactgttc480ceacagcaat ggtttgactt cgctgacccg acacactcgt tctttgtcag tgatttcact540caagtcgata ataatgtgac gaggaacttg cccgggaaat tatttcagga atgggcccag600tgggacgtgg ctatcctgca ttacttgggt cttgaccata tcgggcataa agatggcccg660cattcaaagt ttatggctgc taaacatcaa gaaatggaca gcattctgaa gtcaatatat720gatgaagtgt tggaacatga agatgacgat gatacactga tttgtgttct tggcgaccat780ggaatgaacg aactgggcaa ccatggtggc tcttcagccg gcgaaacatc agcaggattg840ttgtttttgt cacctaagct ggcgcaattt gctaggccag aatcgcaagt aaactacaca900ttgcccatca acgctagtcc ggactggaat ttccagtatt tagagactgt tcaacaaatt960gatatcgtcc ccaccatagc agcactgttt ggtatgccaa tccccatgaa cagtgttggg1020ataataatac ctgacttttt acaactgttg cccaataagt tggcaagtat gaaagaaaat1080tttatgcatt tgtggaaatt atcagaccat cacggcgagg ttgctcttga cgatttcact1140gccgaagata tttatacaaa gatgtacact attcaagaaa cgttaaccaa gtctgcaaca1200aattataatt atcctctttt gacactggct tttgttggtt tcctcataat aacaatcatc1260gccatttatg tattattacg ttattctggg cctgattttt ggcagttgcg cgtttcttcc1320ctgtctgttc tgttagtttc cattatacta ggcgtttcca catttgcaag tagtttcatt1380gaagaggagc accaactgtg gtggtggata gtaactgcat tctcggcggt ccctctgttc1440gtataccgat tgaatgtgct cataatcgtg cgctggttta taatgatggc atgcgtacgc1500tcaatcaagt tttggaataa cagtggccag aaattcattt attctaacgt tatgtccaat1560ctacttaatc agaatccttc ctggaagtgg tgcttaaata tgttgacatt tctagtgctg1620ataatggcat ctgctggttt tcaagtacta cattttattg tcactactat tttggtgggg1680ttgtgtttca cgtacaaaat ctcgtgggaa atcgtcaatg gtaaccaggc agaaataccg1740
ctctttatgc atgatttact ggctaagata gactttgcac caactgaaag taacttgatt1800gtacttgcgc gcgttttctt ccaagcttgg gctattgttg tcatttcaag gttggtcctg1860acgaaattga aagtacttaa caagaactac ctcattaaag atatgaaagt ttatataaca1920attcttttga tgttccaaac ttcttctcag aacataggtc aatttctcgt tttccaaata1980ttagagtccc aaatttttta ctttttccaa aatattccaa ccgcctcatt aacatcaaca2040agtaagattt atttttcgaa tttggtgtcc ttaattttac aaaattttac atttttccaa2100ttcggtggca caaattccat ttctactata gaccttggaa acgcatacca tggtgtttcc2160tcagactaca acatctacgt agtggggata ttaatgtccg ttgccaattt cgcgccggca 2220atatactggt ccatgctacc gtggtcaata aactacgcct ctattccagc acaagttaag2280ttgcaaacgt tcatcagaag taagttacct gccttcacct atcattgtat atttggaact2340tgtttgatga cggcatgcgt cgttttgaga tttcatctet ttatttggtc cgttttcagt 2400ccaaaattat gttattttct tgggtggaat tttgtgatgg gattgctgaa tggctggtta2460cctgaattgg ccctcctttg cgctcttgat taa 2493<210>35<211> 830<212>PRT<213>釀酒酵母(Saccharomyces cerevisiae)<400> 35Met Asn Leu Lys Gln Phe Thr Cys Leu Ser Cys Ala Gln Leu Leu Ala1 5 10 15Ile Leu Leu Phe Ile Phe Ala Phe Phe Pro Arg Lys Ile Val Leu Thr20 25 30Gly Ile Ser Lys Gln Asp Pro Asp Gln Asp Arg Asp Leu Gln Arg Asp35 40 45Arg Pro Phe Gln Lys Leu Val Phe Val Ile Ile Asp Ala Leu Arg Ser50 55 60Asp Phe Leu Phe Asp Ser Gln Ile Ser His Phe Asn Asn Val His Gln65 70 75 80Trp Leu A sn Thr Gly Glu Ala Trp Gly Tyr Thr Ser Phe Ala Asn Pro85 90 95Pro Thr Val Thr Leu Pro Arg Leu Lys Ser Ile Thr Thr Gly Ser Thr100 105 110Pro Ser Phe Ile Asp Leu Leu Leu Asn Val Ala Gln Asp Ile Asp Ser115 120 125
Asn Asp Leu Ser Glu His Asp Ser Trp Leu Gln Gln Phe Ile Gln His130 135 140Asn Asn Thr Ile Arg Phe Met Gly Asp Asp Thr Trp Leu Lys Leu Phe145 150 155 160Pro Gln Gln Trp Phe Asp Phe Ala Asp Pro Thr His Ser Phe Phe Val165 170 175Ser Asp Phe Thr Gln Val Asp Asn Asn Val Thr Arg Asn Leu Pro Gly180 185 190Lys Leu Phe Gln Glu Trp Ala Gln Trp Asp Val Ala Ile Leu His Tyr195 200 205Leu Gly Leu Asp His Ile Gly His Lys Asp Gly Pro Hi s Ser Lys Phe210 215 220Met Ala Ala Lys His Gln Glu Met Asp Ser Ile Leu Lys Ser Ile Tyr225 230 235 240Asp Glu Val Leu Glu Hi s Glu Asp Asp Asp Asp Thr Leu Ile Cys Val245 250 255Leu Gly Asp His Gly Met Asn Glu Leu Gly Asn Hi s Gly Gly Ser Ser260 265 270Ala Gly Glu Thr Ser Ala Gly Leu Leu Phe Leu Ser Pro Lys Leu Ala275 280 285Gln Phe Ala Arg Pro Glu Ser Gln Val Asn Tyr Thr Leu Pro Ile Asn290 295 300Ala Ser Pro Asp Trp Asn Phe Gln Tyr Leu Glu Thr Val Gln Gln Ile305 310 315 320Asp Ile Val Pro Thr Ile Ala Ala Leu Phe Gly Met Pro Ile Pro Met325 330 335Asn Ser Val Gly Ile Ile Ile Pro Asp Phe Leu Gln Leu Leu Pro Asn340 345 350Lys Leu Ala Ser Met Lys Glu Asn Phe Met Hi s Leu Trp Lys Leu Ser355 360 365Asp His His Gly Glu Val Ala Leu Asp Asp Phe Thr Ala Glu Asp Ile370 375 380Tyr Thr Lys Met Tyr Thr Ile Gln Glu Thr Leu Thr Lys Ser Ala Thr385 390 395 400Asn Tyr Asn Tyr Pro Leu Leu Thr Leu Ala Phe Val Gly Phe Leu Ile405 410 415Ile Thr Ile Ile Ala Ile Tyr Val Leu Leu Arg Tyr Ser Gly Pro Asp420 425 430Phe Trp Gln Leu Arg Val Ser Ser Leu Ser Val Leu Leu Val Ser Ile
435 440 445Ile Leu Gly Val Ser Thr Phe Ala Sel Ser Phe Ile Glu Glu Glu His450 455 460Gln Leu Trp Trp Trp Ile Val Thr Ala Phe Ser Ala Val Pro Leu Phe465 470 475 480Val Tyr Arg Leu Asn Val Leu Ile Ile Val Arg Trp Phe Ile Met Met485 490 495Ala Cys Val Arg Ser Ile Lys Phe Trp Asn Asn Ser Gly Gln Lys Phe500 505 510Ile Tyr Ser Asn Val Met Ser Asn Leu Leu Asn Gln Asn Pro Ser Trp515 520 525Lys Trp Cys Leu Asn Met Leu Thr Phe Leu Val Leu Ile Met Ala Ser530 535 540Ala Gly Phe Gln Val Leu His Phe Ile Val Thr Thr Ile Leu Val Gly545 550 555 560Leu Cys Phe Thr Tyr Lys Ile Ser Trp Glu Ile Val A sn Gly Asn Gln565 570 575Ala Glu Ile Pro Leu Phe Met His Asp Leu Leu Ala Lys Ile Asp Phe580 585 590Ala Pro Thr Glu Ser Asn Leu Ile Val Leu Ala Arg Val Phe Phe Gln595 600 605Ala Trp Ala Ile Val Val Ile Ser Arg Leu Val Leu Thr Lys Leu Lys610 615 620Val Leu Asn Lys Asn Tyr Leu Ile Lys Asp Met Lys Val Tyr Ile Thr625 630 635 640Ile Leu Leu Met Phe Gln Thr Ser Ser Gln Asn Ile Gly Gln Phe Leu645 650 655Val Phe Gln Ile Leu Glu Ser Gln Ile Phe Tyr Phe Phe Gln Asn Ile660 665 670Pro Thr Ala Ser Leu Thr Ser Thr Ser Lys Ile Tyr Phe Ser Asn Leu675 680 685Val Ser Leu Ile Leu Gln Asn Phe Thr Phe Phe Gln Phe Gly Gly Thr690 695 700Asn Ser Ile Ser Thr Ile Asp Leu Gly Asn Ala Tyr Hi s Gly Val Ser705 710 715 720Ser Asp Tyr Asn Ile Tyr Val Val Gly Ile Leu Met Ser Val Ala Asn725 730 735Phe Ala Pro Ala Ile Tyr Trp Ser Met Leu Pro Trp Ser Ile Asn Tyr740 745 750
Ala Ser Ile Pro Ala Gln Val Lys Leu Gln Thr Phe Ile Arg Ser Lys755 760 76 5Leu Pro Ala Phe Thr Tyr Hi s Cys Ile Phe Gly Thr Cys Leu Met Thr770 775 780Ala Cys Val Val Leu Arg Phe Hi s Leu Phe Ile Trp Ser Val Phe Ser785 790 795 800Pro Lys Leu Cys Tyr Phe Leu Gly Trp Asn Phe Val Met Gly Leu Leu805 810 815Asn Gly Trp Leu Pro Glu Leu Ala Leu Leu Cys Ala Leu Asp820 825 830<210>36<211> 1605<212>DNA<213>釀酒酵母(Saccharomyces cerevisiae)<400>36atgtccaatg caaatctaag aaaatgggtt ggtttttgct ttgttgccat ttatctcttt 60ttaggtgttc cactgtggta caagctaact acagtttata gagcatcact accaataaat120tacattgagt cacttcaaaa taacaaattc caagatattc atctcgtaat accggtgtat180gttaagtcag atacttacag atttcctgac gttcatgacg ctatccaagt acaagttaac240catttattga attctcagga gcaacgggtc ccttggtctt tacaagttct tccatataat300gagactattg agcagatgga aagtgaaggc aaccagtttc atgtcgttac tttgaagtta360gacgaattta ttggttactc atcagcttac gacaccaaag aaacactagt atattacgac420gatgctgccg ttttaagtaa tgatctaccg ttttttgttg ctcaaacatt ggtagagcac480actttccaat tggaatggac gcatttgaat aaaacgtgtg aaggcgtttc tacaaacaac540gatgtcgcaa tatcttatga tccaaacatt catttaagtg taactttatt gtcaggtgat600gggaatcctg ttgcatggga aattgagcct acattaactg actacttttc accttttagg660aagttcttat caccactggt aaattttaca gtagattcat ccattgttta tcataatgat720ttgaatttgc attcattaaa tggatcatgt acaagcgtta cgtggtttga tctctctcat780actattgatc tttctgaact ttcttcaatg gcctattacc cagaagattc tgcactgaat840ttagccatag tctttcctag tgcttcttea agtcccgatg gtctggcgtt cattaatggc900actcggattt cagacgaaat aaccacatta gattggaata gttatctagt tcctcaatgg960ggggttataa taataaataa aatgccgttg aagccaaatt cagtcattag cgaagattat1020ttagaaccta tgatgtaccg ttttgcgaca gatatttttc aactattggg attaacggag1080
ggctcgcaag atttgttatc accttatatt accatagatt cattcaaaag gttgacaatt 1140ttacagaatc tagataaagc tacggaaaca ttatggtcgt tagtgaaatt aactcaacaa 1200tttcagggca tgtctatccc acgcgaagta tcggataatg ttatcgaagc tttagactta 1260aggctacaga ttattgattt attaaatgat cctggaaagg gtggagatat cgtctggaac 1320aatgccctgc atctaagtaa tgaattggtt aaactatgcg aaaaggcatt tttcaatgga 1380gaaatggttc aacaaaattt cttcccacaa gagcacatga tagctgtgta tttaccttta 1440ttaggcccaa tatcggcagt catgttcttt ggtttctaca acgtgatgaa ggaaaagaat 1500caaaagagta aaaagaatgg aaccgagaga gaagttgcta aagaaaaatt agagttgaaa 1560gaggctcaaa aattacatgc tattgatggt gaagatgaat tatga 1605<210> 37<211>534<212>PRT<213>釀酒酵母(Saccharomyces cerevisiae)<400> 37Met Ser Asn Ala Asn Leu Arg Lys Trp Val Gly Phe Cys Phe Val Ala15 10 15Ile Tyr Leu Phe Leu Gly Val Pro Leu Trp Tyr Lys Leu Thr Thr Val20 25 30Tyr Arg Ala Ser Leu Pro Ile Asn Tyr Ile Glu Ser Leu Gln Asn Asn35 40 45Lys Phe Gln Asp Ile His Leu Val Ile Pro Val Tyr Val Lys Ser Asp50 55 60Thr Tyr Arg Phe Pro Asp Val Hi s Asp Ala Ile Gln Val Gln Val Asn65 70 75 80His Leu Leu Asn Set Gln Glu Gln Arg Val Pro Trp Ser Leu Gln Val85 90 95Leu Pro Tyr Asn Glu Thr Ile Glu Gln Met Glu Ser Glu Gly Asn Gln100 l05 110Phe His Val Val Thr Leu Lys Leu Asp Glu Phe Ile Gly Tyr Ser Ser115 120 125Ala Tyr Asp Thr Lys Glu Thr Leu Val Tyr Tyr Asp Asp Ala Ala Val130 135 140Leu Ser Asn Asp Leu Pro Phe Phe Val Ala Gln Thr Leu Val Glu His145 150 155 160Thr Phe Gln Leu Glu Trp Thr Hi s Leu Asn Lys Thr Cys Glu Gly Val165 170 175
Ser Thr Asn Asn Asp Val Ala Ile Ser Tyr Asp Pro Asn Ile His Leu180 185 190Ser Val Thr Leu Leu Ser Gly Asp Gly Asn Pro Val Ala Trp Glu Ile195 200 205Glu Pro Thr Leu Thr Asp Tyr Phe Ser Pro Phe Arg Lys Phe Leu Ser210 215 220Pro Leu Val Asn Phe Thr Val Asp Ser Ser Ile Val Tyr His Asn Asp225 230 235 240Leu Asn Leu His Ser Leu Asn Gly Ser Cys Thr Ser Val Thr Trp Phe245 250 255Asp Leu Ser His Thr Ile Asp Leu Ser Glu Leu Ser Ser Met Ala Tyr260 265 270Tyr Pro Glu Asp Ser Ala Leu Asn Leu Ala Ile Val Phe Pro Ser Ala275 2 80 285Ser Ser Ser Pro Asp Gly Leu Ala Phe Ile Asn Gly Thr Arg Ile Ser290 295 300Asp Glu Ile Thr Thr Leu Asp Trp Asn Ser Tyr Leu Val Pro Gln Trp305 310 3l5 320Gly Val Ile Ile Ile Asn Lys Met Pro Leu Lys Pro Asn Ser Val Ile325 330 335Ser Glu Asp Tyr Leu Glu Pro Met Met Tyr Arg Phe Ala Thr Asp Ile340 345 350Phe Gln Leu Leu Gly Leu Thr Glu Gly Ser Gln Asp Leu Leu Ser Pro355 360 365Tyr Ile Thr Ile Asp Ser Phe Lys Arg Leu Thr Ile Leu Gln Asn Leu370 375 380Asp Lys Ala Thr Glu Thr Leu Trp Ser Leu Val Lys Leu Thr Gln Gln385 390 395 400Phe Gln Gly Met Ser Ile Pro Arg Glu Val Ser Asp Asn Val Ile Glu405 410 415Ala Leu Asp Leu Arg Leu Gln Ile Ile Asp Leu Leu Asn Asp Pro Gly420 425 430Lys Gly Gly Asp Ile Val Trp Asn Asn Ala Leu Hi s Leu Ser Asn Glu435 440 445Leu Val Lys Leu Cys Glu Ly s Ala Phe Phe Asn Gly Glu Met Val Gln450 455 460Gln Asn Phe Phe Pro Gln Glu Hi s MetIle Ala Val Tyr Leu Pro Leu465 470 475 480
Leu Gly Pro Ile Ser Ala Val Met Phc Phe Gly Phe Tyr Asn Val Met485 490 495Lys Glu Lys Asn Gln Lys Ser Lys Lys Asn Gly Thr Glu Arg Glu Val500 505 51 0Ala Lys Glu Lys Leu Glu Leu Lys Glu Ala Gln Lys Leu His Ala Ile515 520 525Asp Gly Glu Asp Glu Leu530<210> 38<211> 1833<212>DNA<213>釀酒酵母(Saccharomyces cerevisiae)<400> 38atgatcctca cactggccta tttcatgctg ggcactctac tactgggtgt gttcgcagaa 60gacacggtgt cgcagattgg tataaatgac agtttatggt atccgtacga cgaagcactg120gtgttgaagc cgctgcccaa caatgatctg ctactctcat ttgcattcca actgcaatcg180gaaccgtttg acccggccgt atcatccatg tcatatgatg cgtatgagca ctacacgact240ttcccacggg ccatcccacc attgttggaa tctactgcca cgcgtcagtt tcatttaaga300tttaccagag gattctggga tgccctgtcg tggggacagt tgccacatgc tggaaaagag360gcaggtgcct caggtgtgga attgtggtcg caagtgcagg ccatggatca ggaacaggcg420ttccataatt ggaaaaaact gtccaattca ttgageggat tgttttgttc ttctttaaat480tttatcgacg agtcaaggac gacctttccc cggcggtcat atgcttctga tataggagct540cctcttttca atagcaccga gaaactgtac ctgatgcgag catcgttgcc caatgaaccc600atctgtaccg agaacttgac gccgttcata aaactattgc ctactagggg caaatccggt660ttgacatctc tcttggatgg tcataaattg ttcgactctc tatggaatag tatttccttg720gatattgcca ctatttgttc tgaagatgaa gatgctcttt gtcactacga gatggacgca780cgcatagaaa tggtaacaca cgttccctcc gccttggcaa gaggtgagag acctatcccc840aaacctttgg atgggaacac attgcgttgt gacacggata aaccctttga ttcttaccaa900tgcttccctc taccggaacc ttcgcagact cacttcaagc tgtctcagct gtttgccaga960ccaataaaca atggcaacct gtttgctaat aggcccacaa gaatttgtgc agaagttgac1020cgttctacct ggactgcgtt tttgtcagtt gacgatacta ttttcagcac acatgataat1080tgctttgact tatcaaacga tcaaaatgag ggtggttcgg gctacgactt tattttagaa1140tcgacggaca ctactaaagt tactcccata gttcctgtcc caattcacgt aagcagatct1200
ctgactggta atggacaaga tcgtggtgga atgcgtattg ttttccataa cgacaatgat 1260acccctgtga agttgattta tttcgaatca ttgccatggt tcatgagagt ttacctatcc 1320tctcttcaaa ttacttctac tacctctccg caattgcaag aaaacgatat catcttagat 1380aaatactatt tacaagcggc cgatagaaaa agacctggcc acttggagtt cacgatgtta 1440attccagcta atacggacat tgtaatgact tatcaattcg ataaagctct tctgcaattt 1500gccgagtatc caccagatgc aaaccatggt tttgaaatcg atgcagctgt aatcaccgtg 1560ctatctttgg agtcctcatc gtctctttat gaaatgagaa cctctaccct attgttatcc 1620ctgtctacac cggattttag tatgccgtat aacgtcatca ttctaacatc tacaatcatg 1680ggactcatat tcggtatgct atacaatttg atggtgaaga gaatggtcac cgtcgaagag 1740gccgataaga ttacgttgca atctggctta aaatacaaat tgctaaagct aaaggaaaag 1800ttcctaggga aaaaaaagac taaaacagac taa 1833<210> 39<211> 610<212>PRT<213>釀酒酵母(Saccharomyces cerevisiae)<400> 39Met Ile Leu Thr Leu Ala Tyr Phe Met Leu Gly Thr Leu Leu Leu Gly1 5 10 l5Val Phe Ala Glu Asp Thr Val Ser Gln Ile Gly Ile Asn Asp Ser Leu20 25 30Trp Tyr Pro Tyr Asp Glu Ala Leu Val Leu Lys Pro Leu Pro Asn Asn35 40 45Asp Leu Leu Leu Ser Phe Ala Phe Gln Leu Gln Ser Glu Pro Phe Asp50 55 60Pro Ala Val Ser Ser Met Ser Tyr Asp Ala Tyr Glu Hi s Tyr Thr Thr65 70 7580Phe Pro Arg Ala Ile Pro Pro Leu Leu Glu Ser Thr Ala Thr Arg Gln85 90 95Phe His Leu Arg Phe Thr Arg Gly Phe Trp Asp Ala Leu Ser Trp Gly100 105 110Gln Leu Pro Hi s Ala Gly Lys Glu Ala Gly Ala Ser Gly Val Glu Leu115 120 125Trp Ser Gln Val Gln Ala Met Asp Gln Glu Gln Ala Phe Hi s Asn Trp130 135 140Lys Lys Leu Ser Asn Ser Leu Ser Gly Leu Phe Cys Ser Ser Leu Asn
145 150 155 160Phe Ile Asp Glu Ser Arg Thr Thr Phe Pro Arg Arg Ser Tyr Al a Ser165 170 175Asp Ile Gly Ala Pro Leu Phe Asn Ser Thr Glu Lys Leu Tyr Leu Met180 185 190Arg Ala Ser Leu Pro Asn Glu Pro Ile Cy s Thr Glu Asn Leu Thr Pro195 2 00 205Phe Ile Lys Leu Leu Pro Thr Arg Gly Lys Ser Gly Leu Thr Ser Leu210 215 220Leu Asp Gly His Lys Leu Phe Asp Ser Leu Trp Asn Ser Ile Ser Leu225 230 235 240Asp Ile Ala Thr Ile Cys Ser Glu Asp Glu Asp Ala Leu Cys His Tyr245 250 255Glu Met Asp Ala Arg Ile Glu Met Val Thr Hi s Val Pro Ser Ala Leu260 265 270Ala Arg Gly Glu Arg Pro Ile Pro Lys Pro Leu Asp Gly Asn Thr Leu275 280 285Arg Cys Asp Thr Asp Lys Pro Phe Asp Ser Tyr Gln Cy s Phe Pro Leu290 295 300Pro Glu Pro Ser Gln Thr Hi s Phe Lys Leu Ser Gln Leu Phe Ala Arg305 310 315 320Pro Ile Asn Asn Gly Asn Leu Phe Ala Asn Arg Pro Thr Arg Ile Cys325 33 0 335Ala Glu Val Asp Arg Ser Thr Trp Thr Ala Phe Leu Ser Val Asp Asp340 345 350Thr Il e Phe Ser Thr His Asp Asn Cys Phe Asp Leu Ser Asn Asp Gln355 360 365Asn Glu Gly Gly Ser Gly Tyr Asp Phe Ile Leu Glu Ser Thr A sp Thr370 375 380Thr Lys Val Thr Pro Ile Val Pro Val Pro Ile Hi s Val Ser Arg Ser385 390 395 400Leu Thr Gly Asn Gly Gln Asp Arg Gly Gly Met Arg Ile Val Phe His405 41 0 415Asn Asp Asn Asp Thr Pro Val Lys Leu Ile Tyr Phe Glu Ser Leu Pro420 425 430Trp Phe Met Arg Val Tyr Leu Ser Ser Leu Gln Ile Thr Ser Thr Thr435 440 445Ser Pro Gln Leu GlnGlu Asn Asp Ile Ile Leu Asp Lys Tyr Tyr Leu450 455 460
Gln Ala Ala Asp Arg Lys Arg Pro Gly Hi s Leu Glu Phe Thr Met Leu465 470 475 480Ile Pro Ala Asn Thr Asp Ile Val Met Thr Tyr Gln Phe Asp Lys Ala485 490 495Leu Leu Gln Phe Ala Glu Tyr Pro Pro Asp Ala Asn His Gly Phe Glu500 505 510Ile Asp Ala Ala Val Ile Thr Val Leu Ser Leu Glu Ser Ser Ser Ser515 520 525Leu Tyr Glu Met Arg Thr Ser Thr Leu Leu Leu Ser Leu Ser Thr Pro530 535 540Asp Phe Ser Met Pro Tyr Asn Val Ile Ile Leu Thr Ser Thr Ile Met545 550 555 560Gly Leu Ile Phe Gly Met Leu Tyr Asn Leu Met Val Lys Arg Met Val565 570 575Thr Val Glu Glu Ala Asp Lys Ile Thr Leu Gln Ser Gly Leu Lys Tyr580 585 590Lys Leu Leu Lys Leu Lys Glu Lys Phe Leu Gly Lys Lys Lys Thr Lys595 600 605Thr Asp610<210> 40<211>1185<212>DNA<213>釀酒酵母(Saccharomyces cerevisiae)<400>40atggattcca cagcacttaa ggtagctcta ggctgtattg caattcgttt ggctgtgaac 60agcctttttc cctctctaca acaacaactg gaccagtctg tagaattctc aactcccgta120acttcattta ggtcactaca ggaaggtata tacctactgc ggaacaacat ccaagtatat180aatcatgggg ttgttcacca tcctccaatt ttgatttttt ttctttccct ctttaattcc240gacaggttaa tttccctcat atacgcttta attgatggat taattgcgta tcagctgaca300gaggtaacaa aggctttcaa aaacttgaaa ctgaaagttt ggctacctgg acttctttat360gccgtgaatc ctttgaccct tttatcgtgc attagtcggt catcaatcat attcacaaat420tttgctattt catcgtcatt gtattgcata ttagctgaag gaaacgttct tttgtcctct480gttatgattt ctatatctgg atatttgtca gtatacccta ttctcctctt aattccgcta540ttaggtatgc tgaaaagttg gaggcaaaga atattatctg ccattgtttc cataetatct600ttattaattc tgctattatt cagctacagt atattaggca gccaaagttg gtcatttttg660
acacaggttt atggatctat tataaccttt gagaaggttt ttccaaatct gggtttgtgg720tggtacttct tcattgaaat gtttgacacc ttcataccgt tcttcaaggc tgtattcaac780atttttattg cagtattcat tacaccattt actttgcgct atcataagca gccattctac840gcattcattt tatgcattgg gtggattgtc cttacaaagc catatccctc actaggtgac900gctggttttt tcttcagctt cctacctttc ttcacgccac tatttggata tttaagatac960cccatcatat cagcattact gtttttacac gcaattgttt tggcgccaat tttctatcat1020ctttgggttg ttttaggttc agggaatagt aattttttct atgctatttc cctagtttat1080gctctggcta tagcatctat attagttgac ttgaactggg cgatgctgag aattgaatac1140gataacggta tcccaaattt caaattgaag gtaacacaaa tttaa1185<210>41<211>394<212>PRT<213>釀酒酵母(Saccharomyces cerevisiae)<400>41Met Asp Ser Thr Ala Leu Lys Val Ala Leu Gly Cys Ile Ala Ile Arg1 5 10 15Leu Ala Val Asn Ser Leu Phe Pro Ser Leu Gln Gln Gln Leu Asp Gln20 25 30Ser Val Glu Phe Ser Thr Pro Val Thr Ser Phe Arg Ser Leu Gln Glu35 40 45Gly Ile Tyr Leu Leu Arg Asn Asn Ile Gln Val Tyr Asn His Gly Val50 55 60Val His His Pro Pro Ile Leu Ile Phe Phe Leu Ser Leu Phe Asn Ser65 70 75 80Asp Arg Leu Ile Ser Leu Ile Tyr Ala Leu Ile Asp Gly Leu Ile Ala85 90 95Tyr Gln Leu Thr Glu Val Thr Lys Ala Phe Lys Asn Leu Lys Leu Lys100 105 110Val Trp Leu Pro Gly Leu Leu Tyr Ala Val Asn Pro Leu Thr Leu Leu115 120 125Ser Cys Ile Ser Arg Ser Ser Ile Ile Phe Thr Asn Phe Ala Ile Ser130 135 140Ser Ser Leu Tyr Cys Ile Leu Ala Glu Gly Asn Val Leu Leu Ser Ser145 150 155 160Val Met Ile Ser Ile Ser Gly Tyr Leu Ser Val Tyr Pro Ile Leu Leu165 170 175
Leu Ile Pro Leu Leu Gly Met Leu Lys Ser Trp Arg Gln Arg Ile Leu180 185 190Ser Ala Ile Val Ser Ile Leu Ser Leu Leu Ile Leu Leu Leu Phe Ser195 200 205Tyr Ser Ile Leu Gly Ser Gln Ser Trp Ser Phe Leu Thr Gln Val Tyr210 215 220Gly Ser Ile Ile Thr Phe Glu Lys Val Phe Pro Asn Leu Gly Leu Trp225 230 235 240Trp Tyr Phe Phe Ile Glu Met Phe Asp Thr Phe Ile Pro Phe Phe Lys245 250 255Ala Val Phe Asn Ile Phe Ile Ala Val Phe Ile Thr Pro Phe Thr Leu260 265 270Arg Tyr His Lys Gln Pro Phe Tyr Ala Phe Ile Leu Cys Ile Gly Trp275 280 285Ile Val Leu Thr Lys Pro Tyr Pro Ser Leu Gly Asp Ala Gly Phe Phe290 295 300Phe Ser Phe Leu Pro Phe Phe Thr Pro Leu Phe Gly Tyr Leu Arg Tyr305 310 315 320Pro Ile Ile Ser Ala Leu Leu Phe Leu His Ala Ile Val Leu Ala Pro325 330 335Ile Phe Tyr His Leu Trp Val Val Leu Gly Ser Gly Asn Ser Asn Phe340 345 350Phe Tyr Ala Ile Ser Leu Val Tyr Ala Leu Ala Ile Ala Ser Ile Leu355 360 365Val Asp Leu Asn Trp Ala Met Leu Arg Ile Glu Tyr Asp Asn Gly Ile370 375 380Pro Asn Phe Lys Leu Lys Val Thr Gln Ile385 390<210>42<211>255<212>DNA<213>人(Homo sapiens)<400>42atggccacgg ggacagacca ggtggtggga ctcggcctcg tcgccgttag cctgatcatc 60ttcacctact acaccgcctg ggtgattctc ttgccattca tcgacagtca gcatgtcatc120cacaagtatt tcctgccccg agcctatgct gtcgccatcc cactggctgc aggcctcctg180ctgctcctgt ttgtgggact gttcatctcc tatgtgatgc tgaagaccaa gagagtgacc240
aagaaggctc agtga255<210>43<211>84<212>PRT<213>人(Homo sapiens)<400>43Met Ala Thr Gly Thr Asp Gln Val yal Gly Leu Gly Leu Val Ala Val1 5 10 15Ser Leu Ile Ile Phe Thr Tyr Tyr Thr Ala Trp Val Ile Leu Leu Pro20 25 30Phe Ile Asp Ser Gln His Val Ile His Lys Tyr Phe Leu Pro Arg Ala35 40 45Tyr Ala Val Ala Ile Pro Leu Ala Ala Gly Leu Leu Leu Leu Leu Phe50 55 60Val Gly Leu Phe Ile Ser Tyr Val Met Leu Lys Thr Lys Arg Val Thr65 70 75 80Lys Lys Ala Gln<210>44<211>369<212>DNA<213>人(Homo sapiens)<400>44atgctctccg tgggcgggct tcggttgagt ttggtccgct tttcctttct gctcctcagg60ggagcattgc ttccttctct cgcagtgacc atgacgaaat tagcgcagtg gctttgggga120ctagcgatcc tgggctccac ctgggtggcc ctgaccacgg gagccttggg cctggagctg180cccttgtcct gccaggaagt cctgtggcca ctgcccgcct acttgctggt gtccgccggc240tgctatgccc tgggcactgt gggctatcgt gtggccactt ttcatgactg cgaggacgcc300gcacgcgagc tgcagagcca gatacaggag gcccgagccg acttagcccg cagggggctg360cgct tctga 369<210>45<211>122<212>PRT<213>人(Homo sapiens)<400>45Met Leu Ser Val Gly Gly Leu Arg Leu Ser Leu Val Arg Phe Ser Phe1 5 10 15
Leu Leu Leu Arg Gly Ala Leu Leu Pro Ser Leu Ala Val Thr Met Thr20 25 30Lys Leu Ala Gln Trp Leu Trp Gly Leu Ala Ile Leu Gly Ser Thr Trp35 40 45Val Ala Leu Thr Thr Gly Ala Leu Gly Leu Glu Leu Pro Leu Ser Cys50 55 60Gln Glu Val Leu Trp Pro Leu Pro Ala Tyr Leu Leu Val Ser Ala Gly65 70 75 80Cys Tyr Ala Leu Gly Thr Val Gly Tyr Arg Val Ala Thr Phe His Asp85 90 95Cys Glu Asp Ala Ala Arg Glu Leu Gln Ser Gln Ile Gln Glu Ala Arg100 105 110Ala Asp Leu Ala Arg Arg Gly Leu Arg Phe115 120<210>46<211>744<212>DNA<213>人(Homo sapiens)<400>46atggcggccg aggcggacgg accgcttaaa cggctgctcg tgccgattct tttacctgag60aaatgctacg accaactttt cgttcagtgg gacttgcttc acgtcccctg cctcaagatt120ctcctcagca aaggcctggg gctgggcatt gtggctggct cacttctagt aaagctgccc180caggtgttta aaatccgggg agccaagagt gctgaagggt tgagtctcca gtctgtaatg240ctggagctag tggcattgac tgggaccatg gtctacagca tcactaacaa cttcccattc300agctcttggg gtgaagcctt attcctgatg ctccagacga tcaccatctg cttcctggtc360atgcactaca gaggacagac tgtgaaaggt gtcgctttcc tcgcttgcta cggcctggtc420ctgctggtgc ttctctcacc tctgacgccc ttgactgtag tcaccctgct ccaggcctcc480aatgtgcctg ctgtggtggt ggggaggctt ctccaggcag ccaccaacta ccacaacggg540tacacaggcc agctctcagc catcacagtc ttcctgctgt ttgggggctc cctggcccga600atcttcactt ccattcagga aaccggagat cccctgatgg ctgggacctt tgtggtctcc660tctctctgca acggcctcat cgccgcccag ctgctcttct actggaatgc aaagcctccc720cacaagcaga aaaaggcgca gtag 744<210>47<211>247<212>PRT
<213>人(Homo sapiens)<400>47Met Ala Ala Glu Ala Asp Gly Pro Leu Lys Arg Leu Leu Val Pro Ile1 5 10 15Leu Leu Pro Glu Lys Cys Tyr Asp Gln Leu Phe Val Gln Trp Asp Leu20 25 30Leu His Val Pro Cys Leu Lys Ile Leu Leu Ser Lys Gly Leu Gly Leu35 40 45Gly Ile Val Ala Gly Ser Leu Leu Val Lys Leu Pro Gln Val Phe Lys50 55 60Ile Arg Gly Ala Lys Ser Ala Glu Gly Leu Ser Leu Gln Ser Val Met65 70 75 80Leu Glu Leu Val Ala Leu Thr Gly Thr Met Val Tyr Ser Ile Thr Asn85 90 95Asn Phe Pro Phe Ser Ser Trp Gly Glu Ala Leu Phe Leu Met Leu Gln100 105 110Thr Ile Thr Ile Cys Phe Leu Val Met His Tyr Arg Gly Gln Thr Val115 120 125Lys Gly Val Ala Phe Leu Ala Cys Tyr Gly Leu Val Leu Leu Val Leu130 135 140Leu Ser Pro Leu Thr Pro Leu Thr Val Val Thr Leu Leu Gln Ala Ser145 150 155 160Asn Val Pro Ala Val Val Val Gly Arg Leu Leu Gln Ala Ala Thr Asn165 170 175Tyr His Asn Gly Tyr Thr Gly Gln Leu Ser Ala Ile Thr Val Phe Leu180 185 190Leu Phe Gly Gly Ser Leu Ala Arg Ile Phe Thr Ser Ile Gln Glu Thr195 200 205Gly Asp Pro Leu Met Ala Gly Thr Phe Val Val Ser Ser Leu Cys Asn210 215 220Gly Leu Ile Ala Ala Gln Leu Leu Phe Tyr Trp Asn Ala Lys Pro Pro225 230 235 240His Lys Gln Lys Lys Ala Gln245<210>48<211>25<212>DNA<213>人工的<220>
<223>人工合成的引物序列<400>48atgacaatgt ggggaagtca acggg25<210>49<211>30<212>DNA<213>人工的<220>
<223>人工合成的引物序列<400>49tgtgtggtta ccgttctttg aatacataga30<210>50<211> 29<212>DNA<213>人工的<220>
<223>人工合成的引物序列<400>50atagaaaatg atttatggta cagctcaaa 29<210>51<211>30<212>DNA<213>人工的<220>
<223>人工合成的引物序列<400>51agaccaaatt aattatgcct ttacatgtac 30<210>52<211>40<212>DNA<213>人工的<220>
<223>人工合成的引物序列<400>52agaattcacc atgagcaaca tgaatatact tgcgtatctt40<210>53<211>30<212>DNA<213>人工的
<220>
<223>人工合成的引物序列<400>53gaaattccaa tgtattccat attcacttat30<210>54<211>42<212>DNA<213>人工的<220>
<223>人工合成的引物序列<400>54aagatctaat acattaaaac attttagatt aatgaatatg tg42<210>55<211>34<212>DNA<213>人工的<220>
<223>人工合成的引物序列<400>55aggtaccgta cactccactc tatgatgatc attc 3權(quán)利要求
1.由核苷酸序列SEQ ID NO5組成的DNA。
2.載體，其中插入權(quán)利要求1的DNA。
3.轉(zhuǎn)化體，保留可表達(dá)狀態(tài)的權(quán)利要求1所述DNA或者權(quán)利要求2所述載體。
4.權(quán)利要求3所述的轉(zhuǎn)化體，其為GPI合酶基因-缺陷型真菌。
5.權(quán)利要求3所述的轉(zhuǎn)化體，其為GPI合酶基因-缺陷型酵母。
6.一種制備由權(quán)利要求1所述DNA編碼的蛋白的方法，該方法包括培養(yǎng)權(quán)利要求3-5中的任一轉(zhuǎn)化體，并從該轉(zhuǎn)化體或其培養(yǎng)上清液收集表達(dá)的蛋白的步驟。
7.一種篩選具有抗瘧活性的化合物的方法，該方法包括以下步驟(1)將試樣與表達(dá)權(quán)利要求1所述DNA的GPI合酶基因-缺陷型酵母接觸；(2)分析該酵母的生長(zhǎng)，和(3)選出抑制該酵母生長(zhǎng)的被試化合物。
8.一種篩選具有抗瘧活性的化合物的方法，該方法包括以下步驟(1)將試樣與表達(dá)權(quán)利要求1所述DNA的GPI合酶基因-缺陷型酵母接觸，(2)檢測(cè)GPI-錨定蛋白轉(zhuǎn)運(yùn)到酵母細(xì)胞壁的量，和(3)選出使步驟(2)中所檢測(cè)的GPI-錨定蛋白轉(zhuǎn)運(yùn)到酵母細(xì)胞壁的量減少的試樣。
9.一種篩選具有抗瘧活性的化合物的方法，該方法包括以下步驟(1)將權(quán)利要求1所述DNA導(dǎo)入GPI合酶基因-缺陷型酵母，并使該DNA編碼的蛋白表達(dá)，(2)獲得步驟(1)中表達(dá)的蛋白，(3)使所得蛋白與試樣和具有與該蛋白結(jié)合的活性的標(biāo)記化合物接觸，(4)檢測(cè)與該蛋白結(jié)合的標(biāo)記化合物，和(5)選出減少與該蛋白結(jié)合的標(biāo)記化合物的量的試樣。
10.一種篩選具有抗瘧活性的化合物的方法，該方法包括以下步驟(1)將(i)權(quán)利要求1的DNA，或者(ii)插入了權(quán)利要求1的DNA的載體，導(dǎo)入GWT1-缺陷型酵母，且表達(dá)由該DNA編碼的蛋白(2)獲得步驟(1)中表達(dá)的蛋白，(3)加入U(xiǎn)DP-GlcNAc或?；o酶A，(4)使所得蛋白與試樣接觸，(5)檢測(cè)GlcN-(?；?PI，和(6)選出減少GlcN-(?；?PI水平的被試化合物。
全文摘要
本發(fā)明人成功地分離出了GWT1(PfGWT1)，其是一種在惡性瘧原蟲中參與GPI生物合成的酶。此外，本發(fā)明人揭示比編碼PfGWT1蛋白的DNA具有更低AT含量的簡(jiǎn)并突變體DNA，能補(bǔ)償GWT1－缺陷型酵母的表型。在此發(fā)現(xiàn)的基礎(chǔ)上，本發(fā)明提供了瘧原蟲的GWT1蛋白，以及該蛋白在篩選抗瘧藥物的方法中的用途。本發(fā)明還提供了編碼參有GPI生物合成的蛋白的簡(jiǎn)并突變體DNA，該DNA比天然DNA具有更低的AT含量。本發(fā)明還提供了利用該簡(jiǎn)并突變體DNA篩選抗瘧藥物的方法。
文檔編號(hào)G01N33/569GK101092628SQ20071010428
公開(kāi)日2007年12月26日 申請(qǐng)日期2003年11月21日 優(yōu)先權(quán)日2002年11月22日
發(fā)明者畑桂, 小川薰, 塚田格, 中本和孝, 相根康司, 田中圭悟, 塚原克平, 堀井俊宏 申請(qǐng)人:衛(wèi)材R&D管理有限公司