篩選在瘧原蟲中抑制gpi生物合成的化合物的方法

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
技術領域：
本發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：
瘧疾是由寄生性原蟲引起的最常見的傳染性人類疾病。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，包括超過兩百萬的致死例(Gardner等，Nature419498-511，2003)。目前，40％的世界人口生活在瘧疾流行區，在該區域據說每3名新生兒中就有一名死于瘧疾。
已知糖基磷脂酰肌醇(GPI)在寄生蟲生長和感染過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胞時起作用的MSP-1蛋白。GPI-錨定蛋白起寄生蟲抗原的作用，在宿主中引發免疫應答。因此，它們長期以來是疫苗開發的研究對象。
GPI不僅作為將蛋白拴到細胞膜的錨，而且是瘧原蟲細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誘導炎性細胞因子如TNF-α和粘附分子的表達。結果，受染紅細胞粘附毛細管，阻塞血管(隔離作用(sequestration))，尤其是腦血管。它能引起進一步的炎癥反應，而炎癥反應認為能導致腦病。最近，Schofield等報道抗-GPI抗體降低利用鼠科瘧原蟲伯氏瘧原蟲(Plasmodium berghei)創建的體內感染模型系統的致死率，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(Schofield L等，Nature 418785-789，2002)。此發現說明GPI是瘧疾感染致死率的主要因素。
還報道肌醇酰基化對于甘露糖與GPI的結合來說是必需的(Gerold，P.等，Biochem.J.344731-738，1999)，由過量葡糖胺引起的肌醇酰基化的抑制會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酰基化的化合物可以是非常有用的抗瘧藥物。
發明的公開本發明的目的是提供抑制GPI生物合成的抗瘧藥物。更具體地說，本發明提供了編碼GWT1蛋白的瘧原蟲DNA，所述蛋白是參與GPI生物合成的蛋白質(GPI合酶)。本發明還提供了在篩選抗瘧藥物的方法中利用該DNA的方法。本發明還提供了編碼瘧原蟲GPI生物合成蛋白的DNA的簡并突變體DNA。該簡并突變體DNA比天然DNA具有更低的AT含量。本發明還提供了在篩選抗瘧藥物的方法中利用該簡并突變體DNA的方法。
最早發現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。本發明人證實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(Gardner等，Nature 419498-511，2003；Carlton等，Nature 419512-519，2003)。本發明人還發現GWT1基因產物在GPI生物合成途徑中起GlcN-PI酰基轉移酶的作用。PfGWT1基因產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值的抗瘧藥物。
在WO 02/04626中，本發明人發現了一組抑制真菌GWT1基因產物的活性的化合物。抑制PfGWT1基因產物的活性的化合物有望成為抗瘧藥物。
在本發明中，本發明人成功地對幾乎是PfGWT1全長的區域進行了分離。利用瘧原蟲如惡性瘧原蟲的GWT1基因產物，抗瘧藥物的篩選可以通過結合試驗，葡糖胺(酰基)磷脂酰肌醇(PI-GlcN)酰基轉移酶試驗，或利用GPI-錨定蛋白檢測系統來進行。從所述篩選方法獲得的化合物可能是有價值的抗瘧藥物。更進一步地，本發明人揭示簡并突變體DNA(編碼瘧原蟲GPI生物合成蛋白的DNA簡并突變體)，該DNA比天然DNA具有更低AT含量，補償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，該真菌中導入了(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，本發明提供了下述[1]到[25][1](a)到(d)中任一項的DNA，該DNA編碼具有GlcN-PI酰基轉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)在嚴謹條件與包含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/或插入一個或多個氨基酸所得的蛋白的DNA；[2]由項[1]的DNA編碼的蛋白；[3]插入項[1]的DNA的載體；[4]在可表達狀態包含項[1]的DNA或者項[3]的載體的轉化體；[5]一種抗瘧藥物，其包含抑制項[2]的蛋白的活性的化合物作為活性成分；[6]項[5]的抗瘧藥物，其中抑制項[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[2]的蛋白與試樣和具有與該蛋白結合的活性的標記化合物接觸，(2)檢測與該蛋白結合的標記化合物，和(3)選出減少與該蛋白結合的標記化合物的量的試樣；[8]項[7]的方法，其中具有與蛋白結合的活性的標記化合物是通過對選自項[6]的化合物(1)-(5)中的至少一種進行標記而獲得的；[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[2]的蛋白接觸，(2)檢測GlcN-(酰基)PI，和(3)選出減少GlcN-(酰基)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)使試樣與過量表達項[2]的蛋白的細胞接觸，(2)測定GPI-錨定蛋白轉運到該細胞的細胞壁的量，和(3)選出減少步驟(2)中所測定的GPI-錨定蛋白轉運到該細胞的細胞壁的量的試樣；[11]一種治療瘧疾的方法，包括施用抑制項[2]的蛋白的活性的化合物；[12]項[11]的方法，其中抑制項[2]的蛋白的活性的化合物是項[5]的化合物； 一種編碼具有補償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的簡并突變體，其具有少于70％的AT含量；[15]項[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中奇數序列鑒定號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中偶數序列鑒定號中任一所示核苷酸序列的DNA，(c)在嚴謹條件下與包含SEQ ID NOs1和3以及SEQ ID NOs6-47中偶數序列鑒定號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中奇數序列鑒定號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/或插入一個或多個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[13]-[16]中任一的DNA；[18]在可表達狀態包含項[13]-[16]中任一DNA或者項[17]的載體的轉化體；[19]項[18]的轉化體，其為GPI合酶基因-缺陷型真菌；[20]項[18]的轉化體，其為GPI合酶基因-缺陷型酵母；[21]一種制備由項[13]-[16]中任一DNA編碼的蛋白的方法，該方法包括培養項[18]-[20]中的任一轉化體，并從該轉化體或其培養上清液收集表達的蛋白的步驟；[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)將試樣與表達項[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，和(3)選出抑制該真菌生長的被試化合物；[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)將試樣與表達項[13]-[16]中任一DNA的GPI合酶基因-缺陷型真菌接觸，(2)檢測GPI-錨定蛋白轉運到真菌細胞壁的量，和(3)選出使步驟(2)中所檢測的GPI-錨定蛋白轉運到真菌細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[13]-[16]中任一DNA導入GPI合酶基因-缺陷型真菌，并使該DNA編碼的蛋白表達，(2)獲得步驟(1)中表達的蛋白，(3)使所得蛋白與試樣和具有與該蛋白結合的活性的標記化合物接觸，(4)檢測與該蛋白結合的標記化合物，和(5)選出減少與該蛋白結合的標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簡并突變體且表達由該簡并突變體DNA編碼的蛋白的載體，導入GWT1-缺陷型真菌，(2)獲得步驟(1)中表達的蛋白，(3)使所得蛋白與試樣接觸，(4)檢測GlcN-(酰基)PI，和(5)選出減少GlcN-(酰基)PI水平的被試化合物。
本發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，糖基磷脂酰肌醇對瘧原蟲的生長和感染性是必需的。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蛋白來進行篩選。
本發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酰基轉移酶活性。GlcN-PI酰基轉移酶活性可采用Costello和Orlean(J.Biol.Chem.(1992)2678599-8603)或Franzot和Doering(Biochem.J.(1999)34025-32)所報道的方法來進行測定。
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包括在嚴謹條件中與包含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/或插入一個或多個氨基酸。
本發明的DNA可以采用本領域技術人員所熟知的方法來進行分離。所述方法的實例包括利用雜交(Southern E.M.，J.Mol.Biol.98503-517，1975)和聚合酶鏈式反應(PCR)(Saiki R.K.等，Science 2301350-1354，1985；SaikiR.K.等，Science 239487-491，1988)。更具體地，對于本領域技術人員來說，利用SEQ ID NO1或3的DNA或其部分作為探針，或通過利用與包含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是常規實驗。另外，通過雜交或PCR技術分離出的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。
優選，在嚴謹雜交條件利用雜交反應分離出上述DNA。如本文所用，表達“嚴謹雜交條件”是指，例如在65℃于4xSSC中進行雜交，然后在65℃于0.1xSSC中洗滌1小時。可選的嚴謹條件是在42℃于含50％甲酰胺的4xSSC中進行雜交。或者，在65℃于PerfectHybTM(TOYOBO)溶液中雜交2.5小時，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，優選80％或更高，更優選90％或更高，最優選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)來確定。已開發了基于BLAST算法的程序(稱為BLASTN和BLASTX)(Altschul S.F.等，J.Mol.Biol.215403，1990)。當利用BLASTN對核苷酸序列進行分析時，例如，將參數設為得分＝100，字長＝12。另外，當利用BLASTX對氨基酸序列進行分析時，例如，將參數設為得分＝50，字長＝3。當使用BLAST和Gapped BLAST程序時，使用各程序的默認參數。所述分析的具體步驟是已知的(請參見美國國家生物技術信息中心(NationalInstitute of Biotechnology Information)網站http//www.ncbi.nlm.nih.gov)。
本發明的DNA包含基因組DNA、cDNA和化學合成DNA。基因組DNA或cDNA可以根據本領域技術人員所熟知的常規方法來加以制備。例如，基因組DNA可以如下制備(i)提取瘧原蟲基因組DNA；(ii)(利用如質粒、噬菌體、粘粒、BAC或PAC作載體)構建基因組文庫；(iii)涂布該文庫；然后(iv)利用探針進行菌落雜交或噬菌體雜交，所述探針是在編碼本發明的瘧原蟲GWT1蛋白的DNA(例如SEQ ID NO1或3)的基礎上獲得的。另外，利用編碼本發明瘧原蟲GWT1蛋白的DNA(例如SEQ ID NO1或3)的特異性引物，通過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基礎上合成cDNA；(ii)將該合成cDNA插入載體如λZAP中構建cDNA文庫；(iii)涂布該cDNA文庫；和(iv)如上所述，進行菌落雜交或噬菌體雜交。另外，也可以利用PCR制備cDNA。
本發明還提供了與包含SEQ ID NO2或4氨基酸序列的蛋白結構相似的蛋白的編碼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/或添加一個或多個氨基酸殘基的氨基酸序列。對于上述蛋白中的氨基酸突變位點和數量并無限制，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 224，1431-1433；Dalbadie-McFarland，G.等，Proc.Natl.Acad.Sci.USA(1982)79，6409-6413中所述。突變氨基酸的百分數一般為全部氨基酸殘基的10％或更少，優選5％或更少，更優選1％或更少。此外，突變氨基酸的數量通常為30個氨基酸或更少，優選15個氨基酸或更少，更優選5個氨基酸或更少，還更優選3個氨基酸或更少，甚至更優選2個氨基酸或更少。
優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)后保持側鏈性質的氨基酸殘基。氨基酸側鏈性質的例子是疏水性(A，I，L，M，F，P，W，Y，V)和親水性(R，D，N，C，E，Q，G，H，K，S，T)。側鏈包括脂肪族側鏈(G，A，V，L，I，P)；含羥基側鏈(S，T，Y)；含硫原子側鏈(C，M)；含羧酸和酰胺側鏈(D，N，E，Q)；堿性側鏈(R，K，H)；和芳香族側鏈(H，F，Y，W)。
包含瘧原蟲GWT1蛋白的融合蛋白是其中添加一個或多個氨基酸殘基的蛋白實例。融合蛋白可利用本領域技術人員所熟知的技術來進行制備。例如，對此特定技術沒有限制，將編碼本發明瘧原蟲GWT1蛋白的DNA與編碼另一肽或蛋白的DNA聯合，使它們的閱讀框匹配。本發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-標簽，HSV-標簽，E-標簽，SV40T抗原片段，lck標簽，α-微管蛋白片段，B-標簽和蛋白C(Protein C)片段。與本發明蛋白融合的蛋白例子包括谷胱甘肽-S-轉移酶(GST)，HA，免疫球蛋白恒定區，β-半乳糖苷酶和麥芽糖結合蛋白(MBP)。
除利用上述雜交和PCR技術外，本領域技術人員可通過下述方法制得上述DNA，該方法包括例如定點誘變而在該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生突變而使其自然突變。此外，簡并突變體DNA，其核苷酸突變并不會引起蛋白中的氨基酸發生突變(簡并突變體)也包括在本發明內。另外，本發明還包括由本發明上述DNA編碼的蛋白。
本發明提供了包括本發明DNA的載體，包含本發明DNA或載體的轉化體，和利用這些轉化體制備本發明蛋白的方法。
本發明的載體并無限制，只要它能穩定地保留插入載體內的DNA。例如，當大腸桿菌作宿主時，優選用pBluescript載體(Stratagene)作為克隆載體。當使用載體來制備本發明的蛋白時，表達載體是特別有用的。表達載體并無特別的限制，只要它能在體外，在大腸桿菌中，在培養細胞中和在體內表達蛋白。表達載體的優選例子包括用于體外表達的pBEST載體(Promega Corporation)，用于在大腸桿菌中表達的pET載體(Novagen)，用于在培養細胞中表達的pME18S-FL3載體(GenBank登錄號AB009864)，和用于體內表達的pME18S載體(Mol.Cell Biol.8466-472，1988)。可采用常規方法將本發明的DNA插入載體內，例如采用限制性酶位點進行連接酶反應(Current Protocols in Molecular Biology，Ausubel等編輯，John Wiley & Sons，Inc.1987，Section 11.4-11.11)。
導入本發明載體的宿主細胞并無特別地限制，并且可以根據發明目的使用各種宿主細胞。例如，用于表達蛋白的細胞包括但不限于細菌細胞(如鏈球菌(Streptococcus)，葡萄球菌(Staphylococcus)，大腸桿菌，鏈霉菌(Streptomyces)，枯草桿菌(Bacillus subtilis)，真菌細胞(如酵母，曲霉(Aspergillus))，昆蟲細胞(如果蠅(Drosophila)S2，夜蛾(Spodoptera)SF9)，動物細胞(如CHO，COS，HeLa，C127，3T3，BHK，HEK293，Bowes黑素瘤細胞)，以及植物細胞。將載體轉染入宿主細胞可以采用常規方法進行，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。
通過將合適的分泌信號摻入目的蛋白內，可以將在宿主細胞中表達的蛋白分泌到內質網的腔，細胞周圍空腔(cavity)或細胞外環境內。這些信號對于目的蛋白來講可以是內源的或外源的。
當本發明的蛋白分泌到培養基中時，從該培養基中收集蛋白。如果本發明的蛋白是在細胞內產生的，就將該細胞裂解，然后收集蛋白。
利用本領域熟知的方法可從重組的細胞培養物中對本發明蛋白進行收集和純化，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。
包括本發明DNA的化合物是分離的化合物。在此，術語“分離的”是指從原來的環境中分離出來(例如，如果是自然存在，即為天然環境)。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，術語“基本純化的”是指化合物從天然環境分離的狀態，而且該狀態中已除去了至少60％，優選75％，最優選90％的其它共存的天然成分。
本發明提供了抗瘧藥物，該藥物抑制瘧原蟲GWT1基因產物的活性。抑制瘧原蟲GWT1基因產物活性的優選化合物是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基因產物的活性的化合物或其鹽或其水合物可以給藥于哺乳動物(優選人)。還可以利用常規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，滑潤劑，著色劑，調味劑，以及需要時的穩定劑，乳化劑，吸收促進劑，表面活性劑，pH調節劑，防腐劑和抗氧化劑)。藥物制劑可以用常規方法，通過將通常用作藥劑成分的各組分混合來制備。
例如，口服制劑可以如下制備將本發明的化合物或其藥學上可接受的鹽與填充劑，必要時還有粘合劑，崩解劑，潤滑劑，著色劑，調味劑等組合，并通過常規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；高級醇，如十六醇十八醇混合物以及二十二醇；硅氧烷樹脂；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；無機粉末，如硅酸酐，硅酸鋁鎂，以及硅酸鋁；和純水。填充劑的實例子包括乳糖，玉米淀粉，精制白糖，葡萄糖，甘露醇，山梨醇，結晶纖維素，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。對于這些片劑和顆粒劑當然可以使用糖衣，必要時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方法來制備。在該方法中，通過將pH調節劑，增溶劑，等滲劑等，以及必要時的增溶助劑，穩定劑等，加至本發明的化合物或其藥學上可接受的鹽中，來制備。
制備外用制劑的方法沒有限制，而且可以通過常規方法來制備。也就是說，用于制劑的基礎材料可以選自藥物、準藥物(quasi-drug)、化妝品等常用的各種材料。具體地，可使用的基礎材料是，如動物脂肪和植物油，礦物油，酯油，蠟，高級醇，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，可以添加pH調節劑，抗氧化劑，螯合劑，防腐劑和抗真菌劑，著色物質，香料等。但是本發明的外用制劑的基礎材料并不限于此。再者，如果需要，還可以混入具有分化誘導作用的組分，血流促進劑，殺真菌劑，消炎劑，細胞活化劑，維生素，氨基酸，保濕劑，以及角質裂解劑。上述基礎材料按外用制劑常用濃度的量加入。
如本發明所述，術語“鹽”優選包括，如與無機或有機酸的鹽，與無機或有機堿的鹽，或與酸性或堿性氨基酸的鹽。尤其優選是藥學上可接受的鹽。酸和堿以0.1-5個酸或堿分子與1個化合物分子的適當比形成鹽。
與無機酸的鹽的優選例子是與鹽酸，氫溴酸，硫酸，硝酸和磷酸的鹽。與有機酸的鹽優選包括與醋酸，琥珀酸，延胡索酸，馬來酸，酒石酸，檸檬酸，乳酸，硬脂酸，苯甲酸，甲磺酸，對甲苯磺酸的鹽。
與無機堿的鹽的優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。與有機堿的鹽優選包括與二乙胺，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選例子是與天冬氨酸和谷氨酸的鹽，與堿性氨基酸的鹽優選包括與精氨酸，賴氨酸和鳥氨酸的鹽。
施用本發明的化合物或其鹽或其水合物時，對其形式沒有特別的限制，它們可以根據常規方法經口或經非胃腸道途徑給藥。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。本發明的藥物組合物的劑量可以根據癥狀程度，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地選擇。
將治療有效量的本發明的化合物給藥于患者。在此，“治療有效量”是指產生所需藥理效果并且有效地恢復或或緩解所要治療的患者之癥狀的藥劑量。所述劑量根據疾病的類型，癥狀的程度，患者的體重、年齡、性別、對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，優選0.1mg-500mg，更優選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，優選約3-1000μg/kg。
此外，本發明涉及利用瘧原蟲GWT1基因產物篩選抗瘧藥物的方法。所述篩選方法包括但不限于[1]結合試驗，該試驗篩選與標記化合物競爭結合瘧原蟲GWT1基因產物的化合物；[2]GlcN-PI酰基轉移酶分析系統，該系統篩選能抑制瘧原蟲GWT1基因產物的GlcN-PI酰基轉移酶活性的化合物；和[3]GPI-錨定蛋白檢測系統，其中在細胞，優選真菌細胞中表達瘧原蟲GWT1基因產物，然后檢測細胞表面上的GPI-錨定蛋白。本發明并不限于這些方法，而且包括利用瘧原蟲GWT1基因產物篩選抗瘧藥物的任何方法。上面列出的方法[1]-[3]在下面進行詳細描述。
篩選與標記化合物競爭結合瘧原蟲GWTI基因產物的化合物的結合試驗本發明的兩種方法公開如下，即(1)制備瘧原蟲GWT1基因產物(下文中稱為瘧原蟲GWT1蛋白)的方法和(2)涉及標記化合物的結合實驗法(下文中稱為結合試驗)(1制備瘧原蟲GWT1蛋白的方法瘧原蟲GWT1蛋白是從細胞膜級分，優選從真菌細胞，更優選從釀酒酵母(S.cerevisiae)細胞中制得的，其中引入了SEQ ID NO2的瘧原蟲GWT1蛋白的編碼DNA。優選將所述DNA導入GWT1基因-缺陷型細胞中。在結合試驗中，可以在未進行任何其它處理的條件下使用所制的膜級分，或者在使用前對膜級分進行進一步的純化。利用釀酒酵母的方法將在下面進行詳細描述。
(a)瘧原蟲GWT1基因的導入本發明使用的瘧原蟲GWT1基因可以是天然存在的基因，或優選是基于SEQ ID NO2或4的氨基酸序列進行合成的。瘧原蟲GWT1基因富含腺嘌呤和胸腺嘧啶。因此，可以推斷利用常規的基因重組技術將很難對該基因進行操作，且該基因在酵母，細胞等中的表達效率將會很低(inefficient)。因此，優選對核苷酸序列進行設計，將其中與每個相應氨基酸對應的密碼子替換為那些認為可以在酵母，細胞等中能有效表達的密碼子，在該設計序列的基礎上進行DNA合成，制備人工的瘧原蟲GWT1基因，然后在下述實驗中使用該基因。
瘧原蟲GWT1的表達質粒是通過將瘧原蟲GWT1基因插入到釀酒酵母表達載體內來加以制備的，例如，通過將適合的啟動子和終止子，如pKT10-衍生的GAPDH啟動子和GAPDH終止子，插到YEp352的多克隆位點內來制備表達載體(Tanaka等，Mol.Cell Biol.，104303-4313，1990)。將釀酒酵母(如G2-10菌株)培養在合適的培養基(如YPD培養基(酵母提取物-聚蛋白胨-葡萄糖培養基))中，并在適宜的溫度(如30℃)振蕩，在對數生長晚期收獲細胞。洗滌后，采用如乙酸鋰法將GWT1-表達質粒導入釀酒酵母細胞中。該方法描述于User Manual of YEAST MAKERTMYeast Transformation System(BD Biosciences Clontech)。瘧原蟲GWT1-過表達菌株和攜帶陰性對照載體的菌株可通過將轉化細胞于30℃，在SD(ura-)培養基上培養兩天而獲得。
適合除釀酒酵母之外的真菌種類的表達載體和基因轉移法見下述報道適合粟酒裂殖酵母(Schizosaccharomyces Pombe，S.pombe)的表達載體如pcL和其轉移法由Igarashi等描述(Nature 35380-83，1991)，適合白色念珠菌(C.Albicans)的表達載體如pRM10和其轉移法由Pla J.等描述(Yeast，121677-1702，1996)；適合煙曲霉(A.Fumigatus)的表達載體如pAN7-1和其轉移法由Punt P.J.等描述(GENE，56117-124，1987)；適合新生隱球菌(C.Neoformans)的表達載體如pPM8及其轉化法由Monden P.等描述(FEMSMicrobiol.Lett.，18741-45，2000)。
(b)制備膜級分的方法在適合的培養基(如SD(ura-)液體培養基)中培養，并在適合的溫度(如30℃)振蕩導入了瘧原蟲GWT1基因的釀酒酵母細胞。在對數生長中期時收獲真菌細胞，然后用合適量(如真菌細胞的3倍體積)的勻漿緩沖液(如50mMTris-HCl，pH7.5，10mM EDTA，CompleteTM(Roche))懸浮。將合適量的玻璃珠(如真菌細胞的4倍體積)加至懸浮液中。渦旋混合物，并置于冰上。重復此操作數次將真菌細胞破碎。
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胞沉淀。將上清液移到另一試管內。離心該試管，如13,500rpm離心10分鐘，使含有細胞器的膜級分(總膜級分)沉淀。用1ml結合緩沖液(如0.1M磷酸鹽緩沖液，pH7.0，0.05％Tween20，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。然后離心上清液，如15,000rpm離心5分鐘。沉淀物用150-650μl的結合緩沖液重懸浮來制備膜級分。
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分。
另外，可以通過在大腸桿菌，昆蟲和哺乳動物細胞或其他非真菌細胞中表達來制備瘧原蟲GWT1蛋白。
當使用哺乳動物細胞時，將瘧原蟲GWT1基因與含有如CMV啟動子的過表達載體連接，導入哺乳動物細胞中。然后，按照Petaja-Repo等的方法(J.Biol.Chem.，2764416-23，2001)制備膜級分。
表達瘧原蟲GWT1基因的昆蟲細胞(如Sf9細胞)可以利用，例如桿狀病毒表達試劑盒如BAC-TO-BAC桿狀病毒表達系統(Invitrogen)來進行制備。然后，按照Okamoto等的方法(J.Biol.Chem.，276742-751，2001)制備膜級分。
可通過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載體如pGEX載體(Amersham Biosciences Corp.)連接，將此構建體導入大腸桿菌如BL21中。
(2)結合分析法(a)標記化合物的合成標記化合物從已證實結合GWT1蛋白的化合物制備。與GWT1蛋白結合的任何化合物都是可以使用的。標記化合物優選從WO 02/04626中所述的化合物制備，更優選從上述(1)至(5)的化合物制備。
任何標記方法都是可以使用的。化合物優選是用放射性同位素，更優選用3H標記的化合物。采用放射性化合物作為起始物質，用常規制備方法制得放射性標記的化合物。另外，3H標記可以通過3H交換反應獲得。
(b)特異性結合的確定將標記化合物加至制得的膜級分中，將該混合物置于冰上適當長的時間，如1至2小時，直到標記化合物和膜級分間發生結合反應。離心混合物，使膜級分沉淀，如15,000rpm離心3分鐘。沉淀物用結合緩沖液重懸浮，離心懸浮液。適當地重復(兩次)，除去未結合的標記化合物。再用結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。利用液體閃爍計數器測定放射性。
確認標記化合物與GWT1蛋白的特異性結合可以通過下述評估來進行是否標記化合物的結合會被加入的嚴重過量未標記化合物(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GWT1蛋白的真菌細胞制得的膜級分結合。
(c)由試樣引起的標記化合物的結合抑制將試樣和標記化合物加至制得的膜級分中，將該混合物置于冰上適當長的時間，如1至2小時，期間與膜級分發生結合反應。在本發明的篩選方法中使用的待測化合物包括簡單的天然存在化合物，有機化合物，無機化合物，蛋白或肽，以及化合物文庫，基因文庫的表達產物，細胞提取物，細胞培養物上清液，來自發酵細菌的產物，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，使膜級分沉淀。在結合緩沖液中重懸浮沉淀物，離心該懸浮液。適當地重復此步驟(兩次)，從而除去所有未結合的標記化合物。用結合緩沖液懸浮沉淀物。將懸浮液移至閃爍小管，加入閃爍劑。利用液體閃爍計數器測定放射性。
當在試樣存在的條件下，標記化合物與膜級分的結合被抑制，就判定該試樣具有結合瘧原蟲GWT1蛋白的活性。
用于篩選抑制瘧原蟲GWT1蛋白GlcN-PI酰基轉移酶活性的化合物的GlcN-PI酰基轉移酶分析系統酰基到GPI的轉移可以利用下述方法來進行檢測Costello L.C和OrleanP.，J.Biol.Chem.(1992)2678599-8603；或Franzot S.P和Doering T.L.，Biochem.J.(1999)34025-32。該方法的具體實例在下面進行了描述。下述實驗條件優選是對所用的各種瘧原蟲GWT1蛋白進行了優化的。
根據上述部分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，優選含有防止在其它反應中消耗UDP-GIcNAc的抑制劑，如作為幾丁質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長的時間(如24℃孵育15分鐘)。
將已適當標記的，優選放射性標記的GlcN-(酰基)PI前體(如UDP-GlcNAc，Acyl-輔酶A，優選UDP-[14C]GlcNAc)加到混合物中。將所得的混合物孵育適當長的時間(如24℃孵育1小時)。加入氯仿和甲醇(1∶2)的混合物，攪拌所得混合物以使反應停止，提取脂質。將提取的反應產物溶解在合適的溶劑中，優選丁醇。然后，利用如HPLC或薄層層析(TLC)，優選TLC法分離反應產生的GlcN-(酰基)PI。當使用TLC時，展開劑(developing solvent)可以適當地選自，如CHCl3/CH3OH/H2O(65∶25∶4)，CHCl3/CH3OH/1M NH4OH(10∶10∶3)和CHCl3/吡啶/HCOOH(35∶30∶7)。優選的展開劑是CHCl3/CH3OH/H2O(65∶25∶4)。采用對所用標記而言恰當的方法，對分離的GlcNo-(酰基)PI進行定量。當用放射性同位素作標記時，可以基于其放射性對分離的GlcN-(酰基)PI進行定量。
當在試樣存在的條件下，制得的GlcN-(酰基)PI量減少時，就判定該試樣具有抑制由瘧原蟲GWT1蛋白引起的酰基轉移的活性。
包括在細胞中表達瘧原蟲GWT1蛋白然后檢測細胞表面上的GPI-錨定蛋白的GPI-錨定蛋白檢測系統試樣抑制瘧原蟲GWT1蛋白活性的能力可以利用GPI-錨定蛋白檢測系統來進行測定，該系統包括在細胞，優選真菌細胞中表達GWT1蛋白，然后檢測細胞表面上的GPI-錨定蛋白。本發明的真菌是指屬于接合菌門(Zygomycota)，子囊菌門(Ascomycota)，擔子菌門(Basidiomycota)及半知菌綱(Deuteromycete)的真菌，且優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的表達載體導入上述真菌細胞的方法是本領域技術人員所熟知的。
當瘧原蟲GWT1蛋白在真菌細胞中表達時，轉運到真菌細胞壁的GPI-錨定蛋白量可通過下述方法測定(1)利用報告酶的方法；(2)利用與真菌細胞壁表面糖蛋白反應的抗體的方法；(3)利用該蛋白粘附動物細胞的能力；或者(4)在光學顯微鏡或電子顯微鏡下觀察真菌細胞的方法。
(1)-(4)的方法已在WO 02/04626中公開，所述方法在該發明的實施例中進行了詳細描述。利用(1)-(4)的方法，并且優選(1)-(4)的方法的組合可以判斷試樣是否抑制GPI-錨定蛋白到細胞壁上的轉運過程或者GPI-錨定蛋白在真菌細胞表面的表達。
在下文中，將描述方法(1)-(4)。
(1)利用報告酶進行的方法GPI-錨定蛋白到細胞壁的轉運過程可通過示蹤實驗定量如用放射性同位素標記GPI-錨定蛋白，獲得真菌細胞壁級分，并用抗GPI-錨定蛋白的抗體進行免疫沉淀反應。也可以用另一種方法更簡便地定量在各種GPI-錨定蛋白中常見的、被認為起轉運信號作用的C-末端序列可與便于測量的酶(報告酶)表達為融合蛋白，獲得真菌細胞壁級分；然后采用報告系統測量每一級分的酶活性(Van Berkel MAA等，FEBSLetters，349135-138，1994)。在下文中，將要會對一種采用報告酶進行的方法進行描述，但是本發明并不僅限于此。
首先，構建報告基因并將其引入真菌中。報告基因是這樣構建的使在真菌中起作用的啟動子序列，以與閱讀框匹配的方式連接分別編碼信號序列、報告酶和GPI-錨定蛋白C-末端序列的DNA。啟動子序列的實例包括GAL10及ENO1。信號序列的實例包括如α-因子，轉化酶，溶菌酶。報告酶的實例是β-內酰胺酶，溶菌酶，堿性磷酸酶和β-半乳糖苷酶。可以使用易于檢出的綠色螢光蛋白(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，優選將適宜的選擇標記物如LEU2和URA3插到包含所構建的報告基因的載體中。
將所構建的報告基因通過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，利用適于選擇標記物的方法(如適于LEU2的Leu-培養基，以及適于URA3的Ura-培養基)培養真菌，然后挑選其中引入了DNA的真菌。
通過下列方法測定試樣對GPI-錨定蛋白到細胞壁的轉運過程的影響在試樣存在時，于適宜的條件下(如30℃48小時)培養引入了報告基因的真菌。培養之后，離心培養物上清液，并測量培養物上清液級分的報告酶活性。洗滌所得細胞級分，然后通過適當的方法分離細胞壁成分，例如用葡聚糖酶降解細胞壁葡聚糖，然后測量細胞壁級分和細胞質級分的報告酶活性。該試驗可以如下簡單地進行離心確定細胞級分中報告酶的量，然后在不洗滌細胞的情況下按比例計算來源于培養物上清液級分但殘留在細胞級分中的報告酶的量，從細胞級分的報告酶的量中減去此量。
如果試樣顯示出增加培養物上清液級分中報告酶活性的活性(活性/細胞)，或者降低細胞壁級分中報告酶活性的活性(活性/細胞)，那么就可以判定該試樣對GPI-錨定蛋白到細胞壁的轉運過程有影響。
(2)采用與真菌細胞壁表面糖蛋白反應的抗體的方法對于試樣影響GPI-錨定蛋白在真菌表面層的表達的能力，可以通過利用與真菌細胞壁中的GPI-錨定蛋白反應的抗體進行定量來測定。
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測抗原決定簇，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)，合成該區的肽，將其與抗原性物質如載體蛋白結合，然后通過免疫兔子等得到多克隆抗體，或者通過免疫小鼠等得到單克隆抗體。優選是針對Als1p肽的兔多克隆抗體。
在可供選擇的方法中，可以通過用真菌，優選過量表達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，可以進一步用部分純化的GPI-錨定蛋白進行免疫)，并利用ELISA，Western印跡分析等以此制備抗體為基礎選出產生的克隆。
通過下述方法可以檢測試樣對GPI-錨定蛋白到細胞壁的轉運過程的影響，以及試樣對來源于細胞壁中GPI-錨定蛋白的蛋白量的影響。
在試樣存在時，于適宜的條件下如30℃48小時培養真菌。通過離心收集所培養的真菌并將細胞破碎，優選采用玻璃珠。優選用SDS離心提取洗滌過的破碎細胞，然后洗滌沉淀物。提取之后，用降解葡聚糖的酶，優選葡聚糖酶處理破碎的細胞，其經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4℃過夜溫育包被在96-孔板上。用洗滌溶液，優選含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的試劑，優選蛋白如BSA或明膠蛋白，更優選BlockAce(Dainippon Pharmaceutical Co.，Ltd.)進行封閉。用洗滌溶液，優選PBST，再次洗滌之后，加入適當稀釋的GPI-錨定蛋白樣品。在室溫進行適當長時間如2小時的反應。用洗滌溶液，優選PBST，洗滌之后，使針對酶標記的白色念珠菌的抗體，優選HRP-標記的抗-念珠菌抗體，在室溫反應適當長的時間如2小時。標記方法可以是酶標記法或放射性同位素標記法。用洗滌溶液，優選PBST，洗滌之后，通過適于標記類型即酶標記的方法計算GPI-錨定蛋白樣品中Als1p的量，加入底物溶液，然后在終止反應時測量在490nm處的吸光度。
(3)利用粘附動物細胞的能力進行的方法試樣對GPI-錨定蛋白在真菌表面的表達的影響可以通過測量真菌細胞壁中GPI-錨定蛋白的活性，優選通過測量真菌對動物細胞等的粘附能力來進行測定。除了Als1p，Hwp1p等參與粘附動物細胞的活性之外，GPI-錨定蛋白活性還包括α-凝集素參與交配的活性，Flo1p參與酵母聚集的活性等。在下文中，將詳細描述利用真菌對動物細胞的粘附活性進行的方法，但是本發明并不僅限于此。
使用對細胞具有粘附能力的真菌，優選的真菌為白色念珠菌。對于哺乳動物細胞采用粘附真菌的細胞，優選小腸上皮細胞。培養哺乳動物細胞并通過適當的方法，如乙醇固定法將其固定。將試樣和真菌培養適當長的時間，如30℃溫育48小時，然后接種并培養一定長的時間，如在30℃溫育1小時。移出培養物上清液，用緩沖液洗滌，加樣至瓊脂培養基，如Sabouraud Dextrose Agar培養基(Becton Dickinson Company，Ltd.)。30℃過夜培養后，計數菌落的數目，并計算粘附率。
如果與未經該化合物處理的真菌相比，觀察到試樣具有使細胞粘附而形成的菌落數目降低的活性，則可以判定該試樣已經影響了GPI-錨定蛋白到細胞壁的轉運過程。
(4)利用光學顯微鏡或電子顯微鏡觀察真菌的方法可以通過用電子顯微鏡觀察真菌細胞壁的結構來確定試樣對GPI-錨定蛋白在真菌表面的表達的影響。
在試樣存在的條件下，將真菌如白色念珠菌培養一定長的時間，如30℃培養48小時，用透射電子顯微鏡觀察其超微形態結構。在此，利用透射電子顯微鏡進行的觀察可以通過例如根據Electron Microscope Chart Manual(Medical Publishing Center)的方法來進行。真菌細胞最外層的絮狀纖維結構具有高電子密度，并且可通過透射電子顯微鏡觀察到。此結構不受其它現有抗真菌劑的影響，并認為是包括GPI-錨定蛋白作為其構成成分的表面糖蛋白層。與未經處理的細胞相比，當此結構消失，只留下具有高電子密度的微層時，可以判定所述試樣已經影響了GPI-錨定蛋白到細胞壁的轉運過程。
當在透射電子顯微鏡以及光學顯微鏡下的觀察都顯示真菌細胞大量溶脹且出芽(分裂)受到抑制時，可以判定試樣對細胞壁有影響。
本發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個分離位置含六個或更多個連續A殘基的序列段，且這些序列段作為假(pseudo)-poly(A)位點來制備截短的蛋白。由于如上所述的特性，該基因只能少量地在酵母中表達，而且很難利用PCR進行擴增或者在大腸桿菌中進行復制。也很難確定其核苷酸序列。然而，利用該DNA的簡并突變體(SEQID NO5)，其AT含量低于編碼PfGWT1蛋白的DNA，本發明人成功地使PfGWT1蛋白高效表達。本發明人還揭示簡并突變體DNA的引入能拯救GWT1-缺陷型酵母的表型。此項發現說明瘧原蟲的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含量例如對于惡性瘧原蟲的GPI8為79.35％，對于惡性瘧原蟲的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內的平均AT含量為76.3％，可以推斷大多數惡性瘧原蟲基因幾乎不能在其它物種中表達。本發明人成功地使比編碼PfGWT1蛋白的DNA具有更低AT含量的DNA簡并突變體在酵母中表達。因此，通過利用該簡并DNA突變體，可以在瘧原蟲之外的宿主中表達瘧原蟲GPI合酶。此外，GPI-缺陷型酵母和GWT1-缺陷型酵母已知表現出相似的表型，包括致死性等特性。這樣，可以采用上述簡并突變體DNA拯救GPI合酶基因-缺陷型真菌的表型。
已導入了上述簡并突變體DNA的GPI合酶基因-缺陷型真菌的表型由瘧原蟲GPI合酶的活性來決定。相應地，可以利用GPI合酶基因-缺陷型真菌的表型作為指標，經篩選選出抑制瘧原蟲GPI合酶活性的化合物。這樣，事實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可以在本發明的篩選方法中使用。
如本文所用，術語“AT含量”是指GPI合酶基因編碼區完整核苷酸序列中的腺嘌呤和胸腺嘧啶含量。本發明簡并突變體DNA中的AT含量范圍優選從50％到70％，更優選從53％到65％，更加優選從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生物合成的本發明蛋白包括GWT1，GPI1，GPI8，GPI3/PIG-A，GPI10/PIG-B，YJR013W/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中的偶數序列鑒定號中。各相應氨基酸序列示于SEQ ID NO2和SEQ ID NOs6-21中的奇數序列鑒定編號中。此外，間日瘧原蟲GWT1的核苷酸序列示于SEQ ID NO3，相應的氨基酸序列示于SEQ ID NO4。采用本領域技術人員熟知的方法，如采用雜交或PCR，利用包含SEQ ID NO1和3，以及偶數號的SEQ ID NOs6-21中任一核苷酸序列的DNA，可以對其它瘧原蟲的GWT1，GPI1，GPI8，GPI3/PIG-A，GPI10/PIG-B，YJR013W/PIG-M，GPI13/PIG-O，GAA1/GAA-1或DPM1進行克隆。
另外，利用酵母或人GPI合酶基因可以對瘧原蟲的除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中的偶數序列鑒定號中；各相應氨基酸序列示于SEQ ID NOs22-41中的奇數序列鑒定號中。此外，人DPM2，DPM3和SL15的核苷酸序列分別示于SEQ ID NOs42-47中的偶數序列鑒定號中；各相應氨基酸序列示于SEQ ID NOs42-47中的奇數序列鑒定號中。
編碼參與瘧原蟲GPI生物合成的蛋白的簡并突變體DNA的制備由兩步組成設計和合成，所述簡并突變體DNA的AT含量比天然DNA低。在設計步驟中，首先對目的蛋白的氨基酸序列進行反向翻譯，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基因分析軟件(例如DNASIS-Pro；Hitachi Software Engineering Co.，Ltd)來實現。在列出的密碼子中，為各個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的宿主中使用的常規密碼子)。簡并突變體DNA可以通過利用這些選出的密碼子重排目的蛋白的氨基酸序列來對其進行設計。
如此設計的DNA可以通過本領域技術人員所熟知的方法來進行合成。本發明的簡并突變體DNA可以基于設計的核苷酸序列，通過如利用商購DNA合成儀來進行合成。
本發明還提供了插入了上述簡并突變體DNA的載體，以及在可表達狀態包含該DNA或該載體的轉化體(優選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“GPI合酶基因缺陷”是指該基因的功能產物未表達或表達水平減少。本發明的GPI合酶基因-缺陷型真菌可通過破壞GPI基因來加以制備。破壞可以通過在同源重組技術等的基礎上嵌入與該基因無關的DNA如選擇標記來實現。更具體的說，所述突變型真菌可通過將選擇標記盒導入酵母，該選擇標記盒含有利用引物擴增的粟酒裂殖酵母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個核苷酸)同源的核苷酸序列。
本發明的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。
轉化了本發明簡并突變體DNA的GPI合酶基因-缺陷型真菌可以通過將插入了該簡并突變體DNA的載體導入真菌中來進行制備。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含量的簡并突變體DNA。所述“接觸”可通過將試樣加到上述真菌培養物中來實現。當試樣是蛋白時，可以將包含該蛋白的編碼DNA的載體導入上述真菌中。
在本發明的方法中，下一步包括測量上述真菌的生長程度。更具體地說，在常規培養條件下接種真菌，具體地，將真菌接種至液體培養基，如酵母提取物聚蛋白胨葡萄糖培養基(YPD培養基)或瓊脂平板上，然后在25-37℃溫育4-72小時。這樣，就可以對已轉化本發明簡并突變體DNA的GPI合酶基因-缺陷型真菌進行生長評估。生長程度還可以以培養基液體的混濁度，菌落數量，或者瓊脂平板上形成的斑點大小或顏色作為指標來進行測定。在本發明的方法中，下一步包括選出抑制上述真菌生長的化合物。
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，其中該真菌已導入了上述簡并突變體DNA。下一步包括測定轉運到酵母細胞壁上的GPI-錨定蛋白量。該檢測方法包括(1)利用報告酶的方法；(2)利用與真菌細胞壁表面糖蛋白反應的抗體的方法；(3)利用粘附動物細胞的能力的方法；和(4)利用光學顯微鏡或電子顯微鏡觀察真菌的方法。在本發明的方法中，下一步包括選出降低轉運到細胞壁的GPI-錨定蛋白量的樣品。
本發明提供了利用參與GPI生物合成的蛋白篩選抗瘧藥物的方法，該蛋白是利用本發明的簡并突變體DNA制得的。所述方法包括例如結合分析系統，在該系統中進行篩選是為了選出結合參與GPI生物合成的蛋白的化合物，該化合物與結合所述蛋白的標記化合物競爭。具體地說，將本發明的簡并突變體DNA導入GPI合酶基因-缺陷型真菌中，在該真菌中表達該DNA編碼的蛋白，獲得所表達的蛋白。然后，將制得的蛋白與試樣和能結合該蛋白的標記化合物接觸。在下一步中，檢測結合該蛋白的標記化合物，選出降低結合該蛋白的標記化合物量的試樣。
本發明還提供了GlcN-PI酰基轉移酶分析系統。該系統包括使用GWT1蛋白，該蛋白是利用編碼具有補償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簡并突變體，它比天然DNA具有更低的AT含量。具體地，將簡并突變體DNA導入GWT1-缺陷型真菌，在該真菌中表達該簡并突變體DNA編碼的蛋白，獲得所表達的蛋白。然后，將此蛋白與試樣接觸，檢測GlcN-(酰基)PI，選出降低GlcN-(酰基)PI量的試樣。
本文引用的所有專利，專利申請和公開物其全部引入以供參考。
附圖的簡單說明

圖1顯示四分體分析結果的照片。在導入了opfGWT1-過表達質粒后，gwt1遭破壞的菌株存活。將衍生自單個雙倍體細胞的四個孢子垂直點樣。
如果一個拷貝的GWT1基因被破壞，則僅有一半的孢子生長。在這種情況下，[菌落-形成的斑點]∶[未顯示生長的斑點]的比率為2∶2。在用箭頭標記的縱列中，gwt1破壞體(disruptant)的致死表型由于導入了opfGWT1而得到補償，因此4個斑點全部生長，均形成菌落。
圖2的圖表顯示化合物對于表達opfGWT1基因的酵母生長的抑制活性。在GWT1基因遭破壞的酵母中表達酵母GWT1基因或opfGWT1基因。
具有抑制酵母GWT1-依賴性生長活性的化合物也顯示了對于酵母的opfGWT1-依賴性生長的抑制活性，其中opfGWT1在該酵母中表達。
圖3是顯示抗瘧活性的圖表。用惡性瘧原蟲感染人紅細胞。將GWT1-抑制性化合物加至這些紅細胞中，并確定對瘧原蟲感染的抑制。
表現抗真菌活性的5種化合物均抑制了紅細胞的瘧原蟲感染。
實施本發明的最佳方式下面，將利用實施例詳細地描述本發明，但是本發明并不限于此。
惡性瘧原蟲GWT1(PfGWT1)(1)惡性瘧原蟲GWT1(PfGWT1)的核苷酸序列(SEQ ID NO1)已公開于惡性瘧原蟲基因組數據庫(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)限制酶位點對兩部分進行組裝。這樣，就制得全長PfGWT1基因。另外，在編碼區外的限制酶位點也包含在其中，從而可以插入到表達載體內。
(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PCR擴增PfGWT1基因的5’部分。用pf137F(SEQ ID NO50)和pf151R(SEQ ID NO51)作為引物，通過上述相同的方法，擴增PfGWT1基因的3’部分。將擴增出的DNA片斷亞克隆到pT7-Blue載體(Novagen)中，對插入子的核苷酸序列進行測序，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添加到編碼區的外側，從而可以使PfGWT1基因插到表達載體內。用PF15-5作為模板，以及引物pf154FE(SEQID NO52)和pf157R(SEQ ID NO53)，通過PCR將EcoRI切割位點加至5’部分。將擴增的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PCR擴增3’部分。對擴增的DNA片斷進行亞克隆，制得pT7-plasmBK5克隆。
(4)用下述方法制備全長PfGWT1基因。用限制性內切酶EcoRI和KpnI消化酵母表達載體YEp352GAPII。將來源于pT7-plasmN2的EcoRI-XbaI片斷(約1500bp)，和來源于pT7-plasmBK5的XbaI-KpnI片斷(約1100bp)插入載體的切割位點。這樣，就構建成了包含全長PfGWT1的表達載體YEp352GAPII-PfGWT1。
ATGACAATGTGGGGAAGTCAACGGg(SEQ ID NO48)[pf136R]TGTGTGGTTACCGTTCTTTGAATACATAGA(SEQ ID NO49)[pf137F]ATAGAAAATGATTTATGGTACAGCTCAAA(SEQ ID NO50)[pf151R]AGACCAAATTAATTATGCCTTTACATGTAC(SEQ ID NO51)[pf154FE]agaattcaccATGAGCAACATGAATATACTTGCGTATCTT(SEQ IDNO52)[pf157R]GAAATTCCAATGTATTCCATATTCACTTAT(SEQ ID NO53)[pf168BK]AAGATCTAATACATTAAAACATTTTAGATTAATGAATATGTG(SEQ ID NO54)[pf155RK]aggtaccGTACACTCCACTCTATGATGATCATTC(SEQ ID NO55)[實施例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)，因此通常無法使用常規的生物技術(PCR，以大腸桿菌為基礎的基因工程，重組蛋白的表達系統等)(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％，其包含多個連續的A或T序列段。因此，可以推斷該基因將很難在酵母中復制并以蛋白形式表達。實際上，當將連有酵母過表達載體的天然PfGWT1導入GWT1遭破壞的酵母時，PfGWT1根本不能拯救GWT1破壞體的致死表型。為了降低AT含量，用不改變原本氨基酸順序的同義密碼子替換密碼子。
在惡性瘧原蟲GWT1(PfGWT1)核苷酸序列(SEQ ID NO1)的基礎上進行密碼子取代，所述核苷酸序列已在惡性瘧原蟲基因組數據庫(PlasmoDBdatabase，http//plasmodb.org/)中公開。將所得核苷酸序列命名為“優化的PfGWT1(opfGWT1)”(SEQ ID NO5)。
將上述序列設計為編碼區外包含額外的序列；即EcoRI切割位點序列(GAATTC，5’末端)，Kozak序列(ACC，3’末端)，和KpnI切割位點序列(GGTACC，3’末端)。所得序列的合成委托美國的Blue Heron Inc。用這些額外的限制酶位點將完全合成的opfGWT1與YEp352GAPII載體連接，從而構建opfGWT1的過表達質粒。將該構建體引入僅僅單個GWT1基因拷貝被破壞的二倍體細胞(WDG2)中。在含孢子形成培養基的平板上培養獲得的轉化體，形成用于四分體分析的孢子。
新設計的密碼子-修飾opfGWT1的AT含量減少至61.55％。四分體分析結果示于圖1。在導入opfGWT1過表達質粒后，gwt1遭破壞的菌株存活。上述發現表明在通過密碼子修飾而使AT含量降低時，PfGWT1基因可以在酵母細胞中表達。
利用opfGWT1-表達型酵母進行抗瘧活性分析利用opfGWT1-表達型酵母構建抗瘧活性化合物的篩選系統。
通過將釀酒酵母GWT1終止子，釀酒酵母GAPDH啟動子以及多克隆位點插到單拷貝載體pRS316的SacI-KpnI位點來構建表達盒。分別將釀酒酵母GWT1和opfGWT1插到多克隆位點，制備pGAP-ScGWT1和pGAP-opfGWT1質粒。將這些質粒導入GWT1破壞體。用YPAD連續的兩倍稀釋化合物(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-孔板的每個孔中。將含有各種質粒的酵母細胞過夜培養物稀釋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測定培養物混濁度(圖2和表1)[表1]

雖然GWT1破壞體不能存活，但菌株在導入每種質粒后能存活(如0μg/ml的化合物濃度所示)。化合物(1)抑制ScGWT1-表達型酵母的生長，化合物(1)是GWT1-特異性抑制劑。以25μg/ml使用該化合物，約有85％的生長受抑制。當以50μg/ml使用該化合物時，酵母完全不能存活。化合物(1)也抑制opfGWT1-表達型酵母的生長。以25μg/ml使用該化合物，約有50％的生長受抑制。當以50μg/ml使用該化合物時，酵母完全不能存活。由于opfGWT1-表達型酵母的生長是由導入的opfGWT1的活性來決定的，因此生長抑制可歸因于化合物(1)對opfGWT1功能的抑制作用。此發現說明利用該分析系統，通過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。
抗瘧活性利用紅細胞培養系統，對抑制酵母GWT1的典型化合物(1)-(5)進行抗瘧活性分析。
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，將待測化合物溶解在100％DMSO中，用培養基稀釋，然后將稀釋物的80μl等分試樣加到96-孔培養板的每個孔中。惡性瘧原蟲FCR3菌株在37℃用含10％人血清的RPMI1640培養基預培養，然后在每個孔中加入20μl的培養細胞(含10％紅細胞)。此時，0.47％的紅細胞被感染。在5％O2，5％CO2，和90％N2的37℃條件培養48小時后，將瘧原蟲用吉姆薩(Giemsa)染色法染色。測定原蟲-感染的紅細胞數量，以估計感染率(圖3)。結果，顯示化合物(3)具有很強的抗瘧活性。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，這說明抗瘧藥物可以在這些化合物的基礎上進行合成。
工業實用性本發明成功地制得能表達瘧原蟲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，還從未進行過在真菌細胞中表達瘧原蟲基因以及篩選抑制該基因功能的物質的嘗試。本發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代，采用比較基因組進行用于藥物發現的全新篩選方法的可能性。
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tat ttt act ata atc aag ttg ata tgt tct tgt cta gtt tta tat gta 816Tyr 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 gaa 864Thr 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 aaa 912His 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 gaa 960Glu 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 Lys Glu Glu Ile Asp Glu Lys Lys Lys355 360 365aaa 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 390 395 400aaa 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 425 430
atg 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 490 495gtt 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 520 525tta 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 tta1824Phe Asn Ile Ile Gly Ser Val Asn Leu Tyr Leu Phe Ser Phe Ser Leu595 600 605
ttt 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 650 655cgg 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 710715 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 730 735tca 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 775 780
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Val 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 135 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 Gln165 170 175Asn Cys Ile Asn Lys Leu Asp Lys Asn Asp Tyr Asn Ile Leu Lys Ser180185 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 220Phe Leu Tyr Phe Pro Ile Leu Leu Gln Lys Tyr Glu Ile Leu Thr Thr225 230 235 240Leu 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 Val260 265 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 310 315 320
Ile 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 390 395 400Lys 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 Leu530 535 540Leu Lys Arg Ile Arg Tyr Ile Phe Leu Phe Ser Ile Ile Ser Ile Ile545 550 555 560
Leu 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 635640Asn 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 665 670Ile 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 760765Thr Ser Leu Gly Leu Phe Ser Cys Ala Leu Ser Phe Ser Leu Glu Asp770 775 780Ile Leu Leu Arg Tyr Lys Lys Tyr Lys Ile Asn Ile Asp Ile Thr Val785 790 795 800
Leu Asp Lys Ile Asn Lys Asn Thr Leu Ile Val Phe Leu Phe Ser Asn805 810 815Ile Leu Val Gly Met Phe Asn lle 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 Lys850 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)..(1956)<223>
<400>3atg gcg cat ttg aac ctc ctc gtc tac ctc atc atg tgc ccc ttc aat 48Met 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 cca ttc cgg tta gga agc 96Val 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 aca tat aag aag gat gtt240Leu Ala Lys Met Tyr Tyr Lys Ile Val Leu Thr Tyr Lys Lys Asp Val65 70 75 80
agg 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 Cln 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 175cac 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 215 220ttt 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 250 255
atg ggg gag caa acg ggc caa cac acg ggc gat tgg cac acc atc cgc 816Met 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 ttg 864Gly 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 ttc 912Val 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 ttt 960Ala 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 390 395 400cta 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 Thr405 410 415ttt 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 425 430
cgc 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 470 475 480ccg 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 600 605
tct 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 640ggg gag aag gga gag aag cgg gag aag cag caa taa1956Gly Glu Lys Gly Glu Lys Arg Glu Lys Gln Gln645 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 10 15Val 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 45Ash 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 Leu
130 135 140Ser Trp Asp Ser Cys Leu Tyr Lys Leu Asn Ser Asp Asp Tyr Asn Leu145 150 155 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 255Met 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 Thr
370 375 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 505510Val 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 570575Thr 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 Tyr
610 615 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 Gln645650<210>5<211>2625<212>DNA<213>人工的<220>
<223>人工合成的序列<400>5atgagtaaca tgaacatcct ggcctacctg ctgatctgtc cattcaacct gatctacatc 60ttcgacctgc ctagctacat ccctgagcta aacaagaagc tggagaacga cgaagtcttc120atctacggta aggagatccg taagaacgaa tccgcatact ctctacacta cgagaagtac180ctatacgaat tgtcacgaag atactacgag atcatcctga agtacaacaa ggagttggga240gtcaaccaag agaaggaata caacctgatt atctceagag agatcgataa gaagaagaag300aagcagaaga atagtaccca gggtgaatac aataacgacg atgataacaa ttggaagttg360ttceagattt acgagaagga agaacctagg agctatgaat tgatcagggt agagatatac420aagaaggaca ttctgttgat ctacaagaat gagaagacga agtcctctat caagttcatt480atcaagaagc gtaaggatat caagaattac ttctccttgt gttaccagaa ctgtatcaat540aagctggaca agaatgatta caacatcttg aagtctacca tcaacaattc caaggaaaac600attatcaact ctgcatacat ttacatgtac attatcttct tcttcctgtg catatacgtc660gagaagaacc tgttcttgta cttcccaata ttgcttcaga agtatgagat tctcactacg720ttgttcatcc tcttcatccc attgatccta ttcgtattct tctatttcta cttcacgatt780atcaagctga tttgctcatg cttagtccta tacgtgactt tceagttgat ctactatacg840
cagggaatgc ctatttacat ggaacatagt attctcaagc acaaggagaa ggaagagatt 900tgcgatgaga aggaggaaat ctgtgatgaa aaggaagaga tttgcgatga gaaggaagag 960atttgcgatg agaaggaaga gatttgtgat gagaaggaag aaatctgcga tgagaaagaa1020gaaatccttg ataagaagaa gaagatccac gagaagaaga agaagatcca tgataagaaa1080gaggaaatcg atgagaagaa gaagaagatt catgacaaaa aggacgaaag tcatgataag1140aacgaggaca ttacgtatcc agtccagtac aatatcgaga atgacctatg gtattcatcc1200aagaacgtgg acatcaagat gtattcatcc agcaacaagg gtgaagaata cattatccag1260aacacgttga aacatttccg attgatgaac atgtgtatga cgtacatttg tatcttcgct1320gttgacttct acttcttccc taaccatttc tgcaagtcct actattacgg aaatacgttg1380atggacattg gaatcggtgc atccatttct tccagtgcat actcgcagga gatcaagaag1440ttcacgtaca ttaaggagaa gaaacgaatt atcgagttga aacatatcgt gttattcatt1500ctgggaatta gcagatttat cggtatctac ctattcaact ataactacaa catttctgag1560tatggaatcc attggaactt ctttcttacg ctatgtacaa cctttctgat tagcaacatt1620tgtttcatcc tcctcaagag gattcgttat atcttcctat tcagtattat ctcgatcatc1680ttattcgaaa tcgctattta ctacttcgat ctacataatt acatcctcct gaagaatgac1740cgtttaaact tcttctcttc taacaaggag ggtctattca acattatcgg ttccgtgaac1800ctttatttgt tcagtttctc actcttcaag taccttacga agcagcgtac gtatatcacg1860acgtccaata tccctaagaa caagaaagat atgaataact caatgtattc gaagaatgga1920aaccatacta attcgaatat caacaaccgt aaccataaga tcgtcatccg taataaccac1980attaacaagt atgagcagga caacacgaac aagtacatca acaagcaaat caataacaac2040aagaacaaat tggatgagga agagaagctg aagaaactca agaagcttaa gaataagaag2100aagaacctca agaagaaaat caagtactat ctattgtacc ttcagtatat catcaacatc2160
tacaaggagg 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 tcctgttcea 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 catttattat 60aataatatac ctatatttaa aaaaaaaagt ggaaatgaac aaaacataag tgcgctattt120attttatatg atgtaaaaaa gtatgtgtat aatatggttc atgatcacgt aaatacatta180gtcctagaag cttttagaag agaagacata ataaaaaaaa taaaagtaaa agaaaaacaa240aataataatg ataaaaataa agaaagtaat attgaaaaag ataaaaatga acaaaccaaa300tttacagata tatatgatac aaatagtaag agtgacaaag atatacaaaa gaataatatg360aacgatggtg atagtaataa taaaaatagt agtttattta ttgatccttt tgaaagtgat420tcatatgaaa agaataattt tagtaatgaa aaatgtgctt tccaaaatgt cgataaatca480aaaaaagata aagaacatat atattctgaa aatattactc ctagtagtag taataataat540aatgataata ataaagaaaa tgattgtgat aaggaacaat tagataaata taataaagat600aaagaaaata aattaaaatt aaatgataag gatgaatata tttctttcaa ttttattgaa660
gataaattaa 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 cagcttcatg1020aaaaataaac aaaacccatc tgaatattat aaatatagag aaatcttatt aattcgtatt1080atcaatttaa taatcgatat tttcttggga tttctgatat ttttactact ttattttaac1140gtaataaacc tacattatat atcagagaag gctcaaatat tttatggaac cagtacttta1200acttctatct tgggtacctt gttacagaat ccattaggat ttaaattaaa taataatttt1260acgtctttca ttggtagcat ccttgtttcg atattagaca aatgggattt atttacaaat1320accatccctg tgaataatag tacagttttg aattttgttg gatatacatc actgctgggt1380ttttcttttt ttttgtcttt tgttattgat tatttgagat tcgtaacagc acatgttacc1440attatttatt tatttttgaa aaaaatatgt accctttttc ataaaaatat gtattcctta1500tacctattat tcaatgggaa aaaatggaac atactaaaac taagagttga tacaaattac1560tactcaaatg aagaagtact tttaggaacc atattattta ccattttaat ttttttgtac1620cctaccattt ttgttcttgt tttggttttt ggtcttatat atcttataat aaatagaatt1680atataccttt tgtgtgttat ggagaaaata attttgtaca cccctttcta tattttcttt1740attcaaccga attgtaataa atatattagt aagggtttca agtttacaaa atatgaagtt1800ggggaacatg aactgttaaa aaggtatccg actaacagct acttgttgct agaaaagatt1860cattttttat tttttgataa aataaaattg tttataaata ttttccttta ttttaaaaac1920ctcgagtcaa tgtcatcata ttcatatata tttatttctt tttgtttttg tcttttaaaa1980
aaaaaatata tatatatata tataatataa 2010<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 Ash 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 Ser 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 185 190
Gln Leu Asp Lys Tyr Asn Lys Asp Lys Glu Asn Lys Leu Lys Leu Asn195 200 205Asp 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 GIn 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 IIe 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 410415Asn Asn Asn Phe Thr Ser Phe Ile Gly Ser Ile Leu Val Ser Ile Leu
420 425 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 Leu515 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 650 655
Cys 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 ggtgtgtggg60tctgttaatt ttaccgggtt tgataacaaa aatatgatag gtaagcacgt ggaactagaa 120gggaggtata aaaaggaata tatagatagg ttttttttag aagaattaag aaaacacaat 180tatatgaata acaatgttat attattaagt acatcaagac attattttaa ttatagacat 240accactaatt tattgattgc atataaatat cttaaatatt tcggtgatac tatggataag 300aatattttat taatgattcc atttgatcaa gcttgtgatt gtaggaatat aagagaaggt 360caaatatttc gagaatatga attatttcct agtagtcata ataaagaaac aaaaatagaa 420aatataaatt tatatgagaa tttaaatatt gattataaaa ataataatgt acgtgatgaa 480caaattagaa gagtacttag acatagatat gatgctttta cacctaaaaa aaatagatta 540tataataatg gaaataatga gaaaaattta tttctttata tgaccggaca tggtggtgtg 600aattttttaa aaattcaaga atttaatatt attagttctt ctgaatttaa tatatatata 660caagaattac 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 accatctaaa aatataaaag ccttttcatt atataacatt 960ttaaattatt taaaaacaca acatattatg tcagaaccta ctacaaataa ttctaaattt 1020
aattcgtcca 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 Lys1 5 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 105 110
Asp Cys Arg Asn Ile Arg Glu Gly Gln Ile Phe Arg Glu Tyr Glu Leu115 120 125Phe 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 Lys245250 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 345 350
Phe 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 Asn435 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(Plasmodium falciparum)<400>10atggaaagtg cagttagcga atgcaatata tacaaaaagg aaaaagataa aaatataatt60tacaaacaag aaagaaaatg ttgtatttgc atggtgagtg atttttttta tccaaatttg 120ggaggaatag aaactcacat ttttgaattg tctaagaatt tgataaaaaa gggtttcaag 180gttatagttg taacaaattt taataataat aggcatggta taagatggat gggtaatggt 240attaaggttt attatttgcc cttccagcct tttttagatg tagtgagttt tccaaatatt 300
atagggactt taccactatg tagaaatata ttatataggg aaaaagtcga catagtacat360ggtcaccagg ctacgtcagc attagctcat caattcattc ttcatgccaa aaccttagga420ataaaaacta tttatacaga tcactcatta tacagttttt cagacaaagg atgcatacat480gtaaacaaat tattgaaata ttgtataaac gatgttgatc attctatatg tgtttcccat540acgaatagag 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 acacgatatg 1020atgattttag ctaaaccaaa tcatatcgaa ttatgtaaag cagtcgataa agcattaaaa 1080attgtacaaa aggtggactc aaatttattc cacgaaaggg taaacatgag tttattaaca 1140tatgtaaata tatatatata tatatatata tatatatata taatatatat gaataatttt 1200atttataatc 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 ataa 1494<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 IIe VaI VaI50 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 Gin 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 Ash Asp Val Asp His Ser Ile165 170 175Cys Val Ser His Thr Asn Arg Glu Asn Leu Val 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 250255Ile Gly Gly Glu Gly Pro Lys Arg Leu Leu Leu Glu Glu Met Arg Glu260 265270Lys 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 Phe
450 455 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 aattgattca 60aagatattta 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 ttttatatat840
cataaaggaa cacaaaattg taaacaatat gataaaaata tggttgatca aaatgtttgt 900ggtcaaaata tggttgatca cataattcag aacaggaaca atttgtgtag gacacatttt 960tattcctcca 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 tattttttta1680atttcatctt ctctttttac aaatataagt tttttacttc aatttttatg tattcttttt1740ttctgcctta tacataacag atcacctgaa catgtagcct cttattttag aaacttagaa1800acgaaagatg atcaaaatat ttatatattt ataacaaatt gttatgatat acctttatat1860tcgcatatac atagaaaatt caatatagga tttttagact gttctcctta tgacacgagt1920aatgatgaag ctaccaaaaa ttggagaaaa cgtatatatg aagataaatt taaggaacaa1980ttttttaata tttttcaaga aaaaaaaaat aataatcatc atataaatag tacatatgga2040gatacaatta ctccatatat aataccagat aaatcatttt attggtttgg tcatcatcat2100tttaataaaa aaaataattt tcaatatatt tatcaaaaca ttaatttgtc atgtttaaat2160
tatagatttc atataccttt acaaggacaa ttacctactt atatagttac aacaactatt2220gaacttacac atttacaatt atttctgagt acatataatt ataaacttga aacaaagcct2280tttttttctt attttatgat aagtgaagca aaaggaatag ttccagtata tcattacata2340ttcaagaggg ttccttccta a 2361<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 140Asn Glu Lys Asp Glu Asn Asn Gly Ash Asn Asn Asn Asn Asn Asn Asn145 150 155 160
Asn 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 185 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 390 395 400
Tyr 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 460Lys Tyr Lys Thr Ile Asn Ile Tyr Met Tyr Ile Leu Lys Arg Ile Asp465 470 475 480Trp Ile Val Tyr Leu Val Thr His 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 Ser
625 630 635 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 Ser755 760 765Glu Ala Lys Gly Ile Val Pro Val Tyr His Tyr Ile Phe Lys Arg Val770 775 780Pro Ser785<210>14<211>1233<212>DNA<213>惡性瘧原蟲(Plasmodium falciparum)<400>14atggggcata cacataaaga atataaaaac aatgagaaga gtgcaatatt ttttgagtgg 60ttgatttttt ttgtgggtat aataattcgt ataattattt attattatgg aaggtggcaa120gataaaaact ttaatgttaa gtttacagat gtagattatt atgttttttc tgatgctgca180
aaatatgtac ttatgaacaa atcaccatat gaaagatata catatagata tacaccttta240ttagcatata taatgatacc aaattttttt gttcattttt cttttgggaa aatattattt300tcatttatcg atattcttgt tactattctt ataaatcaaa ttataaaaat caaatatact360aattgtaaaa attatatatt ttatacttgt ttatggtttt taaatccatt agtcataatt420atatcccttc gaggtaatgc agatgttatc ceatgcttct 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 ceatgttttt acaaactatt 1020tcttttattg cattgaataa agtgtgcaca tctcagtatt tcatttggtg tattceattt 1080ttaccaatta tactttgtgc cataacctta agcaagagaa atatgtttct tataatatcc 1140tccattttat tttttattgt ggcaaaagtg ggctcaaaaa gatttctttt attatataaa 1200tatgtctttt catatattat ttttttttca tga1233<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 Ile
1 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 Phe
245 250 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 300Lys 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 ctcatttatc120aatggctatt tttatgcaag acaaaaactt gaagaaaaat cagaaaacct tgagcttttt180
agtaggaaag tatttggaga tgaatacgta gaaagtttga aaaagaaaaa aaatactttt240tcgattatta atgcaccata tgataaagtt gttatacttt taatagattc cttaagattt300gattttaccc tatacgatac taattatgaa aaggaattca taggaaaaga aaaaaataca360gatatctaca ataatatatc tagcgaaaaa aaaaatatat caaatgatgg ggaaaaaaaa420aactcattat tttttttaaa taatatgata aatgtacatc acattttaca aaatgaaaaa480aacaacactt tattattccg gtttgatgca gatgccccta caataacaac ttcaaggata540aaatctatat ttatgggtac cataccaaat tatatggaag taaatgaaaa ctttagtcct600acgactagtg ttgaagataa tttttttgaa cagcttcatt taaataataa aaaagtaatt660gctatcggtg ataataccat tactcatttg atgaaacatt tttctaaaga attagtttat720gagagcttta atgtttttga tttttattca ttagatattg ctgcaaagaa acatttttat780gaagaatacg aatcaaatga ttgggatatt atgtatatac atatgttggg agttgatcat840attggacata taaaaacacc caactcaaaa atcatgggag atgccttaaa agattttgat900acattcatat atgatattat aaataaaata aaattagata atcttaaaaa tattagcaca960gaagaaaaag aaaaaatgaa aataataaaa aaaaaaatat ataaaactta tcgaaacggt 1020catcacatac aaaatgaaaa tgatcatatt attgataata tacaaaatga aaatgatcat 1080attattgata atatacaaaa tgaaaatgat catactattg ataatataca aagtgaaaat 1140gatcatacta ttgataatat acaaaatgaa aatgatcata ctattgaaga tatacaaatt 1200gataatgatc atactattga agatattgaa aatcaaagtg aacaaaaaaa tgatgataaa 1260aaaacactgt tcatattttt tggtgatcat ggacagctag atacaggaga tcacggagga 1320tacagcttgg acgaaaccca tagtgctctt tttgcatatt cacccttaaa ctttatatct 1380ttagataatg acatcattca aaataatttt gttttatatg ataaagataa attaaaaaaa 1440aatgtgaata cactgaatga agaaaataat aataatgaga atatagataa ttataaaaaa 1500
tatcattcat 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 taatattatc2580tttattctat ttaaattatt tccgaaaatt ataactaatt catttcaaat attaagaagt2640aattatttcc ttttgtttgt tattatatgg agctgttgtg aaatgtcctt taattatata2700gataaagaaa gatattatat tcattacatt ttaattgtat atgttatatt tgggatgctt2760
aaatggaagt atcaccgtgt ctttaatata ctaaaagcct tcattttgtt ggtgcttcta2820ataattaatg ctttgtatag ccatacaccc gaatattttg accatggtaa agaaaaaata2880tatttaaaag aatctgtact aaaatcagtt tttccaatat catcttatat tcttagctta2940atattaataa atagtggtat taataattta ctgaaaaaac gaataaaaat aataataacc3000caaatatgga cattacaata tatcctcgtt tttttgttct taaataatat ataccataga3060tatattcaat ttataacacc cccaagtatt tatttcttaa ccatttcaac cttcatattc3120atattcaata caaatttagg tgtattattt ctcttttata tgacatttct ttttttctac3180tttattctta taagttccaa ttgttcagaa aatatgatcc aaatgaatga cataacatcg3240acatggataa atgaaaatat acataataga aatgatccta taattacaaa aggaaatttg3300gagaataaag aaaagtgtac ttcatgtaat acatctataa aagaaaaatt ttattataaa3360ttaatgatag aaaaattcaa attattaaat acatgctctg tattaaatga agaagaaata3420atgggaatac aaatttataa attaattaga gatatttctt atttttatat aaatgaaaca3480gatttttata ttttatcatg tgtactttta atatattctt tttttataac gggacataaa3540tttattttaa ataacttacc actagtttca ggttatgttg gattgtataa atatgtgtgg3600ccaataagtc agttttatat ttttaatcat attttttttc cattcttctt ttcgttattt3660tttatcattt atatttataa cataagaagg ataaaaatta taaattcttt taagcaattt3720gatttgtatt atttttatgt ttatccacta atgaactttt ccttcaaggc ctcctttttg3780ttttgttgca agtttattat atctgtatgg gtttcatatt acttgaatct gcacataatg3840gtaaaaatat aa3852<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 Gln35 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 Lys145 150 155 160Asn Asn Thr Leu Leu Phe Arg Phe Asp Ala Asp Ala Pro Thr Ile Thr165 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 Tyr
225 230 235 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 350Asn 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 Lys
465 470475 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 505510Ser 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 Asn 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 670Met 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 Lys
705 710 715 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 935940
Leu Tyr Ser His Thr Pro Glu Tyr Phe Asp His Gly Lys GIu Lys Ile945 950 955 960Tyr Leu Lys Glu Ser Val Leu Lys Ser Val Phe Pro Ile Ser Ser Tyr965 970 975Ile 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 Gln101010151020Phe 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 Ser116011651170
Phe Phe Ile Thr Gly His Lys Phe Ile Leu Asn Asn Leu Pro Leu11751180 1185yal Ser Gly Tyr Val Gly Leu Tyr Lys Tyr Val Trp Pro Ile Ser11901195 1200Gln Phe Tyr Ile Phe Asn His Ile Phe Phe Pro Phe Phe Phe Ser12051210 1215Leu Phe Phe Ile Ile Tyr Ile Tyr Ash Ile Arg Arg Ile Lys Ile12201225 1230Ile Asn Ser Phe Lys Gln Phe Asp Leu Tyr Tyr Phe Tyr Val Tyr12351240 1245Pro Leu Met Asn Phe Ser Phe Lys Ala Ser Phe Leu Phe Cys Cys12501255 1260Lys Phe Ile Ile Ser Val Trp Val Ser Tyr Tyr Leu Asn Leu His12651270 1275Ile Met Val Lys Ile1280<210>18<211>2433<212>DNA<213>惡性瘧原蟲(Plasmodium falciparum)<400>18atgggatttt cagaaaatcc caaattctgc ctgctcataa ataagcttat caagaaatct 60aaagttatag gcgttttctt atcaattatt ggtgtgattt tctttttggt gtttaataag120tttaataaga atgctgaatt agacgctagg acatttaccc aatttgttgg taattcagta180ttgaacaaaa agaatgagaa gttttataat gatacaaatt cttatttcat gaactataca240tatgaaggga aagaagatat aataaagttg atatatgatt atattagaaa aaatatatta300gtaaatgtag agaatgagat ggttaaaata aaattaacgg atagaattga acagaatata360ttgataagta atgtaggatg taaatattgt aataatatgg aaagtttagt tgtggtaata420
aattttgatt ttaaagaaag gaaatatttt catagcgtaa ttatcggttt aacgttaatg480gaacattttt ctaaatgtaa ctatatgagt aaggatgtga cttttttatt taccaataaa540gaattattat attctttagg tgttcaagaa tttatacaaa aatattttta taataatact600aatagaattg gaaaaaaaat tattagatct tctactatta ttgaatttga ttctatttat660ccttcttata ttaaaattaa ttatgaagga ttaaatggta tgttacctaa tcaagactta720atattattat taacaaatga acttcatttt tattctattc ctattaaaat ggagcttact780catggttcca tatttgatat ggccctagaa aagaattatg aaaatggtca catatacttt840ctgaggtaca aaaaaaaata tgaatatata cgtgatgata atgatgaaat caagaacatt900cccgcattta ctgcaacggg gggtagcaaa gtacccataa gaaataaaat gattaatttg960ttcaacttaa 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 1440aatgatcaaa 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 1740
gaaaaaaaaa 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 taa2433<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 5 10 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 Ash Ala Glu Leu Asp35 40 45Ala Arg Thr Phe Thr Gln Phe Val Gly Asn Ser Val Leu Asn Lys Lys50 55 60Asn Glu Lys Phe Tyr Ash Asp Thr Asn Ser Tyr Phe Met Asn Tyr Thr
65 70 75 80Tyr Glu Gly Lys Glu Asp Ile Ile Lys Leu Ile Tyr Asp Tyr Ile Arg85 90 95Lys 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 120 125Tyr 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 295 300
Ala 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 Phe405 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 Val485 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 535 540
Asn 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 680685Ile 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 720Asp Lys His Lys Ile Leu Gln Ser Lys Tyr Phe Pro Glu 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 775780
Ile 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 Tyr115 120 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 215 220
Glu 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 agataattat 60acagatccaa 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 5 10 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 AIa 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 125Leu Lys Ser Leu Ser Lys Thr Thr Ala Ser Asp Ser Ile Trp Thr Leu130 135 140Ser Phe Trp Leu Thr Leu Trp Tyr Ile Phe Val Ile Ser Ser 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 Leu
195 200 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 acctgtatct120aaccgaaagt tcaaaaaagt caagcaaaat gggagggtag agattaacat gggcatacaa180tatcaceaaa ttgtacttat tttactactg aatattttgt tctatgtaat ttgcctaaga240tcaagatttc tcgaacatat taatagaact tttgaagtga caatcgcgcg aagtttccag300atcttaatta taatgggatt gtttgcctta ggtacaatta tacttgtgag gggacctagt360gtggaaactg taacaatttt caaagaaagt ggactacagc tgtccagagt gaagggtatg420gttatatttc ctcaacaatg gaatcggaag ttctttgaac aagtagagtt tatatccaat480gaaagaatta ttgatgtagt gatcaatgaa ggattctgtc ggggatttcg agtgatattc540tatcttgcag caattgtacg taaatcgtct acgcttaagc tattatttcc agtatgtatt600
caagcgaatt taagattgtt tcttacttca acgatctgat tagaaatact aacacaacaa660tgcagtcaaa tttgcccagt atcgatgacc aacgtctaat atacaacata tctagaaaat720atctcagtaa 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 Phe
165 170 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>27
Met 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 ccaaaatcgt ctcccgaaac120aatgcatcat tgcagcatat ttttcctcat aaatatggcg attatgaaat caatttggtc180atagcgcacc ctgacgacga agttatgttt ttttccccca taatttctca actgaattcg240tactttccga gaaccgtccc atttaacata atctgcttat caaagggcaa cgccgaaggt300cttggcgaaa ccagggtaag agaattaaat gagtcggccg ctttattgct acacaatgaa360
agagcagtct ccgtacaggt gatggatttc caggatggta tggacgaaat atgggatatt420gattctataa cttcttctct ttcacaaaag atagatataa agaatcataa cttgaaccag480attatcgtta cctttgattc atatggtgta tcaaatcata tcaaccacaa aagctgttat540gctgccgtta aaaagttggt ggatgattat gctcaaccta agaccaaaag 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 attag 915<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 80Tyr Phe Pro Arg Thr Val Pro Phe Asn Ile Ile Cys Leu Ser Lys Gly
85 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 cerevisiae)<400>30atgtggaaca aaaccagaac gacgcttctg gctgttggtg tcttatttca tttattttac 60ctatggtcta tttttgatat ctatttcatt tcaccgctcg ttcatggtat gagcccatat120caaagtactc caacccctcc tgcaaagaga ttgtttttga ttgtcggtga tggtttacgt180gcagatacca cttttgataa agtcactcat ccagtatccg gaaaaacaga atttctggca240ccttttatta 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 atacttgatt 1020ggtgtgaact atcctaaaaa ttcagttggt gagttaccaa tagcatatat cgatggaaaa 1080gaaagtgaca agcttgccgc attgtacaac aacgcaagaa gcattttaga gcagtactta 1140gtcaagcaag atgaggtaat agactctcaa tttttttata aggaatactt caagtttgtt 1200
gaaaagtctc attcacatta cttagaagag atagaaacct taattcagcg tatatctgaa 1260ggagaaaact atttggaaca agaagcaatc acccttacag aggaattaat gcagataaca 1320ttggaaggtt tacattattt gacaacctat aattggagat tcattagaac tattgttaca 1380tttgggtttg ttgggtggat ctttttttct tttataatat ttttgaaatc attcatatta 1440gagaatgtaa ttgatgacca aaaagcgtca ccattaagcc atgcagtatt tggttccata 1500ggaattttac taaattggat tttgttctac caacattctc cgttcaattt ttacatgtac 1560cttcttttcc cattatactt ttggagctat atttttacaa atagatccgt actacgttca 1620ggtatcaagg aattcttcaa aggtacctct ccttggaaaa gagttttaat aacaatctct 1680attatatcag tttatgaggg aattgtatat ggatttttcc atagatggac gtttacgcta 1740attacaaata tattggcgtt ttacccgttt atttgtgggg tgagagagct atccgtgaat 1800atattgtgga tcataactag tgttctttta tctacattta ccttatttga cgctgttaaa 1860attgaggact tgaaccagat acatctagca gggttattaa tcattctcag tgccttttat 1920gctctttaca aaatacattc caggataaat tcctacacgc gtgctatatt tgccattcaa 1980atttccttgg tggctgccat gttggcggtt actcatcgtt cagttatctc tttacagcta 2040agacaagggt taccaagaga gtcacaggtc gctggatgga taattttttt tgtatctctt 2100tttgtaatgc caattttaca ttataggaag cccaacaatg attacaaagt gagattattg 2160atcatttatt taaccttcgc accatccttt atcattttga ctatatcatt cgaatccctt 2220ttctacttct tgttcactag ttacatggta caatggattg aaattgagaa caaaatcaaa 2280gaaatgaaga cccaaaaaga tgaaaattgg ttacaagtgc taagagtttc agtaatcggg 2340ttctttttac ttcaagtcgc attctttgga actggtaacg tcgcttcaat ctcttcattt 2400tcattggagt ctgtttgtag attgttgcca atttttgatc ctttcctgat gggcgcatta 2460ttgatgttga aattgataat tccctacggg ctattgtcca catgcctagg tatactgaat 2520
ttaaaactta acttcaagga ctacacaatc tcatcattaa ttatttccat gagtgatatt 2580ctgtcgttga attttttcta ccttttaaga acggaggggt cgtggttgga tattggcata 2640accatttcca actattgttt ggcgatccta tcatctttgt tcatgcttat tttggaagta 2700ctcggtcatg tgttgctaaa aaatgtcatc atacaggata aaaccaaaaa aacacaatag 2760<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 Tyr130 135 140Ser Phe Gly Ser Pro Asp Ile Leu Pro Met Phe Lys Asp Gly Ala Ser145150 155 160
Asp 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 Val
385 390 395 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 445Thr 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 Trp Ile Ile Thr Ser Val595 600 605Leu 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 Tyr
625 630 635 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 Glu Met Lys Thr Gln Lys Asp Glu755 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 855 860
Phe Phe Tyr Leu Leu Arg Thr Glu Gly Ser Trp Leu Asp Ile Gly Ile865 870 875880Thr 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 tcgtceacct 120gtgtatgtga ggaaaacccc tctgatgaca tttccatacc atttagtagc actactttat 180tactacgttt ttgtatcttc aaatttcaat acggtgaagt tgctaagttt tttgattcct 240acacaagttg cttatttagt tttacaattc aataaatgca cagtttacgg taacaaaatc 300attaagatca attactcatt gaccattatt tgtctaggtg ttacattttt gttgagcttt 360cccacaatgt tattaactat attatttggt gcgceattaa tggacttatt gtgggaaacc 420tggctgttgt cactgcattt tgcattttta gcataccctg cagtttattc tgtatttaat 480tgtgatttca aagtgggatt atggaagaag tattttatct ttatcgttgt agggggttgg 540attagttgtg ttgtcattcc tttggattgg gatagagatt ggcagaattg gccaattcct 600attgttgttg gaggttattt gggcgctttg gtgggctata ctatcggtgc ctatatataa 660<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 5 10 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 190Asp Trp Gln Asn Trp Pro Ile Pro Ile Val Val Gly Gly Tyr Leu Gly195 200 205Ala Leu Val Gly Tyr Thr Ile Gly Ala Tyr Ile
210 215<210>34<211>2493<212>DNA<213>釀酒酵母(Saccharomyces cerevisiae)<400>34atgaacttga agcagttcac gtgcctatca tgcgctcaat tactcgctat tctgctcttt60atctttgctt ttttccctag aaaaatcgtg ctgacaggta tatcaaagca agatccggat 120caagaccgtg atctccagcg cgataggccc ttccagaaat tggtgtttgt gatcattgat 180gctctcagat cagactttct ttttgattcg cagatttccc acttcaacaa cgtgcaccaa 240tggctcaata cgggcgaagc atggggttac acgtcatttg ctaatccgcc taccgtgacg 300ctgcctagac tcaaaagtat tactacggga tctacaccta gcttcattga cttgctgctg 360aatgtagccc aggacataga ttccaacgat ctttcggagc acgattcctg gctgcagcag 420ttcatccaac ataataacac gattcgtttc atgggcgatg acacctggct gaaactgttc 480ccacagcaat ggtttgactt cgctgacccg acacactcgt tctttgtcag tgatttcact 540caagtcgata ataatgtgac gaggaacttg cccgggaaat tatttcagga atgggcccag 600tgggacgtgg ctatcctgca ttacttgggt cttgaccata tcgggcataa agatggcccg 660cattcaa8gt ttatggctgc taaacatcaa gaaatggaca gcattctgaa gtcaatatat 720gatgaagtgt tggaacatga agatgacgat gatacactga tttgtgttct tggcgaccat 780ggaatgaacg aactgggcaa ccatggtggc tcttcagccg gcgaaacatc agcaggattg 840ttgtttttgt cacctaagct ggcgcaattt gctaggccag aatcgcaagt aaactacaca 900ttgcccatca acgctagtcc ggactggaat ttccagtatt tagagactgt tcaacaaatt 960gatatcgtcc ccaccatagc agcactgttt ggtatgccaa tccccatgaa cagtgttggg 1020ataataatac ctgacttttt acaactgttg cccaataagt tggcaagtat gaaagaaaat 1080
tttatgcatt 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 agaaataccg1740ctctttatgc 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 cgcgccggca2220atatactggt ccatgctacc gtggtcaata aactacgcct ctattccagc acaagttaag2280ttgcaaacgt tcatcagaag taagttacct gccttcacct atcattgtat atttggaact2340tgtttgatga cggcatgcgt cgttttgaga tttcatctct ttatttggtc cgttttcagt2400
ccaaaattat 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 Asn 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 125Asn 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 170 175
Ser 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 200205Leu Gly Leu Asp His Ile Gly His Lys Asp Gly Pro His 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 His Glu Asp Asp Asp Asp Thr Leu Ile Cys Val245 250 255Leu Gly Asp His Gly Met Asn Glu Leu Gly Asn His Gly Gly Ser Ser260 265 270Ala Gly Glu Thr Ser Ala Gly Leu Leu Phe Leu Ser Pro Lys Leu Ala275 280285Gln 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 His 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 Ile
405 410 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 Ile435 440 445Ile Leu Gly Val Ser Thr Phe Ala Ser 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 Asn Gly Asn Gln565570 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 630 635 640
Ile 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 680685Val 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 His 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 750Ala Ser Ile Pro Ala Gln Val Lys Leu Gln Thr Phe Ile Arg Ser Lys755 760 765Leu Pro Ala Phe Thr Tyr His Cys Ile Phe Gly Thr Cys Leu Met Thr770 775 780Ala Cys Val Val Leu Arg Phe His 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 ttatctcttt60
ttaggtgttc 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 tgcttcttca agtcccgatg gtctggcgtt cattaatggc900actcggattt cagacgaaat aaccacatta gattggaata gttatctagt tcctcaatgg960ggggttataa taataaataa aatgccgttg aagccaaatt cagtcattag cgaagattat 1020ttagaaccta tgatgtaccg ttttgcgaca gatatttttc aactattggg attaacggag 1080ggctcgcaag 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 1380
gaaatggttc 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 Ala1 5 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 His Asp Ala Ile Gln Val Gln Val Asn65 70 75 80His Leu Leu Asn Ser 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 105 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 His
145 150 155160Thr Phe Gln Leu Glu Trp Thr His Leu Asn Lys Thr Cys Glu Gly Val165 170 175Ser 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 280 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 310 315 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 375 380
Asp 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 His Leu Ser Asn Glu435 440 445Leu Val Lys Leu Cys Glu Lys Ala Phe Phe Asn Gly Glu Met Val Gln450 455 460Gln Asn Phe Phe Pro Gln Glu His Met Ile Ala Val Tyr Leu Pro Leu465 470 475 480Leu Gly Pro Ile Ser Ala Val Met Phe 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 510Ala 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 ctacacgact240
ttcccacggg ccatcccacc attgttggaa tctactgcca cgcgtcagtt tcatttaaga300tttaccagag gattctggga tgccctgtcg tggggacagt tgccacatgc tggaaaagag360gcaggtgcct caggtgtgga attgtggtcg caagtgcagg ccatggatca ggaacaggcg420ttccataatt ggaaaaaact gtccaattca ttgagcggat 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 agaagttgac 1020cgttctacct ggactgcgtt tttgtcagtt gacgatacta ttttcagcac acatgataat 1080tgctttgact tatcaaacga tcaaaatgag ggtggttcgg gctacgactt tattttagaa 1140tcgacggaca ctactaaagt tactcccata gttcctgtcc caattcacgt aagcagatct 1200ctgactggta 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 1560
ctatctttgg 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 taa1833<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 15Val 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 His Tyr Thr Thr65 70 75 80Phe 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 His 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 His Asn Trp130 135 140
Lys Lys Leu Ser Asn Ser Leu Ser Gly Leu Phe Cys Ser Ser Leu Asn145 150 155 160Phe Ile Asp Glu Ser Arg Thr Thr Phe Pro Arg Arg Ser Tyr Ala 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 Cys Thr Glu Asn Leu Thr Pro195 200 205Phe Ile Lys Leu Leu Pro Thr Arg Gly Lys Ser Gly Leu Thr Ser Leu210 215220Leu 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 His 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 Cys Phe Pro Leu290 295 300Pro Glu Pro Ser Gln Thr His 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 330 335Ala Glu Val Asp Arg Ser Thr Trp Thr Ala Phe Leu Ser Val Asp Asp340 345 350Thr Ile 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 Asp Thr
370 375 380Thr Lys Val Thr Pro Ile Val Pro Val Pro Ile His Val Ser Arg Ser385 390 395 400Leu Thr Gly Asn Gly Gln Asp Arg Gly Gly Met Arg Ile Val Phe His405 410 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 Gln Glu Asn Asp Ile Ile Leu Asp Lys Tyr Tyr Leu450 455 460Gln Ala Ala Asp Arg Lys Arg Pro Gly His 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 570575Thr 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 Asp
610<210>40<211>1185<212>DNA<213>釀酒酵母(Saccharomyces cerevisiae)<400>40atggattcca cagcacttaa ggtagctcta ggctgtattg caattcgttt ggctgtgaac60agcctttttc cctctctaca acaacaactg gaccagtctg tagaattctc aactcccgta 120acttcattta ggtcactaca ggaaggtata tacctactgc ggaacaacat ccaagtatat 180aatcatgggg ttgttcaeca tcctccaatt ttgatttttt ttctttccct ctttaattcc 240gacaggttaa tttccctcat atacgcttta attgatggat taattgcgta tcagctgaca 300gaggtaacaa aggctttcaa aaacttgaaa ctgaaagttt ggctacctgg acttctttat 360gccgtgaatc ctttgaccct tttatcgtgc attagtcggt catcaatcat attcacaaat 420tttgctattt catcgtcatt gtattgcata ttagctgaag gaaacgttct tttgtcctct 480gttatgattt ctatatctgg atatttgtca gtatacccta ttctcctctt aattccgcta 540ttaggtatgc tgaaaagttg gaggcaaaga atattatctg ccattgtttc catactatct 600ttattaattc tgctattatt cagctacagt atattaggca gccaaagttg gtcatttttg 660acacaggttt atggatctat tataaccttt gagaaggttt ttccaaatct gggtttgtgg 720tggtacttct tcattgaaat gtttgacacc ttcataccgt tcttcaaggc tgtattcaac 780atttttattg cagtattcat tacaccattt actttgcgct atcataagca gccattctac 840gcattcattt tatgcattgg gtggattgtc cttacaaagc catatccctc actaggtgac 900gctggttttt tcttcagctt cctacctttc ttcacgccac tatttggata tttaagatac 960cccatcatat cagcattact gtttttacac gcaattgttt tggcgccaat tttctatcat 1020ctttgggttg ttttaggttc agggaatagt aattttttct atgctatttc cctagtttat 1080
gctctggcta tagcatctat attagttgac ttgaactggg cgatgctgag aattgaatac 1140gataacggta tcccaaattt caaattgaag gtaacacaaa tttaa 1185<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 gagagtgacc240aagaaggctc agtga 255<210>43<211>84<212>PRT<213>人(Homo sapiens)<400>43Met Ala Thr Gly Thr Asp Gln Val Val 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 gctcctcagg 60ggagcattgc ttccttctct cgcagtgacc atgacgaaat tagcgcagtg gctttgggga120ctagcgatcc tgggctccac ctgggtggcc ctgaccacgg gagccttggg cctggagctg180cccttgtcct gccaggaagt cctgtggcca ctgcccgcct acttgctggt gtccgccggc240tgctatgccc tgggcactgt gggctatcgt gtggccactt ttcatgactg cgaggacgcc300gcacgcgagc tgcagagcca gatacaggag gcccgagccg acttagcccg cagggggctg360cgcttctga369<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 15Leu 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 Arg
100 105 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 tttacctgag 60aaatgctacg 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>47
Met 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 Pro
225 230 235 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 acggg 25<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 attcacttat 30<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 tg 42
<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權利要求
1.(a)到(d)中任一項的DNA，該DNA編碼具有GlcN-PI酰基轉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)在嚴謹條件中與包含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，且其中添加、缺失、取代和/或插入一個或多個氨基酸而得的蛋白的DNA。
2.由權利要求1所述的DNA編碼的蛋白。
3.插入權利要求1所述的DNA的載體。
4.一種轉化體，其在可表達狀態包含權利要求1所述的DNA或者權利要求3所述的載體。
5.一種抗瘧藥物，其包含抑制權利要求2所述蛋白的活性的化合物作為活性成分。
6.權利要求5所述的抗瘧藥物，其中抑制權利要求2所述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)使權利要求2所述的蛋白與試樣和具有與該蛋白結合的活性的標記化合物接觸，(2)檢測與該蛋白結合的標記化合物，和(3)選出減少與該蛋白結合的標記化合物的量的試樣。
8.權利要求7所述的方法，其中具有與蛋白結合的活性的標記化合物是通過對選自權利要求6所述的化合物(1)-(5)中的至少一種進行標記而獲得的。
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)使試樣與權利要求2所述的蛋白接觸，(2)檢測GlcN-(酰基)PI，和(3)選出減少GlcN-(酰基)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)使試樣與過量表達權利要求2所述蛋白的細胞接觸，(2)測定GPI-錨定蛋白轉運到該細胞的細胞壁的量，和(3)選出減少步驟(2)中所測定的GPI-錨定蛋白轉運到該細胞的細胞壁的量的試樣。
11.一種治療瘧疾的方法，包括施用抑制權利要求2所述蛋白的活性的化合物。
12.權利要求11所述的方法，其中抑制權利要求2所述蛋白的活性的化合物是權利要求5所述的化合物。
13.一種編碼具有補償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的簡并突變體，其具有少于70％的AT含量。
15.權利要求13或14所述的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中奇數序列鑒定號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中偶數序列鑒定號中任一所示核苷酸序列的DNA，(c)在嚴謹條件下與包含SEQ ID NOs1和3以及SEQ ID NOs6-47中偶數序列鑒定號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中奇數序列鑒定號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/或插入一個或多個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，其中插入權利要求13-16中任一的DNA。
18.在可表達狀態包含權利要求13-16中任一DNA或者權利要求17所述載體的轉化體。
19.權利要求18所述的轉化體，其為GPI合酶基因-缺陷型真菌。
20.權利要求18所述的轉化體，其為GPI合酶基因-缺陷型酵母。
21.一種制備由權利要求13-16中任一DNA編碼的蛋白的方法，該方法包括培養權利要求18-20中的任一轉化體，并從該轉化體或其培養上清液收集表達的蛋白的步驟。
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)將試樣與表達權利要求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，和(3)選出抑制該真菌生長的被試化合物。
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)將試樣與表達權利要求13-16中任一DNA的GPI合酶基因-缺陷型真菌接觸，(2)檢測GPI-錨定蛋白轉運到真菌細胞壁的量，和(3)選出使步驟(2)中所檢測的GPI-錨定蛋白轉運到真菌細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)將權利要求13-16中任一DNA導入GPI合酶基因-缺陷型真菌，并使該DNA編碼的蛋白表達，(2)獲得步驟(1)中表達的蛋白，(3)使所得蛋白與試樣和具有與該蛋白結合的活性的標記化合物接觸，(4)檢測與該蛋白結合的標記化合物，和(5)選出減少與該蛋白結合的標記化合物的量的試樣。
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簡并突變體且表達由該簡并突變體DNA編碼的蛋白的載體，導入GWT1-缺陷型真菌，(2)獲得步驟(1)中表達的蛋白，(3)使所得蛋白與試樣接觸，(4)檢測GlcN-(酰基)PI，和(5)選出減少GlcN-(酰基)PI水平的被試化合物。
全文摘要
本發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，本發明人揭示比編碼PfGWT1蛋白的DNA具有更低AT含量的簡并突變體DNA，能補償GWT1－缺陷型酵母的表型。在此發現的基礎上，本發明提供了瘧原蟲的GWT1蛋白，以及該蛋白在篩選抗瘧藥物的方法中的用途。本發明還提供了編碼參有GPI生物合成的蛋白的簡并突變體DNA，該DNA比天然DNA具有更低的AT含量。本發明還提供了利用該簡并突變體DNA篩選抗瘧藥物的方法。
文檔編號C12N15/09GK1742203SQ20038010910
公開日2006年3月1日 申請日期2003年11月21日 優先權日2002年11月22日
發明者畑桂, 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 申請人:衛材株式會社