筛选在疟原虫中抑制gpi生物合成的化合物的方法

专利名称：筛选在疟原虫中抑制gpi生物合成的化合物的方法
技术领域：
本发明涉及筛选在疟原虫中抑制GPI生物合成的化合物的方法。
背景技术：
疟疾是由寄生性原虫引起的最常见的传染性人类疾病。该疾病由于感染疟原虫所致，以蚊子，冈比亚疟纹(Anopheles gambiae)为媒介。每年约有50亿病例感染疟疾，包括超过两百万的致死例(Gardner等，Nature419498-511，2003)。目前，40％的世界人口生活在疟疾流行区，在该区域据说每3名新生儿中就有一名死于疟疾。
已知糖基磷脂酰肌醇(GPI)在寄生虫生长和感染过程中起着重要的作用，所述寄生虫包括疟原虫。在这些寄生虫的细胞表面上有许多GPI-锚定蛋白。GPI-锚定蛋白包括如在寄生虫侵入红细胞时起作用的MSP-1蛋白。GPI-锚定蛋白起寄生虫抗原的作用，在宿主中引发免疫应答。因此，它们长期以来是疫苗开发的研究对象。
GPI不仅作为将蛋白拴到细胞膜的锚，而且是疟原虫细胞膜的丰富糖脂成分。最近的研究揭示GPI有毒，并能促成致死症状。GPI诱导炎性细胞因子如TNF-α和粘附分子的表达。结果，受染红细胞粘附毛细管，阻塞血管(隔离作用(sequestration))，尤其是脑血管。它能引起进一步的炎症反应，而炎症反应认为能导致脑病。最近，Schofield等报道抗-GPI抗体降低利用鼠科疟原虫伯氏疟原虫(Plasmodium berghei)创建的体内感染模型系统的致死率，并且该抗体在体外抑制由恶性疟原虫(Plasmodium falciparum)引起的后期炎症反应(Schofield L等，Nature 418785-789，2002)。此发现说明GPI是疟疾感染致死率的主要因素。
还报道肌醇酰基化对于甘露糖与GPI的结合来说是必需的(Gerold，P.等，Biochem.J.344731-738，1999)，由过量葡糖胺引起的肌醇酰基化的抑制会抑制恶性疟原虫的成熟(Naik，R.S.等，J.Biol.Chem.2782036-2042，2003)。因此，选择地抑制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疟药物。
发明的公开本发明的目的是提供抑制GPI生物合成的抗疟药物。更具体地说，本发明提供了编码GWT1蛋白的疟原虫DNA，所述蛋白是参与GPI生物合成的蛋白质(GPI合酶)。本发明还提供了在筛选抗疟药物的方法中利用该DNA的方法。本发明还提供了编码疟原虫GPI生物合成蛋白的DNA的简并突变体DNA。该简并突变体DNA比天然DNA具有更低的AT含量。本发明还提供了在筛选抗疟药物的方法中利用该简并突变体DNA的方法。
最早发现GWT1基因编码真菌GPI-锚定蛋白合酶(WO 02/04626)，其在从酵母到人类的生物体中是保守的。本发明人证实GWT1同源物(恶性疟原虫(P.falciparum)GWT1的PfGWT1；约氏疟原虫(P.yoelii yoelii)GWT1的PyGWT1)包括在恶性疟原虫和约氏疟原虫的全基因组序列内(Gardner等，Nature 419498-511，2003；Carlton等，Nature 419512-519，2003)。本发明人还发现GWT1基因产物在GPI生物合成途径中起GlcN-PI酰基转移酶的作用。PfGWT1基因产物期望具有相似的活性，这样抑制其活性的化合物就可能是有价值的抗疟药物。
在WO 02/04626中，本发明人发现了一组抑制真菌GWT1基因产物的活性的化合物。抑制PfGWT1基因产物的活性的化合物有望成为抗疟药物。
在本发明中，本发明人成功地对几乎是PfGWT1全长的区域进行了分离。利用疟原虫如恶性疟原虫的GWT1基因产物，抗疟药物的筛选可以通过结合试验，葡糖胺(酰基)磷脂酰肌醇(PI-GlcN)酰基转移酶试验，或利用GPI-锚定蛋白检测系统来进行。从所述筛选方法获得的化合物可能是有价值的抗疟药物。更进一步地，本发明人揭示简并突变体DNA(编码疟原虫GPI生物合成蛋白的DNA简并突变体)，该DNA比天然DNA具有更低AT含量，补偿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为指标，该真菌中导入了(比疟原虫中编码GPI生物合成蛋白的DNA)具有更低AT含量的简并突变体DNA，就有可能筛选出抑制在疟原虫中参与GPI生物合成的蛋白活性的化合物。
更具体地，本发明提供了下述[1]到[25][1](a)到(d)中任一项的DNA，该DNA编码具有GlcN-PI酰基转移酶活性的疟原虫蛋白。
(a)编码包含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)在严谨条件与包含SEQ ID NO1或3所示核苷酸序列的DNA杂交的DNA，和(d)编码包含SEQ ID NO2或4所示氨基酸序列，且其中已添加、缺失、取代和/或插入一个或多个氨基酸所得的蛋白的DNA；[2]由项[1]的DNA编码的蛋白；[3]插入项[1]的DNA的载体；[4]在可表达状态包含项[1]的DNA或者项[3]的载体的转化体；[5]一种抗疟药物，其包含抑制项[2]的蛋白的活性的化合物作为活性成分；[6]项[5]的抗疟药物，其中抑制项[2]的蛋白的活性的化合物是选自下述化合物(1)-(5)中的至少一种化合物
和 [7]一种筛选具有抗疟活性的化合物的方法，其包括下述步骤(1)使项[2]的蛋白与试样和具有与该蛋白结合的活性的标记化合物接触，(2)检测与该蛋白结合的标记化合物，和(3)选出减少与该蛋白结合的标记化合物的量的试样；[8]项[7]的方法，其中具有与蛋白结合的活性的标记化合物是通过对选自项[6]的化合物(1)-(5)中的至少一种进行标记而获得的；[9]一种筛选具有抗疟活性的化合物的方法，其包括下述步骤(1)使试样与项[2]的蛋白接触，(2)检测GlcN-(酰基)PI，和(3)选出减少GlcN-(酰基)PI水平的试样；[10]一种筛选具有抗疟活性的化合物的方法，其包括下述步骤(1)使试样与过量表达项[2]的蛋白的细胞接触，(2)测定GPI-锚定蛋白转运到该细胞的细胞壁的量，和
(3)选出减少步骤(2)中所测定的GPI-锚定蛋白转运到该细胞的细胞壁的量的试样；[11]一种治疗疟疾的方法，包括施用抑制项[2]的蛋白的活性的化合物；[12]项[11]的方法，其中抑制项[2]的蛋白的活性的化合物是项[5]的化合物；[13]一种编码具有补偿GPI合酶基因-缺陷型酵母表型的活性的蛋白的DNA，该DNA是编码在疟原虫中参与GPI生物合成的蛋白的DNA的简并突变体，其比天然DNA具有更低的AT含量；[14]一种编码具有补偿GPI合酶基因-缺陷型酵母表型的活性的蛋白的DNA，该DNA是编码在疟原虫中参与GPI生物合成的蛋白的DNA的简并突变体，其具有少于70％的AT含量；[15]项[13]或[14]的DNA，其中该DNA选自下组(a)编码包含SEQ ID NOs2和4以及SEQ ID NOs6-47中奇数序列鉴定号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中偶数序列鉴定号中任一所示核苷酸序列的DNA，(c)在严谨条件下与包含SEQ ID NOs1和3以及SEQ ID NOs6-47中偶数序列鉴定号中任一所示核苷酸序列的DNA杂交的DNA，和(d)编码包含SEQ ID NOs2和4以及SEQ ID NOs6-47中奇数序列鉴定号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/或插入一个或多个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]一种载体，其中插入项[13]-[16]中任一的DNA；[18]在可表达状态包含项[13]-[16]中任一DNA或者项[17]的载体的转化体；[19]项[18]的转化体，其为GPI合酶基因-缺陷型真菌；[20]项[18]的转化体，其为GPI合酶基因-缺陷型酵母；[21]一种制备由项[13]-[16]中任一DNA编码的蛋白的方法，该方法包括培养项[18]-[20]中的任一转化体，并从该转化体或其培养上清液收集表达的蛋白的步骤；[22]一种筛选具有抗疟活性的化合物的方法，该方法包括以下步骤
(1)将试样与表达项[13]-[16]中任一DNA的GPI合酶基因-缺陷型真菌接触；(2)分析该真菌的生长，和(3)选出抑制该真菌生长的被试化合物；[23]一种筛选具有抗疟活性的化合物的方法，该方法包括以下步骤(1)将试样与表达项[13]-[16]中任一DNA的GPI合酶基因-缺陷型真菌接触，(2)检测GPI-锚定蛋白转运到真菌细胞壁的量，和(3)选出使步骤(2)中所检测的GPI-锚定蛋白转运到真菌细胞壁的量减少的试样，[24]一种筛选具有抗疟活性的化合物的方法，该方法包括以下步骤(1)将项[13]-[16]中任一DNA导入GPI合酶基因-缺陷型真菌，并使该DNA编码的蛋白表达，(2)获得步骤(1)中表达的蛋白，(3)使所得蛋白与试样和具有与该蛋白结合的活性的标记化合物接触，(4)检测与该蛋白结合的标记化合物，和(5)选出减少与该蛋白结合的标记化合物的量的试样；和[25]一种筛选具有抗疟活性的化合物的方法，该方法包括以下步骤(1)将(i)编码具有补偿GWT1-缺陷型酵母表型的活性的蛋白的DNA，其中所述DNA是编码疟原虫GWT1蛋白的DNA的简并突变体，该DNA比天然DNA具有更低的AT含量，或者(ii)插入DNA简并突变体且表达由该简并突变体DNA编码的蛋白的载体，导入GWT1-缺陷型真菌，(2)获得步骤(1)中表达的蛋白，(3)使所得蛋白与试样接触，(4)检测GlcN-(酰基)PI，和(5)选出减少GlcN-(酰基)PI水平的被试化合物。
本发明第一次分离出了编码恶性疟原虫GWT1蛋白的DNA(PfGWT1)。编码PfGWT1蛋白的DNA的核苷酸序列示于SEQ ID NO1，PfGWT1蛋白的氨基酸序列显示于SEQ ID NO2。另外，编码间日疟原虫(Plasmodium vivax)GWT1蛋白(PvGWT1)的DNA的核苷酸序列示于SEQ ID NO3，PvGWT1蛋白的氨基酸序列显示于SEQ ID NO4。
GWT1蛋白参与糖基磷脂酰肌醇(GPI)的生物合成，糖基磷脂酰肌醇对疟原虫的生长和感染性是必需的。因此，抑制疟原虫GWT1蛋白活性的化合物可以用作抗疟药物。所述抗疟药物可以利用此疟原虫GWT1蛋白来进行筛选。
本发明提供了编码疟原虫GWT1蛋白的DNA。所述DNA包括编码包含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。
本发明还提供了与包含SEQ ID NO2或4氨基酸序列的蛋白功能等同的蛋白的编码DNA。在此，表述“功能等同”是指具有与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酰基转移酶活性。GlcN-PI酰基转移酶活性可采用Costello和Orlean(J.Biol.Chem.(1992)2678599-8603)或Franzot和Doering(Biochem.J.(1999)34025-32)所报道的方法来进行测定。
与包含SEQ ID NO2或4氨基酸序列的蛋白功能等同的蛋白的编码DNA包括在严谨条件中与包含SEQ ID NO1或3核苷酸序列的DNA杂交的DNA，和编码包含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/或插入一个或多个氨基酸。
本发明的DNA可以采用本领域技术人员所熟知的方法来进行分离。所述方法的实例包括利用杂交(Southern E.M.，J.Mol.Biol.98503-517，1975)和聚合酶链式反应(PCR)(Saiki R.K.等，Science 2301350-1354，1985；SaikiR.K.等，Science 239487-491，1988)。更具体地，对于本领域技术人员来说，利用SEQ ID NO1或3的DNA或其部分作为探针，或通过利用与包含SEQID NO1或3核苷酸序列的DNA特异性杂交的DNA作为引物，从疟原虫中分离出与包含SEQ ID NO1或3核苷酸序列的DNA高度同源的DNA是常规实验。另外，通过杂交或PCR技术分离出的DNA，以及与包含SEQ IDNO1或3核苷酸序列的DNA杂交的DNA也包括在本发明的DNA之中。所述DNA包括含有SEQ ID NO2或4氨基酸序列的蛋白的疟原虫同源物的编码DNA。疟原虫同源物包括恶性疟原虫，间日疟原虫，三日疟原虫(Plasmodium malariae)和卵形疟原虫(Plasmodium ovale)同源物，其包含SEQID NO2或4的氨基酸序列。
优选，在严谨杂交条件利用杂交反应分离出上述DNA。如本文所用，表达“严谨杂交条件”是指，例如在65℃于4xSSC中进行杂交，然后在65℃于0.1xSSC中洗涤1小时。可选的严谨条件是在42℃于含50％甲酰胺的4xSSC中进行杂交。或者，在65℃于PerfectHybTM(TOYOBO)溶液中杂交2.5小时，然后如下洗涤(1)在25℃，于含0.05％SDS的2xSSC中洗涤5分钟；(2)在25，于含0.05％SDS的2xSSC中洗涤15分钟；及(3)在50℃，于含0.1％SDS的0.1x SSC中洗涤20分钟。如此分离的DNA有望编码与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”是指与完整氨基酸序列有至少70％或更高的序列同一性，优选80％或更高，更优选90％或更高，最优选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)来确定。已开发了基于BLAST算法的程序(称为BLASTN和BLASTX)(Altschul S.F.等，J.Mol.Biol.215403，1990)。当利用BLASTN对核苷酸序列进行分析时，例如，将参数设为得分＝1 00，字长＝12。另外，当利用BLASTX对氨基酸序列进行分析时，例如，将参数设为得分＝50，字长＝3。当使用BLAST和Gapped BLAST程序时，使用各程序的默认参数。所述分析的具体步骤是已知的(请参见美国国家生物技术信息中心(NationalInstitute of Biotechnology Information)网站http://www.ncbi.nlm.nih.gov)。
本发明的DNA包含基因组DNA、cDNA和化学合成DNA。基因组DNA或cDNA可以根据本领域技术人员所熟知的常规方法来加以制备。例如，基因组DNA可以如下制备(i)提取疟原虫基因组DNA；(ii)(利用如质粒、噬菌体、粘粒、BAC或PAC作载体)构建基因组文库；(iii)涂布该文库；然后(iv)利用探针进行菌落杂交或噬菌体杂交，所述探针是在编码本发明的疟原虫GWT1蛋白的DNA(例如SEQ ID NO1或3)的基础上获得的。另外，利用编码本发明疟原虫GWT1蛋白的DNA(例如SEQ ID NO1或3)的特异性引物，通过PCR制备基因组DNA。另一方面，cDNA可以如下制备(i)在提取的疟原虫mRNA基础上合成cDNA；(ii)将该合成cDNA插入载体如λZAP中构建cDNA文库；(iii)涂布该cDNA文库；和(iv)如上所述，进行菌落杂交或噬菌体杂交。另外，也可以利用PCR制备eDNA。
本发明还提供了与包含SEQ ID NO2或4氨基酸序列的蛋白结构相似的蛋白的编码DNA。所述DNA包括那些编码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/或添加一个或多个氨基酸残基的氨基酸序列。对于上述蛋白中的氨基酸突变位点和数量并无限制，只要该突变蛋白仍保留原始蛋白的功能，如Mark，D.F.等，Proc.Natl.Acad.Sci.USA(1984)81，5662-5666；Zoller，M.J.&Smith，M.，Nucleic Acids Research(1982)10，6487-6500；Wang，A.等，Science 224，143 1-1433；Dalbadie-McFarland，G.等，Proc.Natl.Acad.Sci.USA(1982)79，6409-6413中所述。突变氨基酸的百分数一般为全部氨基酸残基的10％或更少，优选5％或更少，更优选1％或更少。此外，突变氨基酸的数量通常为30个氨基酸或更少，优选15个氨基酸或更少，更优选5个氨基酸或更少，还更优选3个氨基酸或更少，甚至更优选2个氨基酸或更少。
优选的突变氨基酸残基是在其突变(已知为保守氨基酸取代法)后保持侧链性质的氨基酸残基。氨基酸侧链性质的例子是疏水性(A，I，L，M，F，P，W，Y，V)和亲水性(R，D，N，C，E，Q，G，H，K，S，T)。侧链包括脂肪族侧链(G，A，V，L，I，P)；含羟基侧链(S，T，Y)；含硫原子侧链(C，M)；含羧酸和酰胺侧链(D，N，E，Q)；碱性侧链(R，K，H)；和芳香族侧链(H，F，Y，W)。
包含疟原虫GWT1蛋白的融合蛋白是其中添加一个或多个氨基酸残基的蛋白实例。融合蛋白可利用本领域技术人员所熟知的技术来进行制备。例如，对此特定技术没有限制，将编码本发明疟原虫GWT1蛋白的DNA与编码另一肽或蛋白的DNA联合，使它们的阅读框匹配。本发明的蛋白可以与许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-标签，HSV-标签，E-标签，SV40T抗原片段，lck标签，α-微管蛋白片段，B-标签和蛋白C(Protein C)片段。与本发明蛋白融合的蛋白例子包括谷胱甘肽-S-转移酶(GST)，HA，免疫球蛋白恒定区，β-半乳糖苷酶和麦芽糖结合蛋白(MBP)。
除利用上述杂交和PCR技术外，本领域技术人员可通过下述方法制得上述DNA，该方法包括例如定点诱变而在该DNA中引入突变(Kramer W.and Fritz H-J.，Methods Enzymol.154350，1987)。蛋白的氨基酸序列还可以由于编码该蛋白的核苷酸序列发生突变而使其自然突变。此外，简并突变体DNA，其核苷酸突变并不会引起蛋白中的氨基酸发生突变(简并突变体)也包括在本发明内。另外，本发明还包括由本发明上述DNA编码的蛋白。
本发明提供了包括本发明DNA的载体，包含本发明DNA或载体的转化体，和利用这些转化体制备本发明蛋白的方法。
本发明的载体并无限制，只要它能稳定地保留插入载体内的DNA。例如，当大肠杆菌作宿主时，优选用pBluescript载体(Stratagene)作为克隆载体。当使用载体来制备本发明的蛋白时，表达载体是特别有用的。表达载体并无特别的限制，只要它能在体外，在大肠杆菌中，在培养细胞中和在体内表达蛋白。表达载体的优选例子包括用于体外表达的pBEST载体(Promega Corporation)，用于在大肠杆菌中表达的pET载体(Novagen)，用于在培养细胞中表达的pME18S-FL3载体(GenBank登录号AB009864)，和用于体内表达的pME18S载体(Mol.Cell Biol.8466-472，1988)。可采用常规方法将本发明的DNA插入载体内，例如采用限制性酶位点进行连接酶反应(Current Protocols in Molecular Biology，Ausubel等编辑，John Wiley&Sons，Inc.1987，Section 11.4-11.11)。
导入本发明载体的宿主细胞并无特别地限制，并且可以根据发明目的使用各种宿主细胞。例如，用于表达蛋白的细胞包括但不限于细菌细胞(如链球菌(Streptococcus)，葡萄球菌(Staphylococcus)，大肠杆菌，链霉菌(Streptomyces)，枯草杆菌(Bacillus subtilis)，真菌细胞(如酵母，曲霉(Aspergillus))，昆虫细胞(如果蝇(Drosophila)S2，夜蛾(Spodoptera)SF9)，动物细胞(如CHO，COS，HeLa，C127，3T3，BHK，HEK293，Bowes黑素瘤细胞)，以及植物细胞。将载体转染入宿主细胞可以采用常规方法进行，如磷酸钙沉淀，电穿孔(Current protocols in Molecular Biology，Ausubel等编辑，John Wiley&Sons，Inc.1987，Section 9.1-9.9)，Lipofectamine法(GIBCO-BRL)和显微注射。
通过将合适的分泌信号掺入目的蛋白内，可以将在宿主细胞中表达的蛋白分泌到内质网的腔，细胞周围空腔(cavity)或细胞外环境内。这些信号对于目的蛋白来讲可以是内源的或外源的。
当本发明的蛋白分泌到培养基中时，从该培养基中收集蛋白。如果本发明的蛋白是在细胞内产生的，就将该细胞裂解，然后收集蛋白。
利用本领域熟知的方法可从重组的细胞培养物中对本发明蛋白进行收集和纯化，所述方法包括但不限于，硫酸铵或乙醇沉淀，酸提取，阴离子或阳离子交换层析，磷酸纤维素层析，疏水作用层析，亲和层析，羟磷灰石层析和外源凝集素(lectin)层析。
包括本发明DNA的化合物是分离的化合物。在此，术语“分离的”是指从原来的环境中分离出来(例如，如果是自然存在，即为天然环境)。样品中的目的化合物非常丰富，和/或样品中的目的化合物是部分或基本纯化的，则该样品中的化合物是“分离的”化合物。在本文中，术语“基本纯化的”是指化合物从天然环境分离的状态，而且该状态中已除去了至少60％，优选75％，最优选90％的其它共存的天然成分。
本发明提供了抗疟药物，该药物抑制疟原虫GWT1基因产物的活性。抑制疟原虫GWT1基因产物活性的优选化合物是WO 02/04626所述的化合物，和包括化合物(1)至(5) 化合物(1)1-(4-丁基苄基)异喹啉 化合物(2)4-[4-(1-异喹啉基甲基)苯基]-3-丁炔-1-醇 化合物(3)5-丁基-2-(1-异喹啉基甲基)苯酚
化合物(4)2-(4-溴-2-氟苄基)-3-甲氧基吡啶 化合物(5)N-[2-(4-丁基苄基)-3-吡啶基]-N-甲胺象这样，抑制疟原虫GWT1基因产物的活性的化合物或其盐或其水合物可以给药于哺乳动物(优选人)。还可以利用常规方法将它配制成片剂，粉剂，微粒剂，颗粒剂，包衣片剂，胶囊，糖浆，锭剂，吸入剂，栓剂，注射剂，软膏剂，眼膏，滴眼剂，滴鼻剂，滴耳剂，糊剂，洗剂等，然后给药。
为配制药物，可以使用药物制剂常用的助剂(例如填充剂，粘合剂，滑润剂，着色剂，调味剂，以及需要时的稳定剂，乳化剂，吸收促进剂，表面活性剂，pH调节剂，防腐剂和抗氧化剂)。药物制剂可以用常规方法，通过将通常用作药剂成分的各组分混合来制备。
例如，口服制剂可以如下制备将本发明的化合物或其药学上可接受的盐与填充剂，必要时还有粘合剂，崩解剂，润滑剂，着色剂，调味剂等组合，并通过常规方法将该混合物配制成粉剂，微粒剂，颗粒剂，片剂，包衣片剂，胶囊等。
这些组分的例子包括动物脂肪和植物油，如大豆油，牛脂(tallow)，以及合成甘油酯；烃，如液体石蜡，角鲨烯，以及固体石蜡；酯油，如肉豆蔻酸辛基十二醇酯和肉豆蔻酸异丙酯；高级醇，如十六醇十八醇混合物以及二十二醇；硅氧烷树脂；硅油；表面活性剂，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，如羟乙基纤维素，聚丙烯酸，羧基乙烯基聚合物，聚乙二醇，聚乙烯基吡咯烷酮，以及甲基纤维素；低级醇，如乙醇和异丙醇；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；无机粉末，如硅酸酐，硅酸铝镁，以及硅酸铝；和纯水。填充剂的实例子包括乳糖，玉米淀粉，精制白糖，葡萄糖，甘露醇，山梨醇，结晶纤维素，以及二氧化硅。粘合剂的例子包括聚乙烯醇，聚乙烯醚，甲基纤维素，乙基纤维素，阿拉伯树胶，黄芪胶，明胶，虫胶，羟丙基甲基纤维素，羟丙基纤维素，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。崩解剂的例子包括淀粉，琼脂，粉状明胶，结晶纤维素，碳酸钙，碳酸氢钠，柠檬酸钙，糊精，果胶，以及羧甲基纤维素钙。润滑剂是硬脂酸镁，滑石，聚乙二醇，二氧化硅，硬化的植物油等。着色剂的例子为那些允许加入药物中的着色剂。调味剂是可可粉，l-薄荷醇，芳香分散剂，薄荷油，冰片，肉桂粉等。对于这些片剂和颗粒剂当然可以使用糖衣，必要时还可以使用其它适宜的包衣。
此外，液体制剂如糖浆和注射剂可以采用常规方法来制备。在该方法中，通过将pH调节剂，增溶剂，等渗剂等，以及必要时的增溶助剂，稳定剂等，加至本发明的化合物或其药学上可接受的盐中，来制备。
制备外用制剂的方法没有限制，而且可以通过常规方法来制备。也就是说，用于制剂的基础材料可以选自药物、准药物(quasi-drug)、化妆品等常用的各种材料。具体地，可使用的基础材料是，如动物脂肪和植物油，矿物油，酯油，蜡，高级醇，脂肪酸，硅油，表面活性剂，磷脂，醇，多元醇，水溶性大分子，粘土矿和纯化水。必要时，可以添加pH调节剂，抗氧化剂，螯合剂，防腐剂和抗真菌剂，着色物质，香料等。但是本发明的外用制剂的基础材料并不限于此。再者，如果需要，还可以混入具有分化诱导作用的组分，血流促进剂，杀真菌剂，消炎剂，细胞活化剂，维生素，氨基酸，保湿剂，以及角质裂解剂。上述基础材料按外用制剂常用浓度的量加入。
如本发明所述，术语“盐”优选包括，如与无机或有机酸的盐，与无机或有机碱的盐，或与酸性或碱性氨基酸的盐。尤其优选是药学上可接受的盐。酸和碱以0.1-5个酸或碱分子与1个化合物分子的适当比形成盐。
与无机酸的盐的优选例子是与盐酸，氢溴酸，硫酸，硝酸和磷酸的盐。与有机酸的盐优选包括与醋酸，琥珀酸，延胡索酸，马来酸，酒石酸，柠檬酸，乳酸，硬脂酸，苯甲酸，甲磺酸，对甲苯磺酸的盐。
与无机碱的盐的优选例子是碱金属盐如钠盐和钾盐；碱土金属盐如钙盐和镁盐；铝盐，和铵盐。与有机碱的盐优选包括与二乙胺，二乙醇胺，甲葡胺和N，N’-二苄基乙二胺的盐。
与酸性氨基酸的盐的优选例子是与天冬氨酸和谷氨酸的盐，与碱性氨基酸的盐优选包括与精氨酸，赖氨酸和鸟氨酸的盐。
施用本发明的化合物或其盐或其水合物时，对其形式没有特别的限制，它们可以根据常规方法经口或经非胃肠道途径给药。它们可以配制成如片剂，粉剂，微粒剂，胶囊，糖浆，锭剂，吸入剂，栓剂，注射剂，软膏剂，眼膏，滴眼剂，滴鼻剂，滴耳剂，糊剂和洗剂剂型。本发明的药物组合物的剂量可以根据症状程度，患者年龄，性别和体重，剂型，盐的类型，疾病的具体类型等适当地选择。
将治疗有效量的本发明的化合物给药于患者。在此，“治疗有效量”是指产生所需药理效果并且有效地恢复或或缓解所要治疗的患者之症状的药剂量。所述剂量根据疾病的类型，症状的程度，患者的体重、年龄、性别、对制剂的敏感性而显著地不同。但通常，成人的日剂量为约0.03mg-1000mg，优选0.1mg-500mg，更优选0.1mg-100mg，并且每日给药一次或几次，或者每若干天给药一次或几次。注射剂量一般为约1-3000μg/kg，优选约3-1000μg/kg。
此外，本发明涉及利用疟原虫GWT1基因产物筛选抗疟药物的方法。所述筛选方法包括但不限于[1]结合试验，该试验筛选与标记化合物竞争结合疟原虫GWT1基因产物的化合物；[2]GlcN-PI酰基转移酶分析系统，该系统筛选能抑制疟原虫GWT1基因产物的GlcN-PI酰基转移酶活性的化合物；和[3]GPI-锚定蛋白检测系统，其中在细胞，优选真菌细胞中表达疟原虫GWT1基因产物，然后检测细胞表面上的GPI-锚定蛋白。本发明并不限于这些方法，而且包括利用疟原虫GWT1基因产物筛选抗疟药物的任何方法。上面列出的方法[1]-[3]在下面进行详细描述。
筛选与标记化合物竞争结合疟原虫GWT1基因产物的化合物的结合试验本发明的两种方法公开如下，即(1)制备疟原虫GWT1基因产物(下文中称为疟原虫GWT1蛋白)的方法和(2)涉及标记化合物的结合实验法(下文中称为结合试验)
(1)制备疟原虫GWT1蛋白的方法疟原虫GWT1蛋白是从细胞膜级分，优选从真菌细胞，更优选从酿酒酵母(S.cerevisiae)细胞中制得的，其中引入了SEQ ID NO2的疟原虫GWT1蛋白的编码DNA。优选将所述DNA导入GWT1基因-缺陷型细胞中。在结合试验中，可以在未进行任何其它处理的条件下使用所制的膜级分，或者在使用前对膜级分进行进一步的纯化。利用酿酒酵母的方法将在下面进行详细描述。
(a)疟原虫GWT1基因的导入本发明使用的疟原虫GWT1基因可以是天然存在的基因，或优选是基于SEQ ID NO2或4的氨基酸序列进行合成的。疟原虫GWT1基因富含腺嘌呤和胸腺嘧啶。因此，可以推断利用常规的基因重组技术将很难对该基因进行操作，且该基因在酵母，细胞等中的表达效率将会很低(inefficient)。因此，优选对核苷酸序列进行设计，将其中与每个相应氨基酸对应的密码子替换为那些认为可以在酵母，细胞等中能有效表达的密码子，在该设计序列的基础上进行DNA合成，制备人工的疟原虫GWT1基因，然后在下述实验中使用该基因。
疟原虫GWT1的表达质粒是通过将疟原虫GWT1基因插入到酿酒酵母表达载体内来加以制备的，例如，通过将适合的启动子和终止子，如pKT10-衍生的GAPDH启动子和GAPDH终止子，插到YEp352的多克隆位点内来制备表达载体(Tanaka等，Mol.Cell Biol.，104303-4313，1990)。将酿酒酵母(如G2-10菌株)培养在合适的培养基(如YPD培养基(酵母提取物-聚蛋白胨-葡萄糖培养基))中，并在适宜的温度(如30℃)振荡，在对数生长晚期收获细胞。洗涤后，采用如乙酸锂法将GWT1-表达质粒导入酿酒酵母细胞中。该方法描述于User Manual of YEAST MAKERTMYeast Transformation System(BD Biosciences Clontech)。疟原虫GWT1-过表达菌株和携带阴性对照载体的菌株可通过将转化细胞于30℃，在SD(ura-)培养基上培养两天而获得。
适合除酿酒酵母之外的真菌种类的表达载体和基因转移法见下述报道适合粟酒裂殖酵母(Schizosaccharomyces Pombe，S.pombe)的表达载体如pcL和其转移法由Igarashi等描述(Nature 35380-83，1991)，适合白色念珠菌(C.Albicans)的表达载体如pRM10和其转移法由Pla J.等描述(Yeast，121677-1702，1996)；适合烟曲霉(A.Fumigatus)的表达载体如pAN7-1和其转移法由Punt P.J.等描述(GENE，56117-124，1987)；适合新生隐球菌(C.Neoformans)的表达载体如pPM8及其转化法由Monden P.等描述(FEMSMicrobiol.Lett.，18741-45，2000)。
(b)制备膜级分的方法在适合的培养基(如SD(ura-)液体培养基)中培养，并在适合的温度(如30℃)振荡导入了疟原虫GWT1基因的酿酒酵母细胞。在对数生长中期时收获真菌细胞，然后用合适量(如真菌细胞的3倍体积)的匀浆缓冲液(如50mMTris-HCl，pH 7.5，10mM EDTA，CompleteTM(Roche))悬浮。将合适量的玻璃珠(如真菌细胞的4倍体积)加至悬浮液中。涡旋混合物，并置于冰上。重复此操作数次将真菌细胞破碎。
将1毫升的匀浆缓冲液加入所得的裂解液中。离心混合物；如2,500rpm离心5分钟，使玻璃珠和未碎的真菌细胞沉淀。将上清液移到另一试管内。离心该试管，如13,500rpm离心10分钟，使含有细胞器的膜级分(总膜级分)沉淀。用1ml结合缓冲液(如0.1M磷酸盐缓冲液，pH 7.0，0.05％Tween 20，CompleteTM(Roche))悬浮沉淀物，然后离心，如2,500rpm离心1分钟，除去未悬浮的物质。然后离心上清液，如15,000rpm离心5分钟。沉淀物用150-650μl的结合缓冲液重悬浮来制备膜级分。
采用适合粟酒裂殖酵母的Yoko-o等的方法(Eur.J.Biochem.257630-637，1998)；适合白色念珠菌的Sentandreu M等的方法(J.Bacteriol.，180282-289，1998)；适合烟曲霉的Mouyna I等的方法(J.Biol.Chem.，27514882-14889，2000)；和适合新生隐球菌的Thompson JR等的方法(J.Bacteriol.，181444-453，1999)可以从除酿酒酵母之外的真菌物种中制备膜级分。
另外，可以通过在大肠杆菌，昆虫和哺乳动物细胞或其他非真菌细胞中表达来制备疟原虫GWT1蛋白。
当使用哺乳动物细胞时，将疟原虫GWT1基因与含有如CMV启动子的过表达载体连接，导入哺乳动物细胞中。然后，按照Petaja-Repo等的方法(J.Biol.Chem.，2764416-23，2001)制备膜级分。
表达疟原虫GWT1基因的昆虫细胞(如Sf9细胞)可以利用，例如杆状病毒表达试剂盒如BAC-TO-BAC杆状病毒表达系统(Invitrogen)来进行制备。然后，按照Okamoto等的方法(J.Biol.Chem.，276742-751，2001)制备膜级分。
可通过下述步骤从大肠杆菌制备疟原虫GWT1蛋白例如将疟原虫GWT1基因与大肠杆菌表达载体如pGEX载体(Amersham Biosciences Corp.)连接，将此构建体导入大肠杆菌如BL21中。
(2)结合分析法(a)标记化合物的合成标记化合物从已证实结合GWT1蛋白的化合物制备。与GWT1蛋白结合的任何化合物都是可以使用的。标记化合物优选从WO 02/04626中所述的化合物制备，更优选从上述(1)至(5)的化合物制备。
任何标记方法都是可以使用的。化合物优选是用放射性同位素，更优选用3H标记的化合物。采用放射性化合物作为起始物质，用常规制备方法制得放射性标记的化合物。另外，3H标记可以通过3H交换反应获得。
(b)特异性结合的确定将标记化合物加至制得的膜级分中，将该混合物置于冰上适当长的时间，如1至2小时，直到标记化合物和膜级分间发生结合反应。离心混合物，使膜级分沉淀，如15,000rpm离心3分钟。沉淀物用结合缓冲液重悬浮，离心悬浮液。适当地重复(两次)，除去未结合的标记化合物。再用结合缓冲液悬浮沉淀物。将所得的悬浮液移至闪烁小管，加入闪烁剂。利用液体闪烁计数器测定放射性。
确认标记化合物与GWT1蛋白的特异性结合可以通过下述评估来进行是否标记化合物的结合会被加入的严重过量未标记化合物(10倍或更多)所抑制，以及该化合物是否是可忽略地与由不表达GWT1蛋白的真菌细胞制得的膜级分结合。
(c)由试样引起的标记化合物的结合抑制将试样和标记化合物加至制得的膜级分中，将该混合物置于冰上适当长的时间，如1至2小时，期间与膜级分发生结合反应。在本发明的筛选方法中使用的待测化合物包括简单的天然存在化合物，有机化合物，无机化合物，蛋白或肽，以及化合物文库，基因文库的表达产物，细胞提取物，细胞培养物上清液，来自发酵细菌的产物，海洋生物体提取物，植物提取物和类似物。
将混合物离心，例如15,000rpm离心3分钟，使膜级分沉淀。在结合缓冲液中重悬浮沉淀物，离心该悬浮液。适当地重复此步骤(两次)，从而除去所有未结合的标记化合物。用结合缓冲液悬浮沉淀物。将悬浮液移至闪烁小管，加入闪烁剂。利用液体闪烁计数器测定放射性。
当在试样存在的条件下，标记化合物与膜级分的结合被抑制，就判定该试样具有结合疟原虫GWT1蛋白的活性。
用于筛选抑制疟原虫GWT1蛋白GlcN-PI酰基转移酶活性的化合物的GlcN-PI酰基转移酶分析系统酰基到GPI的转移可以利用下述方法来进行检测Costello L.C和OrleanP.，J.Biol.Chem.(1992)2678599-8603；或Franzot S.P和Doering T.L.，Biochem.J.(1999)34025-32。该方法的具体实例在下面进行了描述。下述实验条件优选是对所用的各种疟原虫GWT1蛋白进行了优化的。
根据上述部分1所述的方法制备疟原虫GWT1蛋白。将包含疟原虫GWT1蛋白的膜级分加至缓冲液中，所述缓冲液含有合适的金属离子(Mg2+，Mn2+)，ATP，辅酶A，优选含有防止在其它反应中消耗UDP-GlcNAc的抑制剂，如作为几丁质合成抑制剂的尼可霉素(nikkomycin)Z，或作为天冬酰胺-连接的糖基化抑制剂的衣霉素(tunicamycin)。然后，将试样加到该混合物中，将所得的混合物在合适的温度下孵育适当长的时间(如24℃孵育15分钟)。
将已适当标记的，优选放射性标记的GlcN-(酰基)PI前体(如UDP-GlcNAc，Acyl-辅酶A，优选UDP-[14C]GlcNAc)加到混合物中。将所得的混合物孵育适当长的时间(如24℃孵育1小时)。加入氯仿和甲醇(1∶2)的混合物，搅拌所得混合物以使反应停止，提取脂质。将提取的反应产物溶解在合适的溶剂中，优选丁醇。然后，利用如HPLC或薄层层析(TLC)，优选TLC法分离反应产生的GlcN-(酰基)PI。当使用TLC时，展开剂(developing solvent)可以适当地选自，如CHCl3/CH3OH/H2O(65∶25∶4)，CHCl3/CH3OH/1M NH4OH(10∶10∶3)和CHCl3/吡啶/HCOOH(35∶30∶7)。优选的展开剂是CHCl3/CH3OH/H2O(65∶25∶4)。采用对所用标记而言恰当的方法，对分离的GlcN-(酰基)PI进行定量。当用放射性同位素作标记时，可以基于其放射性对分离的GlcN-(酰基)PI进行定量。
当在试样存在的条件下，制得的GlcN-(酰基)PI量减少时，就判定该试样具有抑制由疟原虫GWT1蛋白引起的酰基转移的活性。
包括在细胞中表达疟原虫GWT1蛋白然后检测细胞表面上的GPI-锚定蛋白的GPI-锚定蛋白检测系统试样抑制疟原虫GWT1蛋白活性的能力可以利用GPI-锚定蛋白检测系统来进行测定，该系统包括在细胞，优选真菌细胞中表达GWT1蛋白，然后检测细胞表面上的GPI-锚定蛋白。本发明的真菌是指属于接合菌门(Zygomycota)，子囊菌门(Ascomycota)，担子菌门(Basidiomycota)及半知菌纲(Deuteromycete)的真菌，且优选的真菌是致病性真菌，毛霉属(Mucor)，酵母属(Saccharomyces)，念珠菌属(Candida)，隐球菌属(Cryptococcus)，丝孢酵母属(Trichosporon)，鳞斑霉属(Malassezia)，曲霉属(Aspergillus)，发癣菌属(Trichophyton)，小孢子菌属(Microsporum)，孢子丝菌属(Sporothrix)，芽生酵母属(Blastmyces)，球孢子菌属(Coccidioides)，副球孢子菌属(Paracoccidioides)，青霉属(Penicillinium)和镰孢属(Fusarium)，更优选的真菌为白色念珠菌，光滑念珠菌(C.glabrata)，新生隐球菌和烟曲霉，甚至更优选的真菌是酵母。所述酵母包括酿酒酵母和粟酒裂殖酵母。用于将包含编码疟原虫GWT1蛋白的插入DNA的表达载体导入上述真菌细胞的方法是本领域技术人员所熟知的。
当疟原虫GWT1蛋白在真菌细胞中表达时，转运到真菌细胞壁的GPI-锚定蛋白量可通过下述方法测定(1)利用报告酶的方法；(2)利用与真菌细胞壁表面糖蛋白反应的抗体的方法；(3)利用该蛋白粘附动物细胞的能力；或者(4)在光学显微镜或电子显微镜下观察真菌细胞的方法。
(1)-(4)的方法已在WO 02/04626中公开，所述方法在该发明的实施例中进行了详细描述。利用(1)-(4)的方法，并且优选(1)-(4)的方法的组合可以判断试样是否抑制GPI-锚定蛋白到细胞壁上的转运过程或者GPI-锚定蛋白在真菌细胞表面的表达。
在下文中，将描述方法(1)-(4)。
(1) 利用报告酶进行的方法GPI-锚定蛋白到细胞壁的转运过程可通过示踪实验定量如用放射性同位素标记GPI-锚定蛋白，获得真菌细胞壁级分，并用抗GPI-锚定蛋白的抗体进行免疫沉淀反应。也可以用另一种方法更简便地定量在各种GPI-锚定蛋白中常见的、被认为起转运信号作用的C-末端序列可与便于测量的酶(报告酶)表达为融合蛋白，获得真菌细胞壁级分；然后采用报告系统测量每一级分的酶活性(Van Berkel MAA等，FEBSLetters，349135-138，1994)。在下文中，将要会对一种采用报告酶进行的方法进行描述，但是本发明并不仅限于此。
首先，构建报告基因并将其引入真菌中。报告基因是这样构建的使在真菌中起作用的启动子序列，以与阅读框匹配的方式连接分别编码信号序列、报告酶和GPI-锚定蛋白C-末端序列的DNA。启动予序列的实例包括GAL10及ENO1。信号序列的实例包括如α-因子，转化酶，溶菌酶。报告酶的实例是β-内酰胺酶，溶菌酶，碱性磷酸酶和β-半乳糖苷酶。可以使用易于检出的绿色萤光蛋白(GFP)，即便它不具有酶活性。GPI-锚定蛋白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，优选将适宜的选择标记物如LEU2和URA3插到包含所构建的报告基因的载体中。
将所构建的报告基因通过适宜的方法插到真菌中，如乙酸锂法(Gietz D等，Nucl.Acids Res.201425，1992)。并且在需要时，利用适于选择标记物的方法(如适于LEU2的Leu-培养基，以及适于URA3的Ura-培养基)培养真菌，然后挑选其中引入了DNA的真菌。
通过下列方法测定试样对GPI-锚定蛋白到细胞壁的转运过程的影响在试样存在时，于适宜的条件下(如30℃48小时)培养引入了报告基因的真菌。培养之后，离心培养物上清液，并测量培养物上清液级分的报告酶活性。洗涤所得细胞级分，然后通过适当的方法分离细胞壁成分，例如用葡聚糖酶降解细胞壁葡聚糖，然后测量细胞壁级分和细胞质级分的报告酶活性。该试验可以如下简单地进行离心确定细胞级分中报告酶的量，然后在不洗涤细胞的情况下按比例计算来源于培养物上清液级分但残留在细胞级分中的报告酶的量，从细胞级分的报告酶的量中减去此量。
如果试样显示出增加培养物上清液级分中报告酶活性的活性(活性/细胞)，或者降低细胞壁级分中报告酶活性的活性(活性/细胞)，那么就可以判定该试样对GPI-锚定蛋白到细胞壁的转运过程有影响。
(2) 采用与真菌细胞壁表面糖蛋白反应的抗体的方法对于试样影响GPI-锚定蛋白在真菌表面层的表达的能力，可以通过利用与真菌细胞壁中的GPI-锚定蛋白反应的抗体进行定量来测定。
抗体可以如下制备例如，可以由GPI-锚定蛋白的氨基酸序列预测抗原决定簇，所述GPI-锚定蛋白如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)，合成该区的肽，将其与抗原性物质如载体蛋白结合，然后通过免疫兔子等得到多克隆抗体，或者通过免疫小鼠等得到单克隆抗体。优选是针对Als1p肽的兔多克隆抗体。
在可供选择的方法中，可以通过用真菌，优选过量表达GPI-锚定蛋白，如α-凝集素，Cwp2p及Als1p的真菌免疫小鼠等获得抗GPI-锚定蛋白的单克隆抗体，(在某些情况下，可以进一步用部分纯化的GPI-锚定蛋白进行免疫)，并利用ELISA，Western印迹分析等以此制备抗体为基础选出产生的克隆。
通过下述方法可以检测试样对GPI-锚定蛋白到细胞壁的转运过程的影响，以及试样对来源于细胞壁中GPI-锚定蛋白的蛋白量的影响。
在试样存在时，于适宜的条件下如30℃48小时培养真菌。通过离心收集所培养的真菌并将细胞破碎，优选采用玻璃珠。优选用SDS离心提取洗涤过的破碎细胞，然后洗涤沉淀物。提取之后，用降解葡聚糖的酶，优选葡聚糖酶处理破碎的细胞，其经离心处理的上清液即为GPI-锚定蛋白样品。
将抗-Als1p肽的抗体经4℃过夜温育包被在96-孔板上。用洗涤溶液，优选含0.05％Tween 20的PBS(PBST)洗涤板之后，用封闭96-孔板非特异性吸附位点的试剂，优选蛋白如BSA或明胶蛋白，更优选BlockAce(Dainippon Pharmaceutical Co.，Ltd.)进行封闭。用洗涤溶液，优选PBST，再次洗涤之后，加入适当稀释的GPI-锚定蛋白样品。在室温进行适当长时间如2小时的反应。用洗涤溶液，优选PBST，洗涤之后，使针对酶标记的白色念珠菌的抗体，优选HRP-标记的抗-念珠菌抗体，在室温反应适当长的时间如2小时。标记方法可以是酶标记法或放射性同位素标记法。用洗涤溶液，优选PBST，洗涤之后，通过适于标记类型即酶标记的方法计算GPI-锚定蛋白样品中Als1p的量，加入底物溶液，然后在终止反应时测量在490nm处的吸光度。
(3) 利用粘附动物细胞的能力进行的方法试样对GPI-锚定蛋白在真菌表面的表达的影响可以通过测量真菌细胞壁中GPI-锚定蛋白的活性，优选通过测量真菌对动物细胞等的粘附能力来进行测定。除了Als1p，Hwp1p等参与粘附动物细胞的活性之外，GPI-锚定蛋白活性还包括α-凝集素参与交配的活性，Flo1p参与酵母聚集的活性等。在下文中，将详细描述利用真菌对动物细胞的粘附活性进行的方法，但是本发明并不仅限于此。
使用对细胞具有粘附能力的真菌，优选的真菌为白色念珠菌。对于哺乳动物细胞采用粘附真菌的细胞，优选小肠上皮细胞。培养哺乳动物细胞并通过适当的方法，如乙醇固定法将其固定。将试样和真菌培养适当长的时间，如30℃温育48小时，然后接种并培养一定长的时间，如在30℃温育1小时。移出培养物上清液，用缓冲液洗涤，加样至琼脂培养基，如Sabouraud Dextrose Agar培养基(Becton Dickinson Company，Ltd.)。30℃过夜培养后，计数菌落的数目，并计算粘附率。
如果与未经该化合物处理的真菌相比，观察到试样具有使细胞粘附而形成的菌落数目降低的活性，则可以判定该试样已经影响了GPI-锚定蛋白到细胞壁的转运过程。
(4) 利用光学显微镜或电子显微镜观察真菌的方法可以通过用电子显微镜观察真菌细胞壁的结构来确定试样对GPI-锚定蛋白在真菌表面的表达的影响。
在试样存在的条件下，将真菌如白色念珠菌培养一定长的时间，如30℃培养48小时，用透射电子显微镜观察其超微形态结构。在此，利用透射电子显微镜进行的观察可以通过例如根据Electron Microscope Chart Manual(Medical Publishing Center)的方法来进行。真菌细胞最外层的絮状纤维结构具有高电子密度，并且可通过透射电子显微镜观察到。此结构不受其它现有抗真菌剂的影响，并认为是包括GPI-锚定蛋白作为其构成成分的表面糖蛋白层。与未经处理的细胞相比，当此结构消失，只留下具有高电子密度的微层时，可以判定所述试样已经影响了GPI-锚定蛋白到细胞壁的转运过程。
当在透射电子显微镜以及光学显微镜下的观察都显示真菌细胞大量溶胀且出芽(分裂)受到抑制时，可以判定试样对细胞壁有影响。
本发明还提供了治疗疟疾的方法，该方法包括施用抑制疟原虫GWT1蛋白的活性的化合物的步骤。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点是具有非常高的AT含量(80.41％)，因此其密码子使用是有偏好的(biased)。此外，所述基因包含在23个分离位置含六个或更多个连续A残基的序列段，且这些序列段作为假(pseudo)-poly(A)位点来制备截短的蛋白。由于如上所述的特性，该基因只能少量地在酵母中表达，而且很难利用PCR进行扩增或者在大肠杆菌中进行复制。也很难确定其核苷酸序列。然而，利用该DNA的简并突变体(SEQID NO5)，其AT含量低于编码PfGWT1蛋白的DNA，本发明人成功地使PfGWT1蛋白高效表达。本发明人还揭示简并突变体DNA的引入能拯救GWT1-缺陷型酵母的表型。此项发现说明疟原虫的GPI合酶与真菌如酵母的GPI合酶是可以互换的。
编码疟原虫GPI合酶的基因的AT含量例如对于恶性疟原虫的GPI8为79.35％，对于恶性疟原虫的GPI13为77.89％。其AT含量与PfGWT1的AT含量一样高。由于恶性疟原虫基因组翻译区内的平均AT含量为76.3％，可以推断大多数恶性疟原虫基因几乎不能在其它物种中表达。本发明人成功地使比编码PfGWT1蛋白的DNA具有更低AT含量的DNA简并突变体在酵母中表达。因此，通过利用该简并DNA突变体，可以在疟原虫之外的宿主中表达疟原虫GPI合酶。此外，GPI-缺陷型酵母和GWT1-缺陷型酵母已知表现出相似的表型，包括致死性等特性。这样，可以采用上述简并突变体DNA拯救GPI合酶基因-缺陷型真菌的表型。
已导入了上述简并突变体DNA的GPI合酶基因-缺陷型真菌的表型由疟原虫GPI合酶的活性来决定。相应地，可以利用GPI合酶基因-缺陷型真菌的表型作为指标，经筛选选出抑制疟原虫GPI合酶活性的化合物。这样，事实上并不需要利用疟原虫就能选出靶向GPI生物合成路径的的抗疟药物。
本发明提供了编码具有拯救GPI合酶基因-缺陷型真菌表型的活性的蛋白的简并突变体DNA，其比编码参与GPI生物合成的天然DNA具有更低的AT含量。所述DNA可以在本发明的筛选方法中使用。
如本文所用，术语“AT含量”是指GPI合酶基因编码区完整核苷酸序列中的腺嘌呤和胸腺嘧啶含量。本发明简并突变体DNA中的AT含量范围优选从50％到70％，更优选从53％到65％，更加优选从55％到62％。
GPI合酶基因-缺陷型真菌的表型包括温度敏感性(优选高温敏感性)和致死性。
在疟原虫中参与GPI生物合成的本发明蛋白包括GWT1，GPI1，GPI8，GPI3/PIG-A，GPI10/PIG-B，YJR013 W/PIG-M，GPI13/PIG-O，GAA1/GAA-1，DPM1，GPI2，GPI15，YDR437W，GPI12，MCD4，GPI11，GPI7，GPI17，GPI16，CDC91，DPM2，DPM3和SL15。上述蛋白中，已知GPI1和GPI8存在于疟原虫中，GPI3/PIG-A，GPI10/PIG-B，YJR013W/PIG-M，GPI13/PIG-O，GAA1/GAA-1和DPM1也认为存在于疟原虫中(Delorenzi等，Infect.Immun.704510-4522，2002)。恶性疟原虫GWT1，GPI1，GPI8，GPI3/PIG-A，GPI10/PIG-B，YJR013W/PIG-M，GPI13/PIG-O，GAA1/GAA-1和DPM1的核苷酸序列分别示于SEQ ID NO1和SEQ ID NOs6-21中的偶数序列鉴定号中。各相应氨基酸序列示于SEQ ID NO2和SEQ ID NOs6-21中的奇数序列鉴定编号中。此外，间日疟原虫GWT1的核苷酸序列示于SEQ ID NO3，相应的氨基酸序列示于SEQ ID NO4。采用本领域技术人员熟知的方法，如采用杂交或PCR，利用包含SEQ ID NO1和3，以及偶数号的SEQ ID NOs6-21中任一核苷酸序列的DNA，可以对其它疟原虫的GWT1，GPI1，GPI8，GPI3/PIG-A，GPI10/PIG-B，YJR013W/PIG-M，GPI13/PIG-O，GAA1/GAA-1或DPM1进行克隆。
另外，利用酵母或人GPI合酶基因可以对疟原虫的除GWT1，GPI1，GPI8，GPI3/PIG-A，GPI10/PIG-B，YJR013W/PIG-M，GPI13/PIG-O，GAA1/GAA-1和DPM1之外的GPI合酶基因进行克隆。酵母(酿酒酵母)GPI2，GPI15，YDR437W，GPI12，MCD4，GPI11，GPI7，GPI17，GPI16和CDC91的核苷酸序列分别示于SEQ ID NOs22-41中的偶数序列鉴定号中；各相应氨基酸序列示于SEQ ID NOs22-41中的奇数序列鉴定号中。此外，人DPM2，DPM3和SL1 5的核苷酸序列分别示于SEQ ID NOs42-47中的偶数序列鉴定号中；各相应氨基酸序列示于SEQ ID NOs42-47中的奇数序列鉴定号中。
编码参与疟原虫GPI生物合成的蛋白的简并突变体DNA的制备由两步组成设计和合成，所述简并突变体DNA的AT含量比天然DNA低。在设计步骤中，首先对目的蛋白的氨基酸序列进行反向翻译，然后列出各氨基酸残基的可能密码子。反向翻译可以利用商购基因分析软件(例如DNASIS-Pro；Hitachi Software Engineering Co.，Ltd)来实现。在列出的密码子中，为各个氨基酸选出符合目的的密码子(例如，AT含量较低的密码子和在用于基因表达的宿主中使用的常规密码子)。简并突变体DNA可以通过利用这些选出的密码子重排目的蛋白的氨基酸序列来对其进行设计。
如此设计的DNA可以通过本领域技术人员所熟知的方法来进行合成。本发明的简并突变体DNA可以基于设计的核苷酸序列，通过如利用商购DNA合成仪来进行合成。
本发明还提供了插入了上述简并突变体DNA的载体，以及在可表达状态包含该DNA或该载体的转化体(优选GPI合酶基因-缺陷型真菌)。载体和宿主可以是前面所述的载体和宿主。
如本文所用，表达“GPI合酶基因缺陷”是指该基因的功能产物未表达或表达水平减少。本发明的GPI合酶基因-缺陷型真菌可通过破坏GPI基因来加以制备。破坏可以通过在同源重组技术等的基础上嵌入与该基因无关的DNA如选择标记来实现。更具体的说，所述突变型真菌可通过将选择标记盒导入酵母，该选择标记盒含有利用引物扩增的粟酒裂殖酵母his5基因或卡那霉素抗性基因(Longtine等，Yeast，14953-961，1998)，其均含有与部分该基因(50-70个核苷酸)同源的核苷酸序列。
本发明的GPI合酶基因-缺陷型真菌包括，如GWT1温度敏感突变株gwt1-20，GPI7破坏菌株，GPI8突变株gpi8-1，以及GPI10温度敏感突变株per13-1。
转化了本发明简并突变体DNA的GPI合酶基因-缺陷型真菌可以通过将插入了该简并突变体DNA的载体导入真菌中来进行制备。pRS316，YEp351等可用作酿酒酵母的载体，pcL，pALSK等可用作粟酒裂殖酵母的载体。
本发明还提供了筛选抗疟药物的方法，该方法包括利用上述GPI合酶基因-缺陷型真菌。
在该方法中，第一步包括将试样与GPI合酶基因-缺陷型真菌接触，其中所述真菌转化了比参与疟原虫GPI生物合成的蛋白的编码DNA具有更低AT含量的简并突变体DNA。所述“接触”可通过将试样加到上述真菌培养物中来实现。当试样是蛋白时，可以将包含该蛋白的编码DNA的载体导入上述真菌中。
在本发明的方法中，下一步包括测量上述真菌的生长程度。更具体地说，在常规培养条件下接种真菌，具体地，将真菌接种至液体培养基，如酵母提取物聚蛋白胨葡萄糖培养基(YPD培养基)或琼脂平板上，然后在25-37℃温育4-72小时。这样，就可以对已转化本发明简并突变体DNA的GPI合酶基因-缺陷型真菌进行生长评估。生长程度还可以以培养基液体的混浊度，菌落数量，或者琼脂平板上形成的斑点大小或颜色作为指标来进行测定。在本发明的方法中，下一步包括选出抑制上述真菌生长的化合物。
在另一可选的方法中，第一步包括将试样与GPI合酶基因-缺陷型真菌接触，其中该真菌已导入了上述简并突变体DNA。下一步包括测定转运到酵母细胞壁上的GPI-锚定蛋白量。该检测方法包括(1)利用报告酶的方法；(2)利用与真菌细胞壁表面糖蛋白反应的抗体的方法；(3)利用粘附动物细胞的能力的方法；和(4)利用光学显微镜或电子显微镜观察真菌的方法。在本发明的方法中，下一步包括选出降低转运到细胞壁的GPI-锚定蛋白量的样品。
本发明提供了利用参与GPI生物合成的蛋白筛选抗疟药物的方法，该蛋白是利用本发明的简并突变体DNA制得的。所述方法包括例如结合分析系统，在该系统中进行筛选是为了选出结合参与GPI生物合成的蛋白的化合物，该化合物与结合所述蛋白的标记化合物竞争。具体地说，将本发明的简并突变体DNA导入GPI合酶基因-缺陷型真菌中，在该真菌中表达该DNA编码的蛋白，获得所表达的蛋白。然后，将制得的蛋白与试样和能结合该蛋白的标记化合物接触。在下一步中，检测结合该蛋白的标记化合物，选出降低结合该蛋白的标记化合物量的试样。
本发明还提供了GlcN-PI酰基转移酶分析系统。该系统包括使用GWT1蛋白，该蛋白是利用编码具有补偿GWT1-缺陷型酵母表型的活性的蛋白的DNA制得的，其中所述DNA是编码疟原虫GWT1蛋白的DNA简并突变体，它比天然DNA具有更低的AT含量。具体地，将简并突变体DNA导入GWT1-缺陷型真菌，在该真菌中表达该简并突变体DNA编码的蛋白，获得所表达的蛋白。然后，将此蛋白与试样接触，检测GlcN-(酰基)PI，选出降低GlcN-(酰基)PI量的试样。
本文引用的所有专利，专利申请和公开物其全部引入以供参考。
附图的简单说明

图1显示四分体分析结果的照片。在导入了opfGWT1-过表达质粒后，gwt1遭破坏的菌株存活。将衍生自单个双倍体细胞的四个孢子垂直点样。
如果一个拷贝的GWT1基因被破坏，则仅有一半的孢子生长。在这种情况下，[菌落-形成的斑点]∶[未显示生长的斑点]的比率为2∶2。在用箭头标记的纵列中，gwt1破坏体(disruptant)的致死表型由于导入了opfGWT1而得到补偿，因此4个斑点全部生长，均形成菌落。
图2的图表显示化合物对于表达opfGWT1基因的酵母生长的抑制活性。在GWT1基因遭破坏的酵母中表达酵母GWT1基因或opfGWT1基因。
具有抑制酵母GWT1-依赖性生长活性的化合物也显示了对于酵母的opfGWT1-依赖性生长的抑制活性，其中opfGWT1在该酵母中表达。
图3是显示抗疟活性的图表。用恶性疟原虫感染人红细胞。将GWT1-抑制性化合物加至这些红细胞中，并确定对疟原虫感染的抑制。
表现抗真菌活性的5种化合物均抑制了红细胞的疟原虫感染。
实施本发明的最佳方式下面，将利用实施例详细地描述本发明，但是本发明并不限于此。
恶性疟原虫GWT1(PfGWT1)(1)恶性疟原虫GWT1(PfGWT1)的核苷酸序列(SEQ ID NO1)已公开于恶性疟原虫基因组数据库(PlasmoDB database，http://plasmodb.org/)中。用纯化自恶性疟原虫(3D7菌株)的基因组DNA作为模板，通过PCR克隆PfGWT1基因。将PfGWT1基因的5和3’两部分分别制备，并在XbaI(TCTAGA)限制酶位点对两部分进行组装。这样，就制得全长PfGWT1基因。另外，在编码区外的限制酶位点也包含在其中，从而可以插入到表达载体内。
(2)用恶性疟原虫基因组DNA作为模板，以及引物pf152F(SEQ ID NO48)和pf136R(SEQ ID NO49)，通过PCR扩增PfGWT1基因的5’部分。用pf137F(SEQ ID NO50)和pf151R(SEQ ID NO51)作为引物，通过上述相同的方法，扩增PfGWT1基因的3’部分。将扩增出的DNA片断亚克隆到pT7-Blue载体(Novagen)中，对插入子的核苷酸序列进行测序，以确定其与SEQ ID NO1的同源性。含有PfGWT1基因5’部分的克隆命名为PF15-5克隆。含有PfGWT1基因3’部分的克隆命名为PF20-9克隆。
(3)利用PCR将限制酶的切割位点添加到编码区的外侧，从而可以使PfGWT1基因插到表达载体内。用PF15-5作为模板，以及引物pf154FE(SEQID NO52)和pf157R(SEQ ID NO53)，通过PCR将EcoRI切割位点加至5’部分。将扩增的DNA片断亚克隆到pT7-Blue载体(Novagen)中，制得pT7-plasmN2克隆。同样地，用PF20-9作为模板，以及引物pf168BK(SEQID NO54)和pf155RK(SEQ ID NO55)，通过PCR扩增3’部分。对扩增的DNA片断进行亚克隆，制得pT7-plasmBK5克隆。
(4)用下述方法制备全长PfGWT1基因。用限制性内切酶EcoRI和KpnI消化酵母表达载体YEp352GAPII。将来源于pT7-plasmN2的EcoRI-XbaI片断(约1500bp)，和来源于pT7-plasmBK5的XbaI-KpnI片断(约1100bp)插入载体的切割位点。这样，就构建成了包含全长PfGWT1的表达载体YEp352GAPII-PfGWT1。
ATGACAATGTGGGGAAGTCAACGGg(SEQ ID NO48)[pf136R]TGTGTGGTTACCGTTCTTTGAATACATAGA(SEQ ID NO49)[pf137F]ATAGAAAATGATTTATGGTACAGCTCAAA(SEQ ID NO50)[pf151R]AGACCAAATTAATTATGCCTTTACATGTAC(SEQ ID NO51)[pf154FE]agaattcaccATGAGCA ACATGA ATATACTTGCGTATCTT (SEQ TDNO52)[pf157R]GAAATTCCAATGTATTCCATATTCACTTAT(SEQ ID NO53)[pf168BK]AAGATCTAATACATTAAAACATTTTAGATTAATGAATATGTG(SEQ ID NO54)[pf155RK]aggtaccGTACACTCCACTCTATGATGATCATTC (SEQ ID NO55)[实施例2]全合成的PfGWT1基因恶性疟原虫DNA中的腺嘌呤和胸腺嘧啶(AT)比例非常高(80％或更高)，因此通常无法使用常规的生物技术(PCR，以大肠杆菌为基础的基因工程，重组蛋白的表达系统等)(Sato和Horii；Protein，Nucleic acid，and Enzyme Vol.48，149-155，2003)。同样地，PfGWT1 DNA的AT含量是80.41％，其包含多个连续的A或T序列段。因此，可以推断该基因将很难在酵母中复制并以蛋白形式表达。实际上，当将连有酵母过表达载体的天然PfGWT1导入GWT1遭破坏的酵母时，PfGWT1根本不能拯救GWT1破坏体的致死表型。为了降低AT含量，用不改变原本氨基酸顺序的同义密码子替换密码子。
在恶性疟原虫GWT1(PfGWT1)核苷酸序列(SEQ ID NO1)的基础上进行密码子取代，所述核苷酸序列已在恶性疟原虫基因组数据库(PlasmoDBdatabase，http://plasmodb.org/)中公开。将所得核苷酸序列命名为“优化的PfGWT1(opfGWT1)”(SEQ ID NO5)。
将上述序列设计为编码区外包含额外的序列；即EcoRI切割位点序列(GAATTC，5’末端)，Kozak序列(ACC，3’末端)，和KpnI切割位点序列(GGTACC，3’末端)。所得序列的合成委托美国的Blue Heron Inc。用这些额外的限制酶位点将完全合成的opfGWT1与YEp352GAPII载体连接，从而构建opfGWT1的过表达质粒。将该构建体引入仅仅单个GWT1基因拷贝被破坏的二倍体细胞(WDG2)中。在含孢子形成培养基的平板上培养获得的转化体，形成用于四分体分析的孢子。
新设计的密码子-修饰opfGWT1的AT含量减少至61.55％。四分体分析结果示于图1。在导入opfGWT1过表达质粒后，gwt1遭破坏的菌株存活。上述发现表明在通过密码子修饰而使AT含量降低时，PfGWT1基因可以在酵母细胞中表达。
利用opfGWT1-表达型酵母进行抗疟活性分析利用opfGWT1-表达型酵母构建抗疟活性化合物的筛选系统。
通过将酿酒酵母GWT1终止子，酿酒酵母GAPDH启动子以及多克隆位点插到单拷贝载体pRS316的SacI-KpnI位点来构建表达盒。分别将酿酒酵母GWT1和opfGWT1插到多克隆位点，制备pGAP-ScGWT1和pGAP-opfGWT1质粒。将这些质粒导入GWT1破坏体。用YPAD连续的两倍稀释化合物(1)，使其最高的终浓度为50μg/ml。将稀释化合物的50μl等分试样加到96-孔板的每个孔中。将含有各种质粒的酵母细胞过夜培养物稀释1000倍，然后将稀释物的50μl等分试样加到各孔中。将平板在30℃温育2天，然后在660nm测定培养物混浊度(图2和表1)[表1]

虽然GWT1破坏体不能存活，但菌株在导入每种质粒后能存活(如0μg/ml的化合物浓度所示)。化合物(1)抑制ScGWT1-表达型酵母的生长，化合物(1)是GWT1-特异性抑制剂。以25μg/ml使用该化合物，约有85％的生长受抑制。当以50μg/ml使用该化合物时，酵母完全不能存活。化合物(1)也抑制opfGWT1-表达型酵母的生长。以25μg/ml使用该化合物，约有50％的生长受抑制。当以50μg/ml使用该化合物时，酵母完全不能存活。由于opfGWT1-表达型酵母的生长是由导入的opfGWT1的活性来决定的，因此生长抑制可归因于化合物(1)对opfGWT1功能的抑制作用。此发现说明利用该分析系统，通过筛选化合物可以鉴定出具有恶性疟原虫GWT1-特异性抑制活性GWT1的化合物。
抗疟活性利用红细胞培养系统，对抑制酵母GWT1的典型化合物(1)-(5)进行抗疟活性分析。

化合物(1)1-(4-丁基苄基)异喹啉

化合物(2)4-[4-(1-异喹啉基甲基)苯基]-3-丁炔-1-醇

化合物(3)5-丁基-2-(1-异喹啉基甲基)苯酚
化合物(4)2-(4-溴-2-氟苄基)-3-甲氧基吡啶 化合物(5)N-[2-(4-丁基苄基)-3-吡啶基]-N-甲胺具体地说，将待测化合物溶解在100％DMSO中，用培养基稀释，然后将稀释物的80μl等分试样加到96-孔培养板的每个孔中。恶性疟原虫FCR3菌株在37℃用含10％人血清的RPMI1640培养基预培养，然后在每个孔中加入20μl的培养细胞(含1 0％红细胞)。此时，0.47％的红细胞被感染。在5％O2，5％CO2，和90％N2的37℃条件培养48小时后，将疟原虫用吉姆萨(Giemsa)染色法染色。测定原虫-感染的红细胞数量，以估计感染率(图3)。结果，显示化合物(3)具有很强的抗疟活性。其它4种化合物也显示了抗疟活性。化合物(4)表现的活性最低。这样，抑制酵母GWT1的化合物包括具有抑制恶性疟原虫GWT1活性的化合物，这说明抗疟药物可以在这些化合物的基础上进行合成。
工业实用性本发明成功地制得能表达疟原虫GWT1的真菌。采用该真菌，而不采用疟原虫，能筛选出靶向GPI生物合成途径的抗疟药物。
到目前为止，还从未进行过在真菌细胞中表达疟原虫基因以及筛选抑制该基因功能的物质的尝试。本发明的方法并不需要利用疟原虫，因此本方法证明了在后基因组时代，采用比较基因组进行用于药物发现的全新筛选方法的可能性。
序列表<110>卫材株式会社(Eisai Co.，Ltd.)<120> 筛选在疟原虫中抑制GPI生物合成的化合物的方法<130> E1-A0210P<150> US60/428,589<151> 2002-11-22<160> 55<170> PatentIn version 3.1<210> 1<211> 2625<212> DNA<213>恶性疟原虫(Plasmodium falciparum))<220>
<221>CDS<222> (1)..(2625)<223>
<400> 1atg agc aac atg aat ata ctt gcg tat ctt ttg ata tgt ccc ttt aac48Met Ser Asn Met Asn Ile Leu Ala Tyr Leu Leu Ile Cys Pro Phe Asn1 5 10 15tta ata tat ata ttt gac ctt cct tca tat ata cct gag tta aat aaa96Leu Ile Tyr Ile Phe Asp Leu Pro Ser Tyr Ile Pro Glu Leu Asn Lys20 25 30aag ctg gag aat gac gag gtg ttt ata tat gga aaa gaa ata aga aag144Lys Leu Glu Asn Asp Glu Val Phe Ile Tyr Gly Lys Glu Ile Arg Lys35 40 45aat gaa tct gca tat tct tta cat tat gaa aaa tat tta tat gaa tta192Asn Glu Ser Ala Tyr Ser Leu His Tyr Glu Lys Tyr Leu Tyr Glu Leu50 55 60tca aga aga tat tat gag ata ata tta aaa tat aat aag gag ctc ggg240Ser Arg Arg Tyr Tyr Glu Ile Ile Leu Lys Tyr Asn Lys Glu Leu Gly65 70 75 80gtt aat cag gaa aaa gaa tat aat tta ata ata agt aga gag ata gat288Val Asn Gln Glu Lys Glu Tyr Asn Leu Ile Ile Ser Arg Glu Ile Asp85 90 95aaa aaa aaa aaa aaa caa aaa aat agt aca caa gga gaa tat aat aat336Lys Lys Lys Lys Lys Gln Lys Asn Set Thr Gln Gly Glu Tyr Asn Asn100 105 110gat gat gat aat aat tgg aaa tta ttc caa ata tat gag aag gaa gaa384Asp Asp Asp Asn Asn Trp Lys Leu Phe Gln Ile Tyr Glu Lys Glu Glu115 120 125ccc aga tca tac gaa tta ata cgt gtt gag att tac aaa aaa gat att432
Pro Arg Ser Tyr Glu Leu Ile Arg Val Glu Ile Tyr Lys Lys Asp Ile130 135 140ctt tta att tat aaa aat gaa aaa acc aaa tca tca ata aaa ttt ata480Leu Leu Ile Tyr Lys Asn Glu Lys Thr Lys Ser Ser Ile Lys Phe Ile145 150 155 160ata aag aaa aga aaa gat ata aaa aat tat ttc tca tta tgt tat caa528Ile Lys Lys Arg Lys Asp Ile Lys Asn Tyr Phe Ser Leu Cys Tyr Gln165 170175aat tgt ata aat aaa tta gat aaa aat gat tat aat att tta aaa agt576Asn Cys Ile Asn Lys Leu Asp Lys Asn Asp Tyr Asn Ile Leu Lys Ser180 185 190aca ata aat aat agt aaa gaa aat ata att aat agt gct tat ata tat624Thr Ile Asn Asn Ser Lys Glu Asn Ile Ile Asn Ser Ala Tyr Ile Tyr195 200 205atg tat ata ata ttt ttc ttt tta tgt ata tat gta gaa aaa aat tta672Met Tyr Ile Ile Phe Phe Phe Leu Cys Ile Tyr Val Glu Lys Asn Leu210 215 220ttt tta tac ttt cct ata tta tta caa aag tat gaa ata cta aca aca720Phe Leu Tyr Phe Pro Ile Leu Leu Gln Lys Tyr Glu Ile Leu Thr Thr225 230 235240tta ttt att tta ttt att cct cta att ctt ttt gtt ttt ttt tat ttt768Leu Phe Ile Leu Phe Ile Pro Leu Ile Leu Phe Val Phe Phe Tyr Phe245 250 255tat ttt act ata atc aag ttg ata tgt tct tgt cta gtt tta tat gta816Tyr Phe Thr Ile Ile Lys Leu Ile Cys Ser Cys Leu Val Leu Tyr Val260 265 270aca ttt caa tta att tat tat act caa ggt atg cct ata tat atg gaa864Thr Phe Gln Leu Ile Tyr Tyr Thr Gln Gly Met Pro Ile Tyr Met Glu275 280 285cat agc ata ttg aaa cat aaa gaa aaa gaa gaa att tgt gat gaa aaa912His Ser Ile Leu Lys His Lys Glu Lys Glu Glu Ile Cys Asp Glu Lys290 295 300gaa gaa att tgt gat gaa aaa gaa gaa att tgt gat gaa aaa gaa gaa960Glu Glu Ile Cys Asp Glu Lys Glu Glu Ile Cys Asp Glu Lys Glu Glu305 310 315320att tgt gat gaa aaa gaa gaa att tgt gat gaa aaa gaa gaa att tgt1008Ile Cys Asp Glu Lys Glu Glu Ile Cys Asp Glu Lys Glu Glu Ile Cys325 330 335gat gaa aaa gaa gaa att ctt gat aaa aaa aaa aaa att cat gaa aaa1056Asp Glu Lys Glu Glu Ile Leu Asp Lys Lys Lys Lys Ile His Glu Lys340 345 350aaa aaa aaa att cat gat aaa aaa gaa gaa att gat gaa aaa aaa aaa1104Lys Lys Lys Ile His Asp Lys Ly1s Glu Glu Ile Asp Glu Lys Lys Lys355 360 365
aaa att cat gat aaa aaa gac gaa agt cat gat aaa aat gaa gac ata1152Lys Ile His Asp Lys Lys Asp Glu Ser His Asp Lys Asn Glu Asp Ile370 375 380aca tat cct gtt caa tat aat ata gaa aat gat tta tgg tac agc tca1200Thr Tyr Pro Val Gln Tyr Asn Ile Glu Asn Asp Leu Trp Tyr Ser Ser385 390 395400aaa aat gta gat att aaa atg tat tca tct tca aat aaa gga gaa gaa1248Lys Asn Val Asp Ile Lys Met Tyr Ser Ser Ser Asn Lys Gly Glu Glu405 410 415tat att ata cag aat aca tta aaa cat ttt aga tta atg aat atg tgt1296Tyr Ile Ile Gln Asn Thr Leu Lys His Phe Arg Leu Met Asn Met Cys420 425 430atg aca tat ata tgt atc ttt gct gtt gat ttt tat ttc ttt cca aat1344Met Thr Tyr Ile Cys Ile Phe Ala Val Asp Phe Tyr Phe Phe Pro Asn435 440 445cat ttt tgt aaa tcc tat tat tat gga aat aca cta atg gat ata ggg1392His Phe Cys Lys Ser Tyr Tyr Tyr Gly Asn Thr Leu Met Asp Ile Gly450 455 460ata ggt gct tct ata agc tcc agt gca tat tct caa gaa ata aaa aag1440Ile Gly Ala Ser Ile Ser Ser Ser Ala Tyr Ser Gln Glu Ile Lys Lys465 470 475 480ttt aca tat ata aaa gag aag aaa aga ata att gaa tta aaa cat ata1488Phe Thr Tyr Ile Lys Glu Lys Lys Arg Ile Ile Glu Leu Lys His Ile485 490495gtt tta ttt att tta gga ata tct aga ttt att gga ata tat ctt ttt1536Val Leu Phe Ile Leu Gly Ile Ser Arg Phe Ile Gly Ile Tyr Leu Phe500 505 510aat tat aat tat aat ata agt gaa tat gga ata cat tgg aat ttc ttt1584Asn Tyr Asn Tyr Asn Ile Ser Glu Tyr Gly Ile His Trp Asn Phe Phe515 520525tta aca tta tgt act aca ttt ctt ata tct aat ata tgt ttt att tta1632Leu Thr Leu Cys Thr Thr Phe Leu Ile Ser Asn Ile Cys Phe Ile Leu530 535 540tta aaa agg ata cgt tat att ttt ctt ttt agt ata atc tct ata att1680Leu Lys Arg Ile Arg Tyr Ile Phe Leu Phe Ser Ile Ile Ser Ile Ile545 550 555 560ctt ttt gaa ata gcc ata tac tat ttt gac tta cat aat tat ata tta1728Leu Phe Glu Ile Ala Ile Tyr Tyr Phe Asp Leu His Asn Tyr Ile Leu565 570 575tta aaa aat gat aga tta aat ttc ttt tca tca aac aaa gaa ggc tta1776Leu Lys Asn Asp Arg Leu Asn Phe Phe Ser Ser Asn Lys Glu Gly Leu580 585 590ttt aat att ata ggt tct gtt aat ttg tat tta ttt tct ttc tca tta1824
Phe Asn Ile Ile Gly Ser Val Asn Leu Tyr Leu Phe Ser Phe Ser Leu595 600 605ttt aaa tat tta aca aaa caa agg aca tac ata aca acc tcg aac att1872Phe Lys Tyr Leu Thr Lys Gln Arg Thr Tyr Ile Thr Thr Ser Asn Ile610 615 620cca aaa aat aaa aag gat atg aat aat tct atg tat tca aag aac ggt1920Pro Lys Asn Lys Lys Asp Met Asn Asn Ser Met Tyr Ser Lys Asn Gly625 630 635 640aac cac aca aat agc aat ata aat aat agg aat cat aaa att gta att1968Asn His Thr Asn Ser Asn Ile Asn Asn Arg Asn His Lys Ile Val Ile645 650655cgg aat aat cat ata aat aaa tat gaa caa gat aac aca aat aag tat2016Arg Asn Asn His Ile Asn Lys Tyr Glu Gln Asp Asn Thr Asn Lys Tyr660 665 670att aat aaa caa ata aat aat aat aag aat aaa ctt gat gaa gaa gaa2064Ile Asn Lys Gln Ile Asn Asn Asn Lys Asn Lys Leu Asp Glu Glu Glu675 680 685aaa tta aaa aaa tta aaa aaa tta aaa aac aaa aaa aaa aat tta aaa2112Lys Leu Lys Lys Leu Lys Lys Leu Lys Asn Lys Lys Lys Asn Leu Lys690 695 700aaa aaa att aaa tat tat ttg tta tac ctt caa tat ata ata aat ata2160Lys Lys Ile Lys Tyr Tyr Leu Leu Tyr Leu Gln Tyr Ile Ile Asn Ile705 710 715 720tat aaa gaa gaa tat tat act att tat tat aat ata aaa tta att ata2208Tyr Lys Glu Glu Tyr Tyr Thr Ile Tyr Tyr Asn Ile Lys Leu Ile Ile725 730735tca tct ttt att ttt tat tta tta cat ata att ctt aat tta tat aaa2256Ser Ser Phe Ile Phe Tyr Leu Leu His Ile Ile Leu Asn Leu Tyr Lys740 745 750aat tat agt gtg cgt ata tta tgt aat gca aat tat att ttt tta ata2304Asn Tyr Ser Val Arg Ile Leu Cys Asn Ala Asn Tyr Ile Phe Leu Ile755 760 765aca tca ttg ggt ttg ttt tct tgt gcc ttg agt ttt tcc tta gaa gat2352Thr Ser Leu Gly Leu Phe Ser Cys Ala Leu Ser Phe Ser Leu Glu Asp770 775 780ata tta tta aga tat aaa aaa tat aaa ata aat att gat ata aca gta2400Ile Leu Leu Arg Tyr Lys Lys Tyr Lys Ile Asn Ile Asp Ile Thr Val785 790 795 800ttg gat aag ata aat aaa aat aca ttg ata gtt ttt ctt ttc tcg aat2448Leu Asp Lys Ile Asn Lys Asn Thr Leu Ile Val Phe Leu Phe Ser Asn805 810 815ata ctt gtg ggc atg ttc aat att cta ttt caa act tta tta tta cct2496Ile Leu Val Gly Met Phe Asn Ile Leu Phe Gln Thr Leu Leu Leu Pro820 825830
ctt ata ttt gta ata cct ata ttg gta ttt tat tct ttc tta ata ctg2544Leu Ile Phe Val Ile Pro Ile Leu Val Phe Tyr Ser Phe Leu Ile Leu835 840 845ctt ttt aca aaa tgc tta cca cct tcc ata cgc cat cca aaa aaa aaa2592Leu Phe Thr Lys Cys Leu Pro Pro Ser Ile Arg His Pro Lys Lys Lys850855 860aca cat cat gag gaa aag caa aaa aaa gaa tga2625Thr His His Glu Glu Lys Gln Lys Lys Glu865 870<210> 2<211> 874<212>PRT<213>恶性疟原虫(Plasmodium falciparum)<400> 2Met Ser Asn Met Asn Ile Leu Ala Tyr Leu Leu Ile Cys Pro Phe Asn1 5 10 15Leu Ile Tyr Ile Phe Asp Leu Pro Ser Tyr Ile Pro Glu Leu Asn Lys20 25 30Lys Leu Glu Asn Asp Glu Val Phe Ile Tyr Gly Lys Glu Ile Arg Lys35 40 45Asn Glu Ser Ala Tyr Ser Leu His Tyr Glu Lys Tyr Leu Tyr Glu Leu50 55 60Ser Arg Arg Tyr Tyr Glu Ile Ile Leu Lys Tyr Asn Lys Glu Leu Gly65 70 75 80Val Asn Gln Glu Lys Glu Tyr Asn Leu Ile Ile Ser Arg Glu Ile Asp85 90 95Lys Lys Lys Lys Lys Gln Lys Asn Ser Thr Gln Gly Glu Tyr Asn Asn100 105 110Asp Asp Asp Asn Asn Trp Lys Leu Phe Gln Ile Tyr Glu Lys Glu Glu115 120 125Pro Arg Ser Tyr Glu Leu Ile Arg Val Glu Ile Tyr Lys Lys Asp Ile130 l35 140Leu Leu Ile Tyr Lys Asn Glu Lys Thr Lys Ser Ser Ile Lys Phe Ile145 150 155 160Ile Lys Lys Arg Lys Asp Ile Lys Asn Tyr Phe Ser Leu Cys Tyr Glnl65 170 175Asn Cys Ile Asn Lys Leu Asp Lys Asn Asp Tyr Asn Ile Leu Lys Ser180 185 190Thr Ile Asn Asn Ser Lys Glu Asn Ile Ile Asn Ser Ala Tyr Ile Tyr195 200 205Met Tyr Ile Ile Phe Phe Phe Leu Cys Ile Tyr Val Glu Lys Asn Leu210 215 220
Phe Leu Tyr Phe Pro Ile Leu Leu Gln Lys Tyr Glu Ile Leu Thr Thr225 230 235240Leu Phe Ile Leu Phe Ile Pro Leu Ile Leu Phe Val Phe Phe Tyr Phe245 250 255Tyr Phe Thr Ile Ile Lys Leu Ile Cys Ser Cys Leu Val Leu Tyr Val260265 270Thr Phe Gln Leu Ile Tyr Tyr Thr Gln Gly Met Pro Ile Tyr Met Glu275 280 285His Ser Ile Leu Lys His Lys Glu Lys Glu Glu Ile Cys Asp Glu Lys290 295 300Glu Glu Ile Cys Asp Glu Lys Glu Glu Ile Cys Asp Glu Lys Glu Glu305 310 315320Ile Cys Asp Glu Lys Glu Glu Ile Cys Asp Glu Lys Glu Glu Ile Cys325 330 335Asp Glu Lys Glu Glu Ile Leu Asp Lys Lys Lys Lys Ile His Glu Lys340 345 350Lys Lys Lys Ile His Asp Lys Lys Glu Glu Ile Asp Glu Lys Lys Lys355 360 365Lys Ile His Asp Lys Lys Asp Glu Ser His Asp Lys Asn Glu Asp Ile370 375 380Thr Tyr Pro Val Gln Tyr Asn Ile Glu Asn Asp Leu Trp Tyr Ser Ser385 390 395400Lys Asn Val Asp Ile Lys Met Tyr Ser Ser Ser Asn Lys Gly Glu Glu405 410 415Tyr Ile Ile Gln Asn Thr Leu Lys His Phe Arg Leu Met Asn Met Cys420 425 430Met Thr Tyr Ile Cys Ile Phe Ala Val Asp Phe Tyr Phe Phe Pro Asn435 440 445His Phe Cys Lys Ser Tyr Tyr Tyr Gly Asn Thr Leu Met Asp Ile Gly450 455 460Ile Gly Ala Ser Ile Ser Ser Ser Ala Tyr Ser Gln Glu Ile Lys Lys465 470 475 480Phe Thr Tyr Ile Lys Glu Lys Lys Arg Ile Ile Glu Leu Lys His Ile485 490 495Val Leu Phe Ile Leu Gly Ile Ser Arg Phe Ile Gly Ile Tyr Leu Phe500 505 510Asn Tyr Asn Tyr Asn Ile Ser Glu Tyr Gly Ile His Trp Asn Phe Phe515 520 525Leu Thr Leu Cys Thr Thr Phe Leu Ile Ser Asn Ile Cys Phe Ile Leu
530 535 540Leu Lys Arg Ile Arg Tyr Ile Phe Leu Phe Ser Ile Ile Ser Ile Ile545 550 555 560Leu Phe Glu Ile Ala Ile Tyr Tyr Phe Asp Leu His Asn Tyr Ile Leu565 570 575Leu Lys Asn Asp Arg Leu Asn Phe Phe Ser Ser Asn Lys Glu Gly Leu580 585 590Phe Asn Ile Ile Gly Ser Val Asn Leu Tyr Leu Phe Ser Phe Ser Leu595 600 605Phe Lys Tyr Leu Thr Lys Gln Arg Thr Tyr Ile Thr Thr Ser Asn Ile610 615 620Pro Lys Asn Lys Lys Asp Met Asn Asn Ser Met Tyr Ser Lys Asn Gly625 630 635 640Asn His Thr Asn Ser Asn Ile Asn Asn Arg Asn His Lys Ile Val Ile645 650 655Arg Asn Asn His Ile Asn Lys Tyr Glu Gln Asp Asn Thr Asn Lys Tyr660 665670Ile Asn Lys Gln Ile Asn Asn Asn Lys Asn Lys Leu Asp Glu Glu Glu675 680 685Lys Leu Lys Lys Leu Lys Lys Leu Lys Asn Lys Lys Lys Asn Leu Lys690 695 700Lys Lys Ile Lys Tyr Tyr Leu Leu Tyr Leu Gln Tyr Ile Ile Asn Ile705 710 715 720Tyr Lys Glu Glu Tyr Tyr Thr Ile Tyr Tyr Asn Ile Lys Leu Ile Ile725 730 735Ser Ser Phe Ile Phe Tyr Leu Leu His Ile Ile Leu Asn Leu Tyr Lys740 745 750Asn Tyr Ser Val Arg Ile Leu Cys Asn Ala Asn Tyr Ile Phe Leu Ile755 760 765Thr Ser Leu Gly Leu Phe Ser Cys Ala Leu Ser Phe Ser Leu Glu Asp770 775780Ile Leu Leu Arg Tyr Lys Lys Tyr Lys Ile Asn Ile Asp Ile Thr Val785 790 795 800Leu Asp Lys Ile Asn Lys Asn Thr Leu Ile Val Phe Leu Phe Ser Asn805 810 815Ile Leu Val Gly Met Phe Asn Ile Leu Phe Gln Thr Leu Leu Leu Pro820 825 830Leu Ile Phe Val Ile Pro Ile Leu Val Phe Tyr Ser Phe Leu Ile Leu835 840 845Leu Phe Thr Lys Cys Leu Pro Pro Ser Ile Arg His Pro Lys Lys Lys
850 855 860Thr His His Glu Glu Lys Gln Lys Lys Glu865 870<210> 3<211> 1956<212>DNA<213> 间日疟原虫(Plasmodium vivax))<220>
<221>CDS<222> (1)..(19 56)<223>
<400> 3atg gcg cat ttg aac ctc ctc gtc tac ctc atc atg tgc ccc ttc aat48Met Ala His Leu Asn Leu Leu Val Tyr Leu Ile Met Cys Pro Phe Asn1 5 10 15gtg agg cac atg ctg gat gcg ccc agc ttc cea ttc cgg tta gga agc96Val Arg His Met Leu Asp Ala Pro Ser Phe Pro Phe Arg Leu Gly Ser20 25 30aaa gca gca agt ggt gaa acc ttc acg tat gga gcg act gca aga gag144Lys Ala Ala Ser Gly Glu Thr Phe Thr Tyr Gly Ala Thr Ala Arg Glu35 40 45aac ctg ggg agt tac tct ccc gca cat gac gag cta tac atg tta gag192Asn Leu Gly Ser Tyr Ser Pro Ala His Asp Glu Leu Tyr Met Leu Glu50 55 60tta gcc aaa atg tac tat aaa att gtg tta aea tat aag aag gat gtt240Leu Ala Lys Met Tyr Tyr Lys Ile Val Leu Thr Tyr Lys Lys Asp Val65 70 75 80agg aaa gga cag gag gag agt tac aac ttg gtg gta ggc tcc ttt ggg288Arg Lys Gly Gln Glu Glu Ser Tyr Asn Leu Val Val Gly Ser Phe Gly85 90 95aag gaa gcc aaa ggg gag gtc tcc ctc caa aga gtt ctc atc aca aat336Lys Glu Ala Lys Gly Glu Val Ser Leu Gln Arg Val Leu Ile Thr Asn100 105 110gat gcc gtg tac ctg tcg tac cag gat gtg caa aac gaa cgt ggg atc384Asp Ala Val Tyr Leu Ser Tyr Gln Asp Val Gln Asn Glu Arg Gly Ile115 120 125caa gtt aag ata aaa agg ggg gaa ata tct tcc tat tta gac ctc cta432Gln Val Lys Ile Lys Arg Gly Glu Ile Ser Ser Tyr Leu Asp Leu Leu130 135 140tcg tgg gat tct tgt ttg tat aag ctt aac tca gac gat tat aat tta480Ser Trp Asp Ser Cys Leu Tyr Lys Leu Asn Ser Asp Asp Tyr Asn Leu145 150 155 160atg aag agc gca tcg gat cat agc aag cca atg gtg gtc agc aca tac528Met Lys Ser Ala Ser Asp His Ser Lys Pro Met Val Val Ser Thr Tyr165 170 175
cac ata tac atg ctg ctg ctg gtg ttt tct ctt tgc act tac gtg gag576His Ile Tyr Met Leu Leu Leu Val Phe Ser Leu Cys Thr Tyr Val Glu180 185 190aag agc ctc ctg ctt gaa ttc cct gcg ttg aaa aag tgc caa gta ttt624Lys Ser Leu Leu Leu Glu Phe Pro Ala Leu Lys Lys Cys Gln Val Phe195 200 205cta acc cta tgt ttg gtg tac tgc ccg ata atc agt tac ctt ttt ttt672Leu Thr Leu Cys Leu Val Tyr Cys Pro Ile Ile Ser Tyr Leu Phe Phe210 215220ttt tac tcc cat gtg agc cta ctt ggg gtg tta ctc gtc tat gtg ttt720Phe Tyr Ser His Val Ser Leu Leu Gly Val Leu Leu Val Tyr Val Phe225 230 235 240ttt tgc ggg ctc ttc agg ggc gtc tct tgc aga agg ggg ggg cag cac768Phe Cys Gly Leu Phe Arg Gly Val Ser Cys Arg Arg Gly Gly Gln His245 250 255atg ggg gag caa acg ggc caa cac acg ggc gat tgg cac acc atc cgc816Met Gly Glu Gln Thr Gly Gln His Thr Gly Asp Trp His Thr Ile Arg260 265 270ggc aac cca caa ggt gat gat acg caa gag gag aga cgc aag tgt ttg864Gly Asn Pro Gln Gly Asp Asp Thr Gln Glu Glu Arg Arg Lys Cys Leu275 280 285gtc cat atg agg cta gcc aac ctg tgc atc acc tac ata tgc ata ttc912Val His Met Arg Leu Ala Asn Leu Cys Ile Thr Tyr Ile Cys Ile Phe290 295 300gct gtg gac ttt tat ttt ttc cca agg caa ttt tcc aag tct ttt ttt960Ala Val Asp Phe Tyr Phe Phe Pro Arg Gln Phe Ser Lys Ser Phe Phe305 310 315 320ttt ggt aac act ttg atg gat tta ggg gtg ggg ggg tgc atc aca tcg1008Phe Gly Asn Thr Leu Met Asp Leu Gly Val Gly Gly Cys Ile Thr Ser325 330 335agc gcg tat tct cta aac agt aaa aag ctc cat tct gcg aac cgc aag1056Ser Ala Tyr Ser Leu Asn Ser Lys Lys Leu His Ser Ala Asn Arg Lys340 345 350gga cac cta atc gat tgg aag cat ttc att tta ttt ttc ctt gga ata1104Gly His Leu Ile Asp Trp Lys His Phe Ile Leu Phe Phe Leu Gly Ile355 360 365gct aga tac att gca gtg aag ctt ttc aat tat aat tac agc tta act1152Ala Arg Tyr Ile Ala Val Lys Leu Phe Asn Tyr Asn Tyr Ser Leu Thr370 375 380gag tat ggg atg cac tgg aat ttt ttt ctt act ctc ttt ttt act ctc1200Glu Tyr Gly Met His Trp Asn Phe Phe Leu Thr Leu Phe Phe Thr Leu385 390 395400cta act tgt aac gcc cta ctc tgc ttg ata aga ggg gtt aaa cgc acc1248Leu Thr Cys Asn Ala Leu Leu Cys Leu Ile Arg Gly Val Lys Arg Thr
405410415ttt cac ctg agc tgc gtc ctc atc tgt ttg tat gaa att ata att tgg1296Phe His Leu Ser Cys Val Leu Ile Cys Leu Tyr Glu Ile Ile Ile Trp420 425430cgc ctg gac att acg agt tat tta gtg gtt gac gag gca gaa cgg agc1344Arg Leu Asp Ile Thr Ser Tyr Leu Val Val Asp Glu Ala Glu Arg Ser435 440 445ggc ttt ttt tcg cag aac aga gag ggc ctt atg aac gtc atc ggg tcc1392Gly Phe Phe Ser Gln Asn Arg Glu Gly Leu Met Asn Val Ile Gly Ser450 455 460gtc aat ttg tac ctc ttt tcg ttt tcg cta tgg aat ggc tat gtg ttt1440Val Asn Leu Tyr Leu Phe Ser Phe Ser Leu Trp Asn Gly Tyr Val Phe465 470 475480ccg gat gag ggg cag cag tgg gag cga gga aag gcg gcg cga aga ccg1488Pro Asp Glu Gly Gln Gln Trp Glu Arg Gly Lys Ala Ala Arg Arg Pro485 490 495gat gag gcg gcg cga acg ccg ggg gag gga cat ggc cag cgc tcc cct1536Asp Glu Ala Ala Arg Thr Pro Gly Glu Gly His Gly Gln Arg Ser Pro500 505 510gtc cgc ctc acc ctg aag ttg ctt gcc ctg tcc ctc ctc ttc cac ctg1584Val Arg Leu Thr Leu Lys Leu Leu Ala Leu Ser Leu Leu Phe His Leu515 520 525ctg cac ctg ctg ttg aat tac tac cga aat tac agt gtg cgc atc ctt1632Leu His Leu Leu Leu Asn Tyr Tyr Arg Asn Tyr Ser Val Arg Ile Leu530 535 540tgc aac gcg aac tac ata tgt gtt gtc tcc tcc gtg agt ctc ttc gcg1680Cys Asn Ala Asn Tyr Ile Cys Val Val Ser Ser Val Ser Leu Phe Ala545 550 555 560gct gcc ctg agc tac ctc gta gag aag gta ctc ctc cgc gag aag acc1728Ala Ala Leu Ser Tyr Leu Val Glu Lys Val Leu Leu Arg Glu Lys Thr565 570 575acc acc atc cca gtt ttg caa caa atg aac cgg cac tcc ctg gca gtg1776Thr Thr Ile Pro Val Leu Gln Gln Met Asn Arg His Ser Leu Ala Val580 585 590ttc ctc ttt tgc aac gta aca atg ggc act ttc aac ctc ctc ttt cag1824Phe Leu Phe Cys Asn Val Thr Met Gly Thr Phe Asn Leu Leu Phe Gln595 600 605tct ctc ttg ttt ccc cta ttt ttt gcg tgc ctc gtt ttg gcg gcg tac1872Ser Leu Leu Phe Pro Leu Phe Phe Ala Cys Leu Val Leu Ala Ala Tyr610 615 620tcc tat ggc atg ttg cgc ttc gcc tcc ctg ttg ccc ggc ccc gcg cag1920Ser Tyr Gly Met Leu Arg Phe Ala Ser Leu Leu Pro Gly Pro Ala Gln625 630 635 640
ggg gag aag gga gag aag cgg gag aag cag caa taa1936Gly Glu Lys Gly Glu Lys Arg Glu Lys Gln Gin645 650<210>4<211>651<212>PRT<213>间日疟原虫(Plasmodium vivax)<400>4Met Ala His Leu Asn Leu Leu Val Tyr Leu Ile Met Cys Pro Phe Asn1 5 101 5Val Arg His Met Leu Asp Ala Pro Ser Phe Pro Phe Arg Leu Gly Ser20 25 30Lys Ala Ala Ser Gly Glu Thr Phe Thr Tyr Gly Ala Thr Ala Arg Glu35 40 45Asn Leu Gly Ser Tyr Ser Pro Ala His Asp Glu Leu Tyr Met Leu Glu50 55 60Leu Ala Lys Met Tyr Tyr Lys Ile Val Leu Thr Tyr Lys Lys Asp Val65 70 75 80Arg Lys Gly Gln Glu Glu Ser Tyr Asn Leu Val Val Gly Ser Phe Gly85 90 95Lys Glu Ala Lys Gly Glu Val Ser Leu Gln Arg Val Leu Ile Thr Asn100 105 110Asp Ala Val Tyr Leu Ser Tyr Gln Asp Val Gln Asn Glu Arg Gly Ile115 120 125Gln Val Lys Ile Lys Arg Gly Glu Ile Ser Ser Tyr Leu Asp Leu Leu130 135 140Ser Trp Asp Ser Cys Leu Tyr Lys Leu Asn Ser Asp Asp Tyr Asn Leu145 150 l55 160Met Lys Ser Ala Ser Asp His Ser Lys Pro Met Val Val Ser Thr Tyr165 170 175His Ile Tyr Met Leu Leu Leu Val Phe Ser Leu Cys Thr Tyr Val Glu180 185 190Lys Ser Leu Leu Leu Glu Phe Pro Ala Leu Lys Lys Cys Gln Val Phe195 200 205Leu Thr Leu Cys Leu Val Tyr Cys Pro Ile Ile Ser Tyr Leu Phe Phe210 215 220Phe Tyr Ser His Val Ser Leu Leu Gly Val Leu Leu Val Tyr Val Phe225 230 235 240Phe Cys Gly Leu Phe Arg Gly Val Ser Cys Arg Arg Gly Gly Gln His245 250 255
Met Gly Glu Gln Thr Gly Gln His Thr Gly Asp Trp His Thr Ile Arg260 265 270Gly Asn Pro Gln Gly Asp Asp Thr Gln Glu Glu Arg Arg Lys Cys Leu275 280 285Val His Met Arg Leu Ala Asn Leu Cys Ile Thr Tyr Ile Cys Ile Phe290 295 300Ala Val Asp Phe Tyr Phe Phe Pro Arg Gln Phe Ser Lys Ser Phe Phe305 310 315 320Phe Gly Asn Thr Leu Met Asp Leu Gly Val Gly Gly Cys Ile Thr Ser325 330 335Ser Ala Tyr Ser Leu Asn Ser Lys Lys Leu His Ser Ala Asn Arg Lys340 345 350Gly His Leu Ile Asp Trp Lys His Phe Ile Leu Phe Phe Leu Gly Ile355 360 365Ala Arg Tyr Ile Ala Val Lys Leu Phe Asn Tyr Asn Tyr Ser Leu Thr370 375 380Glu Tyr Gly Met His Trp Asn Phe Phe Leu Thr Leu Phe Phe Thr Leu385 390 395 400Leu Thr Cys Asn Ala Leu Leu Cys Leu Ile Arg Gly Val Lys Arg Thr405 410 415Phe His Leu Ser Cys Val Leu Ile Cys Leu Tyr Glu Ile Ile Ile Trp420 425 430Arg Leu Asp Ile Thr Ser Tyr Leu Val Val Asp Glu Ala Glu Arg Ser435 440 445Gly Phe Phe Ser Gln Asn Arg Glu Gly Leu Met Asn Val Ile Gly Ser450 455 460Val Asn Leu Tyr Leu Phe Ser Phe Ser Leu Trp Asn Gly Tyr Val Phe465 470 475 480Pro Asp Glu Gly Gln Gln Trp Glu Arg Gly Lys Ala Ala Arg Arg Pro485 490 495Asp Glu Ala Ala Arg Thr Pro Gly Glu Gly His Gly Gln Arg Ser Pro500 505 510Val Arg Leu Thr Leu Lys Leu Leu Ala Leu Ser Leu Leu Phe His Leu515 520 525Leu His Leu Leu Leu Asn Tyr Tyr Arg Asn Tyr Ser Val Arg Ile Leu530 535 540Cys Asn Ala Asn Tyr Ile Cys Val Val Ser Ser Val Ser Leu Phe Ala545 550 555 560Ala Ala Leu Ser Tyr Leu Val Glu Lys Val Leu Leu Arg Glu Lys Thr565 570 575
Thr Thr Ile Pro Val Leu Gln Gln Met Asn Arg His Ser Leu Ala Val580 585 590Phe Leu Phe Cys Asn Val Thr Met Gly Thr Phe Asn Leu Leu Phe Gln595 600 605Ser Leu Leu Phe Pro Leu Phe Phe Ala Cys Leu Val Leu Ala Ala Tyr610 615 620Ser Tyr Gly Met Leu Arg Phe Ala Ser Leu Leu Pro Gly Pro Ala Gln625 630 635 640Gly Glu Lys Gly Glu Lys Arg Glu Lys Gln Gln645 650<210>5<211>262 5<212>DNA<213>人工的<220>
<223>人工合成的序列<400> 5atgagtaaca tgaacatcct ggcctacctg ctgatctgtc cattcaacct gatctacatc60ttcgacctgc ctagctacat ccctgagcta aacaagaagc tggagaacga cgaagtcttc 120atctacggta aggagatccg taagaacgaa tccgcatact ctctacacta cgagaagtac 180ctatacgaat tgtcacgaag atactacgag atcatcctga agtacaacaa ggagttggga 240gtcaaccaag agaaggaata caacctgatt atctccagag agatcgataa gaagaagaag 300aagcagaaga atagtaccca gggtgaatac aataacgacg atgataacaa ttggaagttg 360ttccagattt acgagaagga agaacctagg agctatgaat tgatcagggt agagatatac 420aagaaggaca ttctgttgat ctacaagaat gagaagacga agtcctctat caagttcatt 480atcaagaagc gtaaggatat caagaattac ttctccttgt gttaccagaa ctgtatcaat 540aagctggaca agaatgatta caacatcttg aagtctacca tcaacaattc caaggaaaac 600attatcaact ctgcatacat ttacatgtac attatcttct tcttcctgtg catatacgtc 660gagaagaacc tgttcttgta cttcccaata ttgcttcaga agtatgagat tctcactacg 720ttgttcatcc tcttcatccc attgatccta ttcgtattct tctatttcta cttcacgatt 780atcaagctga tttgctcatg cttagtccta tacgtgactt tccagttgat ctactatacg 840cagggaatgc ctatttacat ggaacatagt attctcaagc acaaggagaa ggaagagatt 900tgcgatgaga aggaggaaat ctgtgatgaa aaggaagaga tttgcgatga gaaggaagag 960atttgcgatg agaaggaaga gatttgtgat gagaaggaag aaatctgcga tgagaaagaa 1020
gaaatccttg ataagaagaa gaagatccac gagaagaaga agaagatcca tgataagaaa 1080gaggaaatcg atgagaagaa gaagaagatt catgacaaaa aggacgaaag tcatgataag 1140aacgaggaca ttacgtatcc agtccagtac aatatcgaga atgacctatg gtattcatcc 1200aagaacgtgg acatcaagat gtattcatcc agcaacaagg gtgaagaata cattatccag 1260aacacgttga aacatttccg attgatgaac atgtgtatga cgtacatttg tatcttcgct 1320gttgacttct acttcttccc taaccatttc tgcaagtcct actattacgg aaatacgttg 1380atggacattg gaatcggtgc atccatttct tccagtgcat actcgcagga gatcaagaag 1440ttcacgtaca ttaaggagaa gaaacgaatt atcgagttga aacatatcgt gttattcatt 1500ctgggaatta gcagatttat cggtatctac ctattcaact ataactacaa catttctgag 1560tatggaatcc attggaactt ctttcttacg ctatgtacaa cctttctgat tagcaacatt 1620tgtttcatcc tcctcaagag gattcgttat atcttcctat tcagtattat ctcgatcatc 1680ttattcgaaa tcgctattta ctacttcgat ctacataatt acatcctcct gaagaatgac 1740cgtttaaact tcttctcttc taacaaggag ggtctattca acattatcgg ttccgtgaac 1800ctttatttgt tcagtttctc actcttcaag taccttacga agcagcgtac gtatatcacg 1860acgtccaata tccctaagaa caagaaagat atgaataact caatgtattc gaagaatgga 1920aaccatacta attcgaatat caacaaccgt aaccataaga tcg tcatccg taataaccac 1980attaacaagt atgagcagga caacacgaac aagtacatca acaagcaaat caataacaac 2040aagaacaaat tggatgagga agagaagctg aagaaactca agaagcttaa gaataagaag 2100aagaacctca agaagaaaat caagtactat ctattgtacc ttcagtatat catcaacatc 2160tacaaggagg aatactatac gatctactat aacatcaagc taatcatttc gtcgtttata 2220ttctacctcc ttcacataat ccttaatctg tacaagaact actctgtgcg tattctgtgt 2280aatgcaaact acatcttcct gattacgagt ctgggtctgt tctcctgtgc attatccttc 2340tccctggagg acatcctact ccgttataag aaatacaaga tcaatatcga tattactgtg 2400ctagataaga ttaacaagaa caccttaatc gtgttcctgt ttagtaatat cctagtggga 2460atgttcaaca tcctgttcca aactttgctc ctaccactga tattcgttat ccctattcta 2520gtattctaca gcttcttgat cctgctgttc accaagtgtt tgccaccttc tatccgacat 2580cctaagaaga agacgcatca cgaagagaag cagaagaaag agtga 2625<210>6<211>2010<212>DNA<213>恶性疟原虫(Plasmodium falciparum)
<400>6atgtgcacta caaaaaatga agacaataat aaagtgaatt atctgtatct catttattat60aataatatac ctatatttaa aaaaaaaagt ggaaatgaac aaaacataag tgcgctattt 120attttatatg atgtaaaaaa gtatgtgtat aatatggttc atgatcacgt aaatacatta 180gtcctagaag cttttagaag agaagacata ataaaaaaaa taaaagtaaa agaaaaacaa 240aataataatg ataaaaataa agaaagtaat attgaaaaag ataaaaatga acaaaccaaa 300tttacagata tatatgatac aaatagtaag agtgacaaag atatacaaaa gaataatatg 360aacgatggtg atagtaataa taaaaatagt agtttattta ttgatccttt tgaaagtgat 420tcatatgaaa agaataattt tagtaatgaa aaatgtgctt tccaaaatgt cgataaatca 480aaaaaagata aagaacatat atattctgaa aatattactc ctagtagtag taataataat 540aatgataata ataaagaaaa tgattgtgat aaggaacaat tagataaata taataaagat 600aaagaaaata aattaaaatt aaatgataag gatgaatata tttctttcaa ttttattgaa 660gataaattaa ccgaatcatt tcatatgaat caaataattc atttaattaa taaaaaatgt 720gtatttacca aatgcctaga aaattataaa aatagatatt ttgtactgaa aaaagaagag 780attttaaaaa aaaaaaaaaa gcaaaaaaaa atgtctatat tttcatatat tgtatcaatc 840atattatttt ttacatatat catatcactt ataaatagtt gtttatatta tataatatgt 900acaccaaaat tgttcagtga atatatattt tcaaaaaaat gtgatggata tctgcagaat 960tcggcttacc ctaaatttat atttccttct gaatggcata atatattccg cagcttcatg 1020aaaaataaac aaaacccatc tgaatattat aaatatagag aaatcttatt aattcgtatt 1080atcaatttaa taatcgatat tttcttggga tttctgatat ttttactact ttattttaac 1140gtaataaacc tacattatat atcagagaag gctcaaatat tttatggaac cagtacttta 1200acttctatct tgggtacctt gttacagaat ccattaggat ttaaattaaa taataatttt 1260acgtctttca ttggtagcat ccttgtttcg atattagaca aatgggattt atttacaaat 1320accatccctg tgaataatag tacagttttg aattttgttg gatatacatc actgctgggt 1380ttttcttttt ttttgtcttt tgttattgat tatttgagat tcgtaacagc acatgttacc 1440attatttatt tatttttgaa aaaaatatgt accctttttc ataaaaatat gtattcctta 1500tacctattat tcaatgggaa aaaatggaac atactaaaac taagagttga tacaaattac 1560tactcaaatg aagaagtact tttaggaacc atattattta ccattttaat ttttttgtac 1620cctaccattt ttgttcttgt tttggttttt ggtcttatat atcttataat aaatagaatt 1680
atataccttt tgtgtgttat ggagaaaata attttgtaca cccctttcta tattttcttt 1740attcaaccga attgtaataa atatattagt aagggtttca agtttacaaa atatgaagtt 1800ggggaacatg aactgttaaa aaggtatccg actaacagct acttgttgct agaaaagatt 1860cattttttat tttttgataa aataaaattg tttataaata ttttccttta ttttaaaaac 1920ctcgagtcaa tgtcatcata ttcatatata tttatttctt tttgtttttg tcttttaaaa 1980aaaaaatata tatatatata tataatataa2010<210>7<211>669<212>PRT<213>恶性疟原虫(Plasmodium falciparum)<400>7Met Cys Thr Thr Lys Asn Glu Asp Asn Asn Lys Val Asn Tyr Leu Tyr1 5 10 15Leu Ile Tyr Tyr Asn Asn Ile Pro Ile Phe Lys Lys Lys Ser Gly Asn20 25 30Glu Gln Asn Ile Ser Ala Leu Phe Ile Leu Tyr Asp Val Lys Lys Tyr35 40 45Val Tyr Asn Met Val His Asp His Val Asn Thr Leu Val Leu Glu Ala50 55 60Phe Arg Arg Glu Asp Ile Ile Lys Lys Ile Lys Val Lys Glu Lys Gln65 70 75 80Asn Asn Asn Asp Lys Asn Lys Glu Ser Asn Ile Glu Lys Asp Lys Asn85 90 95Glu Gln Thr Lys Phe Thr Asp Ile Tyr Asp Thr Asn Set Lys Ser Asp100 105 110Lys Asp Ile Gln Lys Asn Asn Met Asn Asp Gly Asp Ser Asn Asn Lys115 120 125Asn Ser Ser Leu Phe Ile Asp Pro Phe Glu Ser Asp Ser Tyr Glu Lys130 135 140Asn Asn Phe Ser Asn Glu Lys Cys Ala Phe Gln Asn Val Asp Lys Ser145 150 155 160Lys Lys Asp Lys Glu His Ile Tyr Ser Glu Asn Ile Thr Pro Ser Ser165 170 175Ser Asn Asn Asn Asn Asp Asn Asn Lys Glu Asn Asp Cys Asp Lys Glu180 185 190Gln Leu Asp Lys Tyr Asn Lys Asp Lys Glu Asn Lys Leu Lys Leu Asn195 200 205
Asp Lys Asp Glu Tyr Ile Ser Phe Asn Phe Ile Glu Asp Lys Leu Thr210 215 220Glu Ser Phe His Met Asn Gln Ile Ile His Leu Ile Asn Lys Lys Cys225 230 235 240Val Phe Thr Lys Cys Leu Glu Asn Tyr Lys Asn Arg Tyr Phe Val Leu245 250 255Lys Lys Glu Glu Ile Leu Lys Lys Lys Lys Lys Gln Lys Lys Met Ser260 265 270Ile Phe Ser Tyr Ile Val Ser Ile Ile Leu Phe Phe Thr Tyr Ile Ile275 280 285Ser Leu Ile Asn Ser Cys Leu Tyr Tyr Ile Ile Cys Thr Pro Lys Leu290 295 300Phe Ser Glu Tyr Ile Phe Ser Lys Lys Cys Asp Gly Tyr Leu Gln Asn305 310 315 320Ser Ala Tyr Pro Lys Phe Ile Phe Pro Ser Glu Trp His Asn Ile Phe325 330 335Arg Ser Phe Met Lys Asn Lys Gln Asn Pro Ser Glu Tyr Tyr Lys Tyr340 345 350Arg Glu Ile Leu Leu Ile Arg Ile Ile Asn Leu Ile Ile Asp Ile Phe355 360 365Leu Gly Phe Leu Ile Phe Leu Leu Leu Tyr Phe Asn Val Ile Asn Leu370 375 380His Tyr Ile Ser Glu Lys Ala Gln Ile Phe Tyr Gly Thr Ser Thr Leu385 390 395 400Thr Ser Ile Leu Gly Thr Leu Leu Gln Asn Pro Leu Gly Phe Lys Leu405 410 415Asn Asn Asn Phe Thr Ser Phe Ile Gly Ser Ile Leu Val Ser Ile Leu420 425 430Asp Lys Trp Asp Leu Phe Thr Asn Thr Ile Pro Val Asn Asn Ser Thr435 440 445Val Leu Asn Phe Val Gly Tyr Thr Ser Leu Leu Gly Phe Ser Phe Phe450 455 460Leu Ser Phe Val Ile Asp Tyr Leu Arg Phe Val Thr Ala His Val Thr465 470 475 480Ile Ile Tyr Leu Phe Leu Lys Lys Ile Cys Thr Leu Phe His Lys Asn485 490 495Met Tyr Ser Leu Tyr Leu Leu Phe Asn Gly Lys Lys Trp Asn Ile Leu500 505 510Lys Leu Arg Val Asp Thr Asn Tyr Tyr Ser Asn Glu Glu Val Leu Leu
515 520 525Gly Thr Ile Leu Phe Thr Ile Leu Ile Phe Leu Tyr Pro Thr Ile Phe530 535 540Val Leu Val Leu Val Phe Gly Leu Ile Tyr Leu Ile Ile Asn Arg Ile545 550 555 560Ile Tyr Leu Leu Cys Val Met Glu Lys Ile Ile Leu Tyr Thr Pro Phe565 570 575Tyr Ile Phe Phe Ile Gln Pro Asn Cys Asn Lys Tyr Ile Ser Lys Gly580 585 590Phe Lys Phe Thr Lys Tyr Glu Val Gly Glu His Glu Leu Leu Lys Arg595 600 605Tyr Pro Thr Asn Ser Tyr Leu Leu Leu Glu Lys Ile His Phe Leu Phe610 615 620Phe Asp Lys Ile Lys Leu Phe Ile Asn Ile Phe Leu Tyr Phe Lys Asn625 630 635 640Leu Glu Ser Met Ser Ser Tyr Ser Tyr Ile Phe Ile Ser Phe Cys Phe645 650 655Cys Leu Leu Lys Lys Lys Tyr Ile Tyr Ile Tyr Ile Ile660 665<210> 8<211>1482<212>DNA<213>恶性疟原虫(Plasmodium falciparum)<400>8atggggataa aaattataat ttatatattt tttctatcgt gggcgaaatg ggtgtgtggg 60tctgttaatt ttaccgggtt tgataacaaa aatatgatag gtaagcacgt ggaactagaa120gggaggtata aaaaggaata tatagatagg ttttttttag aagaattaag aaaacacaat180tatatgaata acaatgttat attattaagt acatcaagac attattttaa ttatagacat240accactaatt tattgattgc atataaatat cttaaatatt tcggtgatac tatggataag300aatattttat taatgattcc atttgatcaa gcttgtgatt gtaggaatat aagagaaggt360caaatatttc gagaatatga attatttcct agtagtcata ataaagaaac aaaaatagaa420aatataaatt tatatgagaa tttaaatatt gattataaaa ataataatgt acgtgatgaa480caaattagaa gagtacttag acatagatat gatgctttta cacctaaaaa aaatagatta540tataataatg gaaataatga gaaaaattta tttctttata tgaccggaca tggtggtgtg600aattttttaa aaattcaaga atttaatatt attagttctt ctgaatttaa tatatatata660
caagaattac ttatcaaaaa tttttataaa tatatattcg taattattga tacgtgtcaa 720ggatatagtt tttatgatga tattctaaat tttgtatata aaaaaaaaat taataatatc 780ttctttttat catcttctaa aagaaatgaa aatagttata gtttattttc cagtagttat 840ttaagcgttt caacggtcga cagatttaca taccattttt ttaattatct tcaacaaata 900cataaaatat atgaaaaaga acgatctaaa aatataaaag ccttttcatt atataacatt 960ttaaattatt taaaaacaca acatattatg tcagaaccta ctacaaataa ttctaaattt 1020aattcgtcca tttttttaca tgataaaaat attcttttct tcaattctaa tttgttaatt 1080atacataaag atgatgtttc tataatatat caagataaac aaacacacaa tcacaaatat 1140atatgtttgg ataatctatc taaatgtggt catataaaaa ataatgtaca taaaaaaatg 1200caaactctat atgaacaaac gttatattat aataataatc aacagaattt cttttctaat 1260catatgtcta attttacaga ttattttttt acacatgata tatataatat atataatata 1320tataatgtat ataatatata taatgtatat aatatatata atgtatatga tatatataat 1380gtatattctt ttcttatatt attgctctct ttatttttta ttatgtgttc tcttcttaca 1440tattatattg ttttttttac agaaaaggct aaaatgacat aa 1482<210>9<211>493<212>PRT<213>恶性疟原虫(Plasmodium falciparum)<400>9Met Gly Ile Lys Ile Ile Ile Tyr Ile Phe Phe Leu Ser Trp Ala Lys15 10 15Trp Val Cys Gly Ser Val Asn Phe Thr Gly Phe Asp Asn Lys Asn Met20 25 30Ile Gly Lys His Val Glu Leu Glu Gly Arg Tyr Lys Lys Glu Tyr Ile35 40 45Asp Arg Phe Phe Leu Glu Glu Leu Arg Lys His Asn Tyr Met Asn Asn50 55 60Asn Val Ile Leu Leu Ser Thr Ser Arg His Tyr Phe Asn Tyr Arg His65 70 75 80Thr Thr Asn Leu Leu Ile Ala Tyr Lys Tyr Leu Lys Tyr Phe Gly Asp85 90 95Thr Met Asp Lys Asn Ile Leu Leu Met Ile Pro Phe Asp Gln Ala Cys100 105 110Asp Cys Arg Asn Ile Arg Glu Gly Gln Ile Phe Arg Glu Tyr Glu Leu115 120 125
Phe Pro Ser Ser His Asn Lys Glu Thr Lys Ile Glu Asn Ile Asn Leu130 135 140Tyr Glu Asn Leu Asn Ile Asp Tyr Lys Asn Asn Asn Val Arg Asp Glu145 150 155 160Gln Ile Arg Arg Val Leu Arg His Arg Tyr Asp Ala Phe Thr Pro Lys165 170 175Lys Asn Arg Leu Tyr Asn Asn Gly Asn Asn Glu Lys Asn Leu Phe Leu180 185 190Tyr Met Thr Gly His Gly Gly Val Asn Phe Leu Lys Ile Gln Glu Phe195 200 205Asn Ile Ile Ser Ser Ser Glu Phe Asn Ile Tyr Ile Gln Glu Leu Leu210 215 220Ile Lys Asn Phe Tyr Lys Tyr Ile Phe Val Ile Ile Asp Thr Cys Gln225 230 235 240Gly Tyr Ser Phe Tyr Asp Asp Ile Leu Asn Phe Val Tyr Lys Lys Lys245 250 255Ile Asn Asn Ile Phe Phe Leu Ser Ser Ser Lys Arg Asn Glu Asn Ser260 265 270Tyr Ser Leu Phe Ser Ser Ser Tyr Leu Ser Val Ser Thr Val Asp Arg275 280 285Phe Thr Tyr His Phe Phe Asn Tyr Leu Gln Gln Ile His Lys Ile Tyr290 295 300Glu Lys Glu Pro Ser Lys Asn Ile Lys Ala Phe Ser Leu Tyr Asn Ile305 310 315 320Leu Asn Tyr Leu Lys Thr Gln His Ile Met Ser Glu Pro Thr Thr Asn325 330 335Asn Ser Lys Phe Asn Ser Ser Ile Phe Leu His Asp Lys Asn Ile Leu340 345 350Phe Phe Asn Ser Asn Leu Leu Ile Ile His Lys Asp Asp Val Ser Ile355 360 365Ile Tyr Gln Asp Lys Gln Thr His Asn His Lys Tyr Ile Cys Leu Asp370 375 380Asn Leu Ser Lys Cys Gly His Ile Lys Asn Asn Val His Lys Lys Met385 390 395 400Gln Thr Leu Tyr Glu Gln Thr Leu Tyr Tyr Asn Asn Asn Gln Gln Asn405 410 415Phe Phe Ser Asn His Met Ser Asn Phe Thr Asp Tyr Phe Phe Thr His420 425 430Asp Ile Tyr Asn Ile Tyr Asn Ile Tyr Asn Val Tyr Asn Ile Tyr Asn
435 440 445Val Tyr Asn Ile Tyr Asn Val Tyr Asp Ile Tyr Asn Val Tyr Ser Phe450 455 460Leu Ile Leu Leu Leu Ser Leu Phe Phe Ile Met Cys Ser Leu Leu Thr465 470 475 480Tyr Tyr Ile Val Phe Phe Thr Glu Lys Ala Lys Met Thr485 490<210>10<211>1494<212>DNA<213>恶性疟原虫(Pla smodium falciparum)<400>10atggaaagtg cagttagcga atgcaatata tacaaaaagg aaaaagataa aaatataatt60tacaaacaag aaagaaaatg ttgtatttgc atggtgagtg atttttttta tccaaatttg120ggaggaatag aaactcacat ttttgaattg tctaagaatt tgataaaaaa gggtttcaag180gttatagttg taacaaattt taataataat aggcatggta taagatggat gggtaatggt240attaaggttt attatttgcc cttccagcct tttttagatg tagtgagttt tccaaatatt300atagggactt taccactatg tagaaatata ttatataggg aaaaagtcga catagtacat360ggtcaccagg ctacgtcagc attagctcat caattcattc ttcatgccaa aaccttagga420ataaaaacta tttatacaga tcactcatta tacagttttt cagacaaagg atgcatacat480gtaaacaaat tattgaaata ttgtataaac gatgttgatc attctatatg tgttteccat540acgaatagag aaaatttagt tttgagaaca gaaagtaatc catataaaac gtcagttata600ggaaatgccc ttgatactac aaaatttgtt ccttgtatta gtaaaagacc aaagtttcca660agaataaata ttattgttat aagtaggtta acatatagaa aaggtataga tttgatagtt720aaggtaatac cattagtatg tcaaaaatat ccattcataa aatttattat agggggagaa780ggtcctaaaa gattgttatt agaagaaatg agagaaaagt atcacttaca taattctgtt840gtattattag gaaaagtaaa acaagaaaat gtaaaaaata ttttacaaac tggtcatata900ttcttaaata catctttaac agaagctttt tgtatagcca taattgaagc agctagttgt960ggtttgcttg tcatatctac ggatgtaggt ggaatatctg aagttttacc acacgatatg1020atgattttag ctaaaccaaa tcatatcgaa ttatgtaaag cagtcgataa agcattaaaa1080attgtacaaa aggtggactc aaatttattc cacgaaaggg taaacatgag tttattaaca1140tatgtaaata tatatatata tatatatata tatatatata taatatatat gaataatttt1200
atttataatc taaccaaaat gtactcttgg gaaaaagtag cggaaaagac ggtaaaatca 1260cataatacat atattatgaa tattgaaaag gtgtatatga acgtattaaa ttatgcaaat 1320ccaagcttgt ttaatagaat aagaaaaata tacgaaatta atacacctat tcatgtttcc 1380tttttttttt ttttagacaa aatattgaag aggtcgttag ctttcctcat ttttatgatg 1440acgaaaataa aaatgaaaaa taagagtttt aaaatgtcga tatataccac ataa1494<210>11<211>497<212>PRT<213>恶性疟原虫(Plasmodium falciparum)<400>11Met Glu Ser Ala Val Ser Glu Cys Asn Ile Tyr Lys Lys Glu Lys Asp1 5 10 15Lys Asn Ile Ile Tyr Lys Gln Glu Arg Lys Cys Cys Ile Cys Met Val20 25 30Ser Asp Phe Phe Tyr Pro Asn Leu Gly Gly Ile Glu Thr His Ile Phe35 40 45Glu Leu Ser Lys Asn Leu Ile Lys Lys Gly Phe Lys Val Ile Val Val50 55 60Thr Asn Phe Asn Asn Asn Arg His Gly Ile Arg Trp Met Gly Asn Gly65 70 75 80Ile Lys Val Tyr Tyr Leu Pro Phe Gln Pro Phe Leu Asp Val Val Ser85 90 95Phe Pro Asn Ile Ile Gly Thr Leu Pro Leu Cys Arg Asn Ile Leu Tyr100 105 110Arg Glu Lys Val Asp Ile Val His Gly His Gln Ala Thr Ser Ala Leu115 120 125Ala His Gln Phe Ile Leu His Ala Lys Thr Leu Gly Ile Lys Thr Ile130 135 140Tyr Thr Asp His Ser Leu Tyr Ser Phe Ser Asp Lys Gly Cys Ile His145 150 155 160Val Asn Lys Leu Leu Lys Tyr Cys Ile Asn Asp Val Asp His Ser Ile165 170 175Cys Val Ser His Thr A sn Arg Glu Asn Leu yal Leu Arg Thr Glu Ser180 185 190Asn Pro Tyr Lys Thr Ser Val Ile Gly Asn Ala Leu Asp Thr Thr Lys195 200 205Phe Val Pro Cys Ile Ser Lys Arg Pro Lys Phe Pro Arg Ile Asn Ile210 215 220
Ile Val Ile Ser Arg Leu Thr Tyr Arg Lys Gly Ile Asp Leu Ile Val225 230 235 240Lys Val Ile Pro Leu Val Cys Gln Lys Tyr Pro Phe Ile Lys Phe Ile245 250 255Ile Gly Gly Glu Gly Pro Lys Arg Leu Leu Leu Glu Glu Met Arg Glu260 265 270Lys Tyr His Leu His Asn Ser Val Val Leu Leu Gly Lys Val Lys Gln275 280 285Glu Asn Val Lys Asn Ile Leu Gln Thr Gly His Ile Phe Leu Asn Thr290 295 300Ser Leu Thr Glu Ala Phe Cys Ile Ala Ile Ile Glu Ala Ala Ser Cys305 310 315 320Gly Leu Leu Val Ile Ser Thr Asp Val Gly Gly Ile Ser Glu Val Leu325 330 335Pro His Asp Met Met Ile Leu Ala Lys Pro Asn His Ile Glu Leu Cys340 345 350Lys Ala Val Asp Lys Ala Leu Lys Ile Val Gln Lys Val Asp Ser Asn355 360 365Leu Phe His Glu Arg Val Asn Met Ser Leu Leu Thr Tyr Val Asn Ile370 375 380Tyr Ile Tyr Ile Tyr Ile Tyr Ile Tyr Ile Ile Tyr Met Asn Asn Phe385 390 395 400Ile Tyr Asn Leu Thr Lys Met Tyr Ser Trp Glu Lys Val Ala Glu Lys405 410 415Thr Val Lys Ser His Asn Thr Tyr Ile Met Asn Ile Glu Lys Val Tyr420 425 430Met Asn Val Leu Asn Tyr Ala Asn Pro Ser Leu Phe Asn Arg Ile Arg435 440 445Lys Ile Tyr Glu Ile Asn Thr Pro Ile His Val Ser Phe Phe Phe Phe450 455 460Leu Asp Lys Ile Leu Lys Arg Ser Leu Ala Phe Leu Ile Phe Met Met465 470 475 480Thr Lys Ile Lys Met Lys Asn Lys Ser Phe Lys Met Ser Ile Tyr Thr485 490 495Thr<210>12<211>2361<212>DNA<213>恶性疟原虫(Plasmodium falciparum)
<400>12atgatttaca atgacatttt aacattatgt gcaaataaat taagaggact aattgattca60aagatattta tttggttgtt aatatttttt agaatattta attgtttatt tgttgtaaca120tcattttatc ctgatgaata ttttcaatct gtagaaattg ctcatttttg ggcttatgga180tatggacata tgtcatggga atgggaacct tgtgtagcat taagatcagt gataactcct240tttatttatt atgtattatt tttattttta aaacttatta acatagatca tcctgtttgt300gtattataca ttcctaaatt atgtcatggt atatgtgctg ctttgtgtga tttaggtatt360tataaattat tgatatattg gtatgttgaa ttgtataatg acgcatggat aaatgaagat420aatataaaac gtaatgagaa agatgaaaat aatggaaata acaacaacaa taataataat480aacaataata ataacaataa taataataat tattattatc ataataatat attatacaac540acaaatgaca ttatttctac aattctatgt tgtcattttt tttgttggtt ttatttttat600tccatatgta ggacatcctc gcattccttt gaatgcttgt ttaatatatg gggtgtatat660tttttatcac aaaattatta ccccttgaaa aatcaatcaa acaaaataga aaagatagac720ttattattac agaacgatgt aatcatacaa aaaggaaaga aacatttaaa tgaatggaca780aatttaaaag aaagaaggaa tgatcatcat tttgatacat acgaaaataa ttttatatat840cataaaggaa cacaaaattg taaacaatat gataaaaata tggttgatca aaatgtttgt900ggtcaaaata tggttgatca cataattcag aacaggaaca atttgtgtag gacacatttt960tattcctcca aatttaacaa aatacaagag gcaaaaaatt tattatttag tttattcttt1020agctccttgt ctgttatatt tcgtccaaac gctttagtat tttggttatc tttatatata1080ctatatatta taaagaatat atttgaaaaa caaaataaaa taaattataa agaaatattc1140aaaataggta ttacgtacac tttttttttc ttaactatta ttattataat tgattcttat1200tattttgggc acattacatt tcccttttgg aatttttttg tttataattt tttaagtgga1260aacaataaat attttggagg gcattctttt tttttttact ttgtatgtgt tataccttct1320atatatttaa ctttaacacc ttttttgttt tatggttatt acattatata taataatata1380ttgaataagg tgaagtataa gacaattaat atatatatgt atatattgaa acgaattgat1440tggatagtat atttagttac acacttagaa atcctatctt tatctttcag taagcataag1500gaacataaaa ttgttatagg atatataccg tttcttacaa tttttgttgg atatgcatta1560tatataataa aattacatta taaaaaatat aatggcaaaa atggaaagaa tatatataat1620aataataaaa tccaatatgg taatataacc ataaagggaa gaaataaata tattttttta1680
atttcatctt ctctttttac aaatataagt tttttacttc aatttttatg tattcttttt 1740ttctgcctta tacataacag atcacctgaa catgtagcct cttattttag aaacttagaa 1800acgaaagatg atcaaaatat ttatatattt ataacaaatt gttatgatat acctttatat 1860tcgcatatac atagaaaatt caatatagga tttttagact gttctcctta tgacacgagt 1920aatgatgaag ctaccaaaaa ttggagaaaa cgtaratatg aagataaatt taaggaacaa 1980ttttttaata tttttcaaga aaaaaaaaat aataatcatc atataaatag tacatatgga 2040gatacaatta ctccatatat aataccagat aaatcatttt attggtttgg tcatcatcat 2100tttaataaaa aaaataattt tcaatatatt tatcaaaaca ttaatttgtc atgtttaaat 2160tatagatttc atataccttt acaaggacaa ttacctactt atatagttac aacaactatt 2220gaacttacac atttacaatt atttctgagt acatataatt ataaacttga aacaaagcct 2280tttttttctt attttatgat aagtgaagca aaaggaatag ttccagtata tcattacata 2340ttcaagaggg ttccttccta a2361<210>13<211>786<212>PRT<213>恶性疟原虫(Plasmodium falciparum)<400>13Met Ile Tyr Asn Asp Ile Leu Thr Leu Cys Ala Asn Lys Leu Arg Gly1 5 10 15Leu Ile Asp Ser Lys Ile Phe Ile Trp Leu Leu Ile Phe Phe Arg Ile20 25 30Phe Asn Cys Leu Phe Val Val Thr Ser Phe Tyr Pro Asp Glu Tyr Phe35 40 45Gln Ser Val Glu Ile Ala His Phe Trp Ala Tyr Gly Tyr Gly His Met50 55 60Ser Trp Glu Trp Glu Pro Cys Val Ala Leu Arg Ser Val Ile Thr Pro65 70 75 80Phe Ile Tyr Tyr Val Leu Phe Leu Phe Leu Lys Leu Ile Asn Ile Asp85 90 95His Pro Val Cys Val Leu Tyr Ile Pro Lys Leu Cys His Gly Ile Cys100 105 110Ala Ala Leu Cys Asp Leu Gly Ile Tyr Lys Leu Leu Ile Tyr Trp Tyr115 120 125Val Glu Leu Tyr Asn Asp Ala Trp Ile Asn Glu Asp Asn Ile Lys Arg130 135 140
Asn Glu Lys Asp Glu Asn Asn Gly Asn Asn Asn Asn Asn Asn Asn Asn145 150 155 160Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Tyr Tyr Tyr His Asn Asn165 170 175Ile Leu Tyr Asn Thr Asn Asp Ile Ile Ser Thr Ile Leu Cys Cys His180 l85 190Phe Phe Cys Trp Phe Tyr Phe Tyr Ser Ile Cys Arg Thr Ser Ser His195 200 205Ser Phe Glu Cys Leu Phe Asn Ile Trp Gly Val Tyr Phe Leu Ser Gln210 215 220Asn Tyr Tyr Pro Leu Lys Asn Gln Ser Asn Lys Ile Glu Lys Ile Asp225 230 235 240Leu Leu Leu Gln Asn Asp Val Ile Ile Gln Lys Gly Lys Lys His Leu245 250 255Asn Glu Trp Thr Asn Leu Lys Glu Arg Arg Asn Asp His His Phe Asp260 265 270Thr Tyr Glu Asn Asn Phe Ile Tyr His Lys Gly Thr Gln Asn Cys Lys275 280 285Gln Tyr Asp Lys Asn Met Val Asp Gln Asn Val Cys Gly Gln Asn Met290 295 300Val Asp His Ile Ile Gln Asn Arg Asn Asn Leu Cys Arg Thr His Phe305 310 315 320Tyr Ser Ser Lys Phe Asn Lys Ile Gln Glu Ala Lys Asn Leu Leu Phe325 330 335Ser Leu Phe Phe Ser Ser Leu Ser Val Ile Phe Arg Pro Asn Ala Leu340 345 350Val Phe Trp Leu Ser Leu Tyr Ile Leu Tyr Ile Ile Lys Asn Ile Phe355 360 365Glu Lys Gln Asn Lys Ile Asn Tyr Lys Glu Ile Phe Lys Ile Gly Ile370 375 380Thr Tyr Thr Phe Phe Phe Leu Thr Ile Ile Ile Ile Ile Asp Ser Tyr385 390 395 400Tyr Phe Gly His Ile Thr Phe Pro Phe Trp Asn Phe Phe Val Tyr Asn405 410 415Phe Leu Ser Gly Asn Asn Lys Tyr Phe Gly Gly His Ser Phe Phe Phe420 425 430Tyr Phe Val Cys Val Ile Pro Ser Ile Tyr Leu Thr Leu Thr Pro Phe435 440 445Leu Phe Tyr Gly Tyr Tyr Ile Ile Tyr Asn Asn Ile Leu Asn Lys Val450 455 460
Lys Tyr Lys Thr Ile Asn Ile Tyr Met Tyr Ile Leu Lys Arg Ile Asp465 470 475 480Trp Ile yal Tyr Leu Val Thr Hi s Leu Glu Ile Leu Ser Leu Ser Phe485 490 495Ser Lys His Lys Glu His Lys Ile Val Ile Gly Tyr Ile Pro Phe Leu500 505 510Thr Ile Phe Val Gly Tyr Ala Leu Tyr Ile Ile Lys Leu His Tyr Lys515 520 525Lys Tyr Asn Gly Lys Asn Gly Lys Asn Ile Tyr Asn Asn Asn Lys Ile530 535 540Gln Tyr Gly Asn Ile Thr Ile Lys Gly Arg Asn Lys Tyr Ile Phe Leu545 550 555 560Ile Ser Ser Ser Leu Phe Thr Asn Ile Ser Phe Leu Leu Gln Phe Leu565 570 575Cys Ile Leu Phe Phe Cys Leu Ile His Asn Arg Ser Pro Glu His Val580 585 590Ala Ser Tyr Phe Arg Asn Leu Glu Thr Lys Asp Asp Gln Asn Ile Tyr595 600 605Ile Phe Ile Thr Asn Cys Tyr Asp Ile Pro Leu Tyr Ser His Ile His610 615 620Arg Lys Phe Asn Ile Gly Phe Leu Asp Cys Ser Pro Tyr Asp Thr Ser625 630 635 640Asn Asp Glu Ala Thr Lys Asn Trp Arg Lys Arg Ile Tyr Glu Asp Lys645 650 655Phe Lys Glu Gln Phe Phe Asn Ile Phe Gln Glu Lys Lys Asn Asn Asn660 665 670His His Ile Asn Ser Thr Tyr Gly Asp Thr Ile Thr Pro Tyr Ile Ile675 680 685Pro Asp Lys Ser Phe Tyr Trp Phe Gly His His His Phe Asn Lys Lys690 695 700Asn Asn Phe Gln Tyr Ile Tyr Gln Asn Ile Asn Leu Ser Cys Leu Asn705 710 715 720Tyr Arg Phe His Ile Pro Leu Gln Gly Gln Leu Pro Thr Tyr Ile Val725 730 735Thr Thr Thr Ile Glu Leu Thr His Leu Gln Leu Phe Leu Ser Thr Tyr740 745 750Asn Tyr Lys Leu Glu Thr Lys Pro Phe Phe Ser Tyr Phe Met Ile Ser755760 765Glu Ala Lys Gly Ile Val Pro Val Tyr His Tyr Ile Phe Lys Arg Val
770 775 780Pro Ser785<210>14<211>1233<212>DNA<213>恶性疟原虫(Plasmodium falciparum)<400>14atggggcata cacataaaga atataaaaac aatgagaaga gtgcaatatt ttttgagtgg60ttgatttttt ttgtgggtat aataattcgt ataattattt attattatgg aaggtggcaa120gataaaaact ttaatgttaa gtttacagat gtagattatt atgttttttc tgatgctgca180aaatatgtac ttatgaacaa atcaccatat gaaagatata catatagata tacaccttta240ttagcatata taatgatacc aaattttttt gttcattttt cttttgggaa aatattattt300tcatttatcg atattcttgt tactattctt ataaatcaaa ttataaaaat caaatatact360aattgtaaaa attatatatt ttatacttgt ttatggtttt taaatccatt agtcataatt420atatcccttc gaggtaatgc agatgttatc ccatgcttct taataatagt aacaatcttt480tgtatatata aaaaacatat ctttttgtct tcgatttttt atggactagc tgtgaacttt540aaaatatata caattattta tgcactacca ttcatgttat atttaaataa aaattattta600cttggggaaa atatttttca attaaatgaa aaaaaaaaaa aaaaaaaaaa tgacttccta660ttaaacacat ttttttatat ttttcgtatt atatctaatt tttttgtgga attatttaaa720ttaaattatg aacagttttt atttgccata tgtagttcct cggtatttct aattttaaac780tgtgtattct atatcatata tggatatgaa tttttgtatg agtcttttat atatcatatt840attagacgtg atcataggca taacttttcc cttttttttt accttatgta tttaagtatt900gagaagaatt caaagattat tcccttaata acctttgtac cacaaataat tttagtagcc960ttatttggat ttaaatatgc aagaacgaat ttggaattat ccatgttttt acaaactatt1020tcttttattg cattgaataa agtgtgcaca tctcagtatt tcatttggtg tattccattt1080ttaccaatta tactttgtgc cataacctta agcaagagaa atatgtttct tataatatcc1140tccattttat tttttattgt ggcaaaagtg ggctcaaaaa gatttctttt attatataaa1200tatgtctttt catatattat ttttttttca tga 1233<210>15<211>410<212>PRT
<213>恶性疟原虫(Plasmodium falciparum)<400>15Met Gly His Thr His Lys Glu Tyr Lys Asn Asn Glu Lys Ser Ala Ile1 5 10 15Phe Phe Glu Trp Leu Ile Phe Phe Val Gly Ile Ile Ile Arg Ile Ile20 25 30Ile Tyr Tyr Tyr Gly Arg Trp Gln Asp Lys Asn Phe Asn Val Lys Phe35 40 45Thr Asp Val Asp Tyr Tyr Val Phe Ser Asp Ala Ala Lys Tyr Val Leu50 55 60Met Asn Lys Ser Pro Tyr Glu Arg Tyr Thr Tyr Arg Tyr Thr Pro Leu65 70 75 80Leu Ala Tyr Ile Met Ile Pro Asn Phe Phe Val His Phe Ser Phe Gly85 90 95Lys Ile Leu Phe Ser Phe Ile Asp Ile Leu Val Thr Ile Leu Ile Asn100 105 110Gln Ile Ile Lys Ile Lys Tyr Thr Asn Cys Lys Asn Tyr Ile Phe Tyr115 120 125Thr Cys Leu Trp Phe Leu Asn Pro Leu Val Ile Ile Ile Ser Leu Arg130 135 140Gly Asn Ala Asp Val Ile Pro Cys Phe Leu Ile Ile Val Thr Ile Phe145 150 155 160Cys Ile Tyr Lys Lys His Ile Phe Leu Ser Ser Ile Phe Tyr Gly Leu165 170 175Ala Val Asn Phe Lys Ile Tyr Thr Ile Ile Tyr Ala Leu Pro Phe Met180 185 190Leu Tyr Leu Asn Lys Asn Tyr Leu Leu Gly Glu Asn Ile Phe Gln Leu195 200 205Asn Glu Lys Lys Lys Lys Lys Lys Asn Asp Phe Leu Leu Asn Thr Phe210 215 220Phe Tyr Ile Phe Arg Ile Ile Ser Asn Phe Phe Val Glu Leu Phe Lys225 230 235 240Leu Asn Tyr Glu Gln Phe Leu Phe Ala Ile Cys Ser Ser Ser Val Phe245 250 255Leu Ile Leu Asn Cys Val Phe Tyr Ile Ile Tyr Gly Tyr Glu Phe Leu260 265 270Tyr Glu Ser Phe Ile Tyr His Ile Ile Arg Arg Asp His Arg His Asn275 280 285Phe Ser Leu Phe Phe Tyr Leu Met Tyr Leu Ser Ile Glu Lys Asn Ser290 295 300
Lys Ile Ile Pro Leu Ile Thr Phe Val Pro Gln Ile Ile Leu Val Ala305 310 315 320Leu Phe Gly Phe Lys Tyr Ala Arg Thr Asn Leu Glu Leu Ser Met Phe325 330 335Leu Gln Thr Ile Ser Phe Ile Ala Leu Asn Lys Val Cys Thr Ser Gln340 345 350Tyr Phe Ile Trp Cys Ile Pro Phe Leu Pro Ile Ile Leu Cys Ala Ile355 360 365Thr Leu Ser Lys Arg Asn Met Phe Leu Ile Ile Ser Ser Ile Leu Phe370 375 380Phe Ile Val Ala Lys Val Gly Ser Lys Arg Phe Leu Leu Leu Tyr Lys385 390 395 400Tyr Val Phe Ser Tyr Ile Ile Phe Phe Ser405 410<210>16<211>3852<212>DNA<213>恶性疟原虫(Plasmodium falciparum)<400>16atgaaaatta aaaataatat taaaaaaaaa aatgataacc tacatcattt cataagtaat 60catacgctaa tattttctat tatcctactt ataaatctat tgattttttt ctcatttatc 120aatggctatt tttatgcaag acaaaaactt gaagaaaaat cagaaaacct tgagcttttt 180agtaggaaag tatttggaga tgaatacgta gaaagtttga aaaagaaaaa aaatactttt 240tcgattatta atgcaccata tgataaagtt gttatacttt taatagattc cttaagattt 300gattttaccc tatacgatac taattatgaa aaggaattca taggaaaaga aaaaaataca 360gatatctaca ataatatatc tagcgaaaaa aaaaatatat caaatgatgg ggaaaaaaaa 420aactcattat tttttttaaa taatatgata aatgtacatc acattttaca aaatgaaaaa 480aacaacactt tattattccg gtttgatgca gatgccccta caataacaac ttcaaggata 540aaatctatat ttatgggtac cataccaaat tatatggaag taaatgaaaa ctttagtcct 600acgactagtg ttgaagataa tttttttgaa cagcttcatt taaataataa aaaagtaatt 660gctatcggtg ataataccat tactcatttg atgaaacatt tttctaaaga attagtttat 720gagagcttta atgtttttga tttttattca ttagatattg ctgcaaagaa acatttttat 780gaagaatacg aatcaaatga ttgggatatt atgtatatac atatgttggg agttgatcat 840attggacata taaaaacacc caactcaaaa atcatgggag atgccttaaa agattttgat 900
acattcatat atgatattat aaataaaata aaattagata atcttaaaaa tattagcaca960gaagaaaaag aaaaaatgaa aataataaaa aaaaaaatat ataaaactta tcgaaacggt1020catcacatac aaaatgaaaa tgatcatatt attgataata tacaaaatga aaatgatcat1080attattgata atatacaaaa tgaaaatgat catactattg ataatataca aagtgaaaat1140gatcatacta ttgataatat acaaaatgaa aatgatcata ctattgaaga tatacaaatt1200gataatgatc atactattga agatattgaa aatcaaagtg aacaaaaaaa tgatgataaa1260aaaacactgt tcatattttt tggtgatcat ggacagctag atacaggaga tcacggagga1320tacagcttgg acgaaaccca tagtgctctt tttgcatatt cacccttaaa ctttatatct1380ttagataatg acatcattca aaataatttt gttttatatg ataaagataa attaaaaaaa1440aatgtgaata cactgaatga agaaaataat aataatgaga atatagataa ttataaaaaa1500tatcattcat atttaaaaga tagaaataaa aaatattctt accattataa tgttaaatat1560acaaaacaag taaatttaat gagtacctta tccttattaa ttggatcaac attaccttat1620ggaaatatag gaaatattat tatggatttc attcccaatg catatataaa aaataataat1680aaaaaaaaaa ataataataa taataataat aataataatt cttcattacc taatgaacag1740acaaatttat attatgatct tttaaattta cattatattg ctgaattaaa ttatgctaac1800ttatggcaat tgaatagata cctgaatgaa tatgaaaaaa agtataatat aataaaaaat1860gaagattatc attttattaa atcctcatgg catattatac aaaaagacaa aaaagaattg1920ttttttcaac caaataaaaa atttattaaa aatgacattt tattaaaaaa agaaaaagaa1980tcatatatag aattcataaa tgaaatgaca actctaatgg atatcacaca aaaatatttt2040tactatatat tcaacataaa agaaaaatat ttcttaattt tatctattgt tttaaatata2100ttcttattat tatttttaaa acatttttat tattattcta aattaaatta ttaccataaa2160ttaattaaag taacctttaa tgattttaat aaaaatattt atttattact ttgtatatgt2220gcattacttt tatatttttt tatcttctgt ttatcaatta aagaatataa agatatattc2280cgaatatttt ctcatgctaa aatcattttc ataagcaata atatagatat gataatacct2340agtataaaaa attaccatat gagcgctaaa cgtaacatga atattacgaa taatgacacc2400taccatacat cacataagga tcggaaatca ttcacaaata aagaagagaa acaaaataat2460acacttatga atatattcta taatattatt tatttcttaa gaataattcg aaaaaaaatt2520gttcagtata taatctttat acatttgaca atatacaatc taactatagg taatattatc2580
tttattctat ttaaattatt tccgaaaatt ataactaatt catttcaaat attaagaagt 2640aattatttcc ttttgtttgt tattatatgg agctgttgtg aaatgtcctt taattatata 2700gataaagaaa gatattatat tcattacatt ttaattgtat atgttatatt tgggatgctt 2760aaatggaagt atcaccgtgt ctttaatata ctaaaagcct tcattttgtt ggtgcttcta 2820ataattaatg ctttgtatag ccatacaccc gaatattttg accatggtaa agaaaaaata 2880tatttaaaag aatctgtact aaaatcagtt tttccaatat catcttatat tcttagctta 2940atattaataa atagtggtat taataattta ctgaaaaaac gaataaaaat aataataacc 3000caaatatgga cattacaata tatcctcgtt tttttgttct taaataatat ataccataga 3060tatattcaat ttataacacc cccaagtatt tatttcttaa ccatttcaac cttcatattc 3120atattcaata caaatttagg tgtattattt ctcttttata tgacatttct ttttttctac 3180tttattctta taagttccaa ttgttcagaa aatatgatcc aaatgaatga cataacatcg 3240acatggataa atgaaaatat acataataga aatgatccta taattacaaa aggaaatttg 3300gagaataaag aaaagtgtac ttcatgtaat acatctataa aagaaaaatt ttattataaa 3360ttaatgatag aaaaattcaa attattaaat acatgctctg tattaaatga agaagaaata 3420atgggaatac aaatttataa attaattaga gatatttctt atttttatat aaatgaaaca 3480gatttttata ttttatcatg tgtactttta atatattctt tttttataac gggacataaa 3540tttattttaa ataacttacc actagtttca ggttatgttg gattgtataa atatgtgtgg 3600ccaataagtc agttttatat ttttaatcat attttttttc cattcttctt ttcgttattt 3660tttatcattt atatttataa cataagaagg ataaaaatta taaattcttt taagcaattt 3720gatttgtatt atttttatgt ttatccacta atgaactttt ccttcaaggc ctcctttttg 3780ttttgttgca agtttattat atctgtatgg gtttcatatt acttgaatct gcacataatg 3840gtaaaaatat aa 3852<210>17<211>1283<212>PRT<213>恶性疟原虫(Plasmodium falciparum)<400>17Met Lys Ile Lys Asn Asn Ile Lys Lys Lys Asn Asp Asn Leu His His1 5 10 15Phe Ile Ser Asn His Thr Leu Ile Phe Ser Ile Ile Leu Leu Ile Asn20 25 30Leu Leu Ile Phe Phe Ser Phe Ile Asn Gly Tyr Phe Tyr Ala Arg Gln
35 40 45Lys Leu Glu Glu Lys Ser Glu Asn Leu Glu Leu Phe Ser Arg Lys Val50 55 60Phe Gly Asp Glu Tyr Val Glu Ser Leu Lys Lys Lys Lys Asn Thr Phe65 70 75 80Ser Ile Ile Asn Ala Pro Tyr Asp Lys Val Val Ile Leu Leu Ile Asp85 90 95Ser Leu Arg Phe Asp Phe Thr Leu Tyr Asp Thr Asn Tyr Glu Lys Glu100 105 110Phe Ile Gly Lys Glu Lys Asn Thr Asp Ile Tyr Asn Asn Ile Ser Ser115 120 125Glu Lys Lys Asn Ile Ser Asn Asp Gly Glu Lys Lys Asn Ser Leu Phe130 135 140Phe Leu Asn Asn Met Ile Asn Val His His Ile Leu Gln Asn Glu Lysl45 150 155 160Asn Asn Thr Leu Leu Phe Arg Phe Asp Ala Asp Ala Pro Thr Ile Thrl65 170 175Thr Ser Arg Ile Lys Ser Ile Phe Met Gly Thr Ile Pro Asn Tyr Met180 185 190Glu Val Asn Glu Asn Phe Ser Pro Thr Thr Ser Val Glu Asp Asn Phe195 200 205Phe Glu Gln Leu His Leu Asn Asn Lys Lys Val Ile Ala Ile Gly Asp210 215 220Asn Thr Ile Thr His Leu Met Lys His Phe Ser Lys Glu Leu Val Tyr225 230 235 240Glu Ser Phe Asn Val Phe Asp Phe Tyr Ser Leu Asp Ile Ala Ala Lys245 250 255Lys His Phe Tyr Glu Glu Tyr Glu Ser Asn Asp Trp Asp Ile Met Tyr260 265 270Ile His Met Leu Gly Val Asp His Ile Gly His Ile Lys Thr Pro Asn275 280 285Ser Lys Ile Met Gly Asp Ala Leu Lys Asp Phe Asp Thr Phe Ile Tyr290 295 300Asp Ile Ile Asn Lys Ile Lys Leu Asp Asn Leu Lys Asn Ile Ser Thr305 310 315 320Glu Glu Lys Glu Lys Met Lys Ile Ile Lys Lys Lys Ile Tyr Lys Thr325 330 335Tyr Arg Asn Gly His His Ile Gln Asn Glu Asn Asp His Ile Ile Asp340 345 350
Asn Ile Gln Asn Glu Asn Asp His Ile Ile Asp Asn Ile Gln Asn Glu355 360 365Asn Asp His Thr Ile Asp Asn Ile Gln Ser Glu Asn Asp His Thr Ile370 375 380Asp Asn Ile Gln Asn Glu Asn Asp His Thr Ile Glu Asp Ile Gln Ile385 390 395 400Asp Asn Asp His Thr Ile Glu Asp Ile Glu Asn Gln Ser Glu Gln Lys405 410 415Asn Asp Asp Lys Lys Thr Leu Phe Ile Phe Phe Gly Asp His Gly Gln420 425 430Leu Asp Thr Gly Asp His Gly Gly Tyr Ser Leu Asp Glu Thr His Ser435 440 445Ala Leu Phe Ala Tyr Ser Pro Leu Asn Phe Ile Ser Leu Asp Asn Asp450 455 460Ile Ile Gln Asn Asn Phe Val Leu Tyr Asp Lys Asp Lys Leu Lys Lys465 470 475 480Asn Val Asn Thr Leu Asn Glu Glu Asn Asn Asn Asn Glu Asn Ile Asp485 490 495Asn Tyr Lys Lys Tyr His Ser Tyr Leu Lys Asp Arg Asn Lys Lys Tyr500 505 510Ser Tyr His Tyr Asn Val Lys Tyr Thr Lys Gln Val Asn Leu Met Ser515 520 525Thr Leu Ser Leu Leu Ile Gly Ser Thr Leu Pro Tyr Gly Asn Ile Gly530 535 540Asn Ile Ile Met Asp Phe Ile Pro Asn Ala Tyr Ile Lys Asn Asn Asn545 550 555 560Lys Lys Lys Asn Asn Asn Asn Asn Asn Asn Asn Asn Asn Ser Ser Leu565 570 575Pro Asn Glu Gln Thr A sn Leu Tyr Tyr Asp Leu Leu Asn Leu His Tyr580 585 590Ile Ala Glu Leu Asn Tyr Ala Asn Leu Trp Gln Leu Asn Arg Tyr Leu595 600 605Asn Glu Tyr Glu Lys Lys Tyr Asn Ile Ile Lys Asn Glu Asp Tyr His610 615 620Phe Ile Lys Ser Ser Trp His Ile Ile Gln Lys Asp Lys Lys Glu Leu625 630 635 640Phe Phe Gln Pro Asn Lys Lys Phe Ile Lys Asn Asp Ile Leu Leu Lys645 650 655Lys Glu Lys Glu Ser Tyr Ile Glu Phe Ile Asn Glu Met Thr Thr Leu660 665 670
Met Asp Ile Thr Gln Lys Tyr Phe Tyr Tyr Ile Phe Asn Ile Lys Glu675 680 685Lys Tyr Phe Leu Ile Leu Ser Ile Val Leu Asn Ile Phe Leu Leu Leu690 695 700Phe Leu Lys His Phe Tyr Tyr Tyr Ser Lys Leu Asn Tyr Tyr His Lys705 710 715 720Leu Ile Lys Val Thr Phe Asn Asp Phe Asn Lys Asn Ile Tyr Leu Leu725 730 735Leu Cys Ile Cys Ala Leu Leu Leu Tyr Phe Phe Ile Phe Cys Leu Ser740 745 750Ile Lys Glu Tyr Lys Asp Ile Phe Arg Ile Phe Ser His Ala Lys Ile755 760 765Ile Phe Ile Ser Asn Asn Ile Asp Met Ile Ile Pro Ser Ile Lys Asn770 775 780Tyr His Met Ser Ala Lys Arg Asn Met Asn Ile Thr Asn Asn Asp Thr785 790 795 800Tyr His Thr Ser His Lys Asp Arg Lys Ser Phe Thr Asn Lys Glu Glu805 810 815Lys Gln Asn Asn Thr Leu Met Asn Ile Phe Tyr Asn Ile Ile Tyr Phe820 825 830Leu Arg Ile Ile Arg Lys Lys Ile Val Gln Tyr Ile Ile Phe Ile His835 840 845Leu Thr Ile Tyr Asn Leu Thr Ile Gly Asn Ile Ile Phe Ile Leu Phe850 855 860Lys Leu Phe Pro Lys Ile Ile Thr Asn Ser Phe Gln Ile Leu Arg Ser865 870 875 880Asn Tyr Phe Leu Leu Phe Val Ile Ile Trp Ser Cys Cys Glu Met Ser885 890 895Phe Asn Tyr Ile Asp Lys Glu Arg Tyr Tyr Ile His Tyr Ile Leu Ile900 905 910Val Tyr Val Ile Phe Gly Met Leu Lys Trp Lys Tyr His Arg Val Phe915 920 925Asn Ile Leu Lys Ala Phe Ile Leu Leu Val Leu Leu Ile Ile Asn Ala930 935 940Leu Tyr Ser His Thr Pro Glu Tyr Phe Asp His Gly Lys Glu Lys Ile945 950 955 960Tyr Leu Lys Glu Ser Val Leu Lys Ser Val Phe Pro Ile Ser Ser Tyr965 970 975
Ile Leu Ser Leu Ile Leu Ile Asn Ser Gly Ile Asn Asn Leu Leu Lys980 985 990Lys Arg Ile Lys Ile Ile Ile Thr Gln Ile Trp Thr Leu Gln Tyr Ile995 10001005Leu Val Phe Leu Phe Leu Asn Asn Ile Tyr His Arg Tyr Ile Gln10101015 1020Phe Ile Thr Pro Pro Ser Ile Tyr Phe Leu Thr Ile Ser Thr Phe102510301035Ile Phe Ile Phe Asn Thr Asn Leu Gly Val Leu Phe Leu Phe Tyr104010451050Met Thr Phe Leu Phe Phe Tyr Phe Ile Leu Ile Ser Ser Asn Cys105510601065Ser Glu Asn Met Ile Gln Met Asn Asp Ile Thr Ser Thr Trp Ile107010751080Asn Glu Asn Ile His Asn Arg Asn Asp Pro Ile Ile Thr Lys Gly108510901095Asn Leu Glu Asn Lys Glu Lys Cys Thr Ser Cys Asn Thr Ser Ile110011051110Lys Glu Lys Phe Tyr Tyr Lys Leu Met Ile Glu Lys Phe Lys Leu111511201125Leu Asn Thr Cys Ser Val Leu Asn Glu Glu Glu Ile Met Gly Ile113011351140Gln Ile Tyr Lys Leu Ile Arg Asp Ile Ser Tyr Phe Tyr Ile Asn114511501155Glu Thr Asp Phe Tyr Ile Leu Ser Cys Val Leu Leu Ile Tyr Ser116011651170Phe Phe Ile Thr Gly His Lys Phe Ile Leu Asn Asn Leu Pro Leu117511801185Val Ser Gly Tyr Val Gly Leu Tyr Lys Tyr Val Trp Pro Ile Ser119011951200Gln Phe Tyr Ile Phe Asn His Ile Phe Phe Pro Phe Phe Phe Ser120512101215Leu Phe Phe Ile Ile Tyr Ile Tyr Asn Ile Arg Arg Ile Lys Ile122012251230Ile Asn Ser Phe Lys Gln Phe Asp Leu Tyr Tyr Phe Tyr Val Tyr123512401245Pro Leu Met Asn Phe Ser Phe Lys Ala Ser Phe Leu Phe Cys Cys125012551260Lys Phe Ile Ile Ser Val Trp Val Ser Tyr Tyr Leu Asn Leu His126512701275
Ile Met Val Lys Ile1280<210>18<211>2433<212>DNA<213>恶性疟原虫(Plasmodium falciparum)<400>18atgggatttt cagaaaatcc caaattctgc ctgctcataa ataagcttat caagaaatct60aaagttatag gcgttttctt atcaattatt ggtgtgattt tctttttggt gtttaataag 120tttaataaga atgctgaatt agacgctagg acatttaccc aatttgttgg taattcagta 180ttgaacaaaa agaatgagaa gttttataat gatacaaatt cttatttcat gaactataca 240tatgaaggga aagaagatat aataaagttg atatatgatt atattagaaa aaatatatta 300gtaaatgtag agaatgagat ggttaaaata aaattaacgg atagaattga acagaatata 360ttgataagta atgtaggatg taaatattgt aataatatgg aaagtttagt tgtggtaata 420aattttgatt ttaaagaaag gaaatatttt catagcgtaa ttatcggttt aacgttaatg 480gaacattttt ctaaatgtaa ctatatgagt aaggatgtga cttttttatt taccaataaa 540gaattattat attctttagg tgttcaagaa tttatacaaa aatattttta taataatact 600aatagaattg gaaaaaaaat tattagatct tctactatta ttgaatttga ttctatttat 660ccttcttata ttaaaattaa ttatgaagga ttaaatggta tgttacctaa tcaagactta 720atattattat taacaaatga acttcatttt tattctattc ctattaaaat ggagcttact 780catggttcca tatttgatat ggccctagaa aagaattatg aaaatggtca catatacttt 840ctgaggtaca aaaaaaaata tgaatatata cgtgatgata atgatgaaat caagaacatt 900cccgcattta ctgcaacggg gggtagcaaa gtacccataa gaaataaaat gattaatttg 960ttcaacttaa ccaaagcatt acaaagttat ttaagaagtc agagtaatac acatgaaggc 1020ttttgtcatt cttcaaattt ttattttttt aatacattca gaaggcatat accaataagt 1080atatattgtt atagtgttta tttaatatgc gcatatagca taatgaaatt atttaaatca 1140acgatattta gaagctacat aaatttttta acaggtttct acacttattt gattacaata 1200ttaattattt ccctacctat atatttaatt tcaacaaata aaaaatttta tgagttgttg 1260aattttgaag aaaattatat tccttcatgt tatgaatggc atcctgataa ttttgataaa 1320tatataaaaa ttgcaaatat atggtggaat gttttatttt tctcaatttt tggtgcattt 1380ttttttaatt tatttatttc ttttttagtt aataaaaaaa gaaaagttat accaaaaaaa 1440
aatgatcaaa atgaatcatt tgatggctat aaaaaggtag agaaggtaga acgaatttta 1500atattagaaa aaatcaaaga attgcaaaat gaaataatga aacgaaaagg tattacaaat 1560aatcataata atattaaaaa ttataatatt tatacaaatg aaaatatata caataataat 1620ataaataata taaataataa taataatatt tatgaaaact tatatgataa tggagaagta 1680aaaaaaaaca ttctggttaa accaaaaatt ataaatagcg atgatgaaga ttttcttctt 1740gaaaaaaaaa attccgaatt cattaaaaaa atagaaaaac aaatagaaat attagaagaa 1800aaattggaat ttttaagtaa tgatgaaaat gtaaaatata ttttttataa taattctata 1860gcaccattta atacaatgat gatatatatg aatatttttt atttcatatt agtagcgcta 1920ttaagttcgt tatataattg gtcctattct gtattattta gcctgctatt tgtaattcct 1980atatcaattt tacataattt aaaaacaaaa ccagtcagaa tttttaagaa gattattctt 2040tcacttttta ttctttgtat gtttatttat atgtatccta atgataatca tctttggaat 2100ataagacaga agctaactaa tttatttagg aataatatat caaaatgttg taaatattta 2160gacaagcaca aaatattaca aagcaaatat ttcccagaaa gtttgcaatt tatttgttcg 2220aataggttat ttgattcatt ttactcaaat aaatattttt tggataatct aaatattaaa 2280tttagttatg tcttggatat tcaaaatgga ttcttattaa ctttatacaa tttagcaaga 2340aatcattttt gtattggtac agctacctat cctttaatat gctttacctt attcccaata 2400gtattttata tagttttttt atttttttgt taa 2433<210>19<211>810<212>PRT<213>恶性疟原虫(Plasmodium falciparum)<400>19Met Gly Phe Ser Glu Asn Pro Lys Phe Cys Leu Leu Ile Asn Lys Leu1 510 15Ile Lys Lys Ser Lys Val Ile Gly Val Phe Leu Ser Ile Ile Gly Val20 25 30Ile Phe Phe Leu Val Phe Asn Lys Phe Asn Lys Asn Ala Glu Leu Asp35 40 45Ala Arg Thr Phe Thr Gln Phe Val Gly Asn Set Val Leu Asn Lys Lys50 55 60Asn Glu Lys Phe Tyr Asn Asp Thr Asn Ser Tyr Phe Met Asn Tyr Thr65 70 75 80Tyr Glu Gly Lys Glu Asp Ile Ile Lys Leu Ile Tyr Asp Tyr Ile Arg85 90 95
Lys Asn Ile Leu Val Asn Val Glu Asn Glu Met Val Lys Ile Lys Leu100 105 110Thr Asp Arg Ile Glu Gln Asn Ile Leu Ile Ser Asn Val Gly Cys Lys115 120125Tyr Cys Asn Asn Met Glu Ser Leu Val Val Val Ile Asn Phe Asp Phe130 135 140Lys Glu Arg Lys Tyr Phe His Ser Val Ile Ile Gly Leu Thr Leu Met145 150 155 160Glu His Phe Ser Lys Cys Asn Tyr Met Ser Lys Asp Val Thr Phe Leu165 170 175Phe Thr Asn Lys Glu Leu Leu Tyr Ser Leu Gly Val Gln Glu Phe Ile180 185 190Gln Lys Tyr Phe Tyr Asn Asn Thr Asn Arg Ile Gly Lys Lys Ile Ile195 200 205Arg Ser Ser Thr Ile Ile Glu Phe Asp Ser Ile Tyr Pro Ser Tyr Ile210 215 220Lys Ile Asn Tyr Glu Gly Leu Asn Gly Met Leu Pro Asn Gln Asp Leu225 230 235 240Ile Leu Leu Leu Thr Asn Glu Leu His Phe Tyr Ser Ile Pro Ile Lys245 250 255Met Glu Leu Thr His Gly Ser Ile Phe Asp Met Ala Leu Glu Lys Asn260 265 270Tyr Glu Asn Gly His Ile Tyr Phe Leu Arg Tyr Lys Lys Lys Tyr Glu275 280 285Tyr Ile Arg Asp Asp Asn Asp Glu Ile Lys Asn Ile Pro Ala Phe Thr290 295 300Ala Thr Gly Gly Ser Lys Val Pro Ile Arg Asn Lys Met Ile Asn Leu305 310 315 320Phe Asn Leu Thr Lys Ala Leu Gln Ser Tyr Leu Arg Ser Gln Ser Asn325 330 335Thr His Glu Gly Phe Cys His Ser Ser Asn Phe Tyr Phe Phe Asn Thr340 345 350Phe Arg Arg His Ile Pro Ile Ser Ile Tyr Cys Tyr Ser Val Tyr Leu355 360 365Ile Cys Ala Tyr Ser Ile Met Lys Leu Phe Lys Ser Thr Ile Phe Arg370 375 380Ser Tyr Ile Asn Phe Leu Thr Gly Phe Tyr Thr Tyr Leu Ile Thr Ile385 390 395 400Leu Ile Ile Ser Leu Pro Ile Tyr Leu Ile Ser Thr Asn Lys Lys Phe
405 410 415Tyr Glu Leu Leu Asn Phe Glu Glu Asn Tyr Ile Pro Ser Cys Tyr Glu420 425 430Trp His Pro Asp Asn Phe Asp Lys Tyr Ile Lys Ile Ala Asn Ile Trp435 440 445Trp Asn Val Leu Phe Phe Ser Ile Phe Gly Ala Phe Phe Phe Asn Leu450 455 460Phe Ile Ser Phe Leu Val Asn Lys Lys Arg Lys Val Ile Pro Lys Lys465 470 475 480Asn Asp Gln Asn Glu Ser Phe Asp Gly Tyr Lys Lys Val Glu Lys yal485 490 495Glu Arg Ile Leu Ile Leu Glu Lys Ile Lys Glu Leu Gln Asn Glu Ile500 505 510Met Lys Arg Lys Gly Ile Thr Asn Asn His Asn Asn Ile Lys Asn Tyr515 520 525Asn Ile Tyr Thr Asn Glu Asn Ile Tyr Asn Asn Asn Ile Asn Asn Ile530 535 540Asn Asn Asn Asn Asn Ile Tyr Glu Asn Leu Tyr Asp Asn Gly Glu Val545 550 555 560Lys Lys Asn Ile Leu Val Lys Pro Lys Ile Ile Asn Ser Asp Asp Glu565 570 575Asp Phe Leu Leu Glu Lys Lys Asn Ser Glu Phe Ile Lys Lys Ile Glu580 585 590Lys Gln Ile Glu Ile Leu Glu Glu Lys Leu Glu Phe Leu Ser Asn Asp595 600 605Glu Asn Val Lys Tyr Ile Phe Tyr Asn Asn Ser Ile Ala Pro Phe Asn610 615 620Thr Met Met Ile Tyr Met Asn Ile Phe Tyr Phe Ile Leu Val Ala Leu625 630 635 640Leu Ser Ser Leu Tyr Asn Trp Ser Tyr Ser Val Leu Phe Ser Leu Leu645 650 655Phe Val Ile Pro Ile Ser Ile Leu His Asn Leu Lys Thr Lys Pro Val660 665 670Arg Ile Phe Lys Lys Ile Ile Leu Ser Leu Phe Ile Leu Cys Met Phe675 680 685Ile Tyr Met Tyr Pro Asn Asp Asn His Leu Trp Asn Ile Arg Gln Lys690 695 700Leu Thr Asn Leu Phe Arg Asn Asn Ile Ser Lys Cys Cys Lys Tyr Leu705 710 715 720
Asp Lys His Lys Ile Leu Gln Ser Lys Tyr Phe Pro G1u Ser Leu Gln725 730 735Phe Ile Cys Ser Asn Arg Leu Phe Asp Ser Phe Tyr Ser Asn Lys Tyr740 745 750Phe Leu Asp Asn Leu Asn Ile Lys Phe Ser Tyr Val Leu Asp Ile Gln755 760 765Asn Gly Phe Leu Leu Thr Leu Tyr Asn Leu Ala Arg Asn His Phe Cys770 775 780Ile Gly Thr Ala Thr Tyr Pro Leu Ile Cys Phe Thr Leu Phe Pro Ile785 790 795 800Val Phe Tyr Ile Val Phe Leu Phe Phe Cys805 810<210>20<211>780<212>DNA<213>恶性疟原虫(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>恶性疟原虫(Plasmodium falciparum)
<400>21Met Val Ile Arg Phe Phe Leu Phe Val Ile Thr Leu Leu Gly Leu Cys1 5 10 15Ile Asn Met Val Cys Cys Asn Phe Lys Tyr Ser Ile Ile Leu Pro Thr20 25 30Tyr Asn Glu Lys Glu Asn Leu Pro Tyr Leu Ile Tyr Met Ile Ile Asp35 40 45Glu Leu Asn Lys His Glu Ile Lys Phe Glu Ile Ile Val Ile Asp Asp50 55 60Asn Ser Gln Asp Gly Thr Ala Asp Val Tyr Lys Lys Leu Gln Asn Ile65 70 75 80Phe Lys Asp Glu Glu Leu Leu Leu Ile Gln Arg Lys Gly Lys Leu Gly85 90 95Leu Gly Ser Ala Tyr Met Glu Gly Leu Lys Asn Val Thr Gly Asp Phe100 105 110Val Ile Ile Met Asp Ala Asp Leu Ser His His Pro Lys Tyr Ile Tyr115120 125Asn Phe Ile Lys Lys Gln Arg Glu Lys Asn Cys Asp Ile Val Thr Gly130 135 140Thr Arg Tyr Lys Asn Gln Gly Gly Ile Ser Gly Trp Ser Phe Asn Arg145 150 155 160Ile Ile Ile Ser Arg Val Ala Asn Phe Leu Ala Gln Phe Leu Leu Phe165 170 175Ile Asn Leu Ser Asp Leu Thr Gly Ser Phe Arg Leu Tyr Lys Thr Asn180 185 190Val Leu Lys Glu Leu Met Gln Ser Ile Asn Asn Thr Gly Tyr Val Phe195 200 205Gln Met Glu Val Leu Val Arg Ala Tyr Lys Met Gly Lys Ser Ile Glu210 215 220Glu Val Gly Tyr Val Phe Val Asp Arg Leu Phe Gly Lys Ser Lys Leu225 230 235 240Glu Thr Thr Asp Ile Leu Gln Tyr Leu Ser Gly Leu Phe Lys Leu Phe245 250 255Trp Ser Ile<210> 22<211> 843<212>DNA<213>酿酒酵母(Saccharomyces cerevisiae)<400>22atgacaagat ctccctggaa gcgcctacta tggttgaaac aggagtaccc agataattat60
acagatccaa gttttattga gttgagagca agacaaaagg ctgagagtaa ccagaagtct120gatagaaaat tatcagaagc tgctcgcgct caaattaggt tggattttat aagtttctac180caaaccatat tgaacacttc tttcatttac atcactttta catatattta ctattatggc240ttcgatccta ttccgccaac tattttcctt tcatttataa cattgattat atcaaggacg300aaagtcgacc ctctattgtc ctcattcatg gacgtaaagt cttcgctgat tatcacattt360gcaatgttga ctctctctcc agtcctcaaa tctctttcta aaacaactgc atctgattcc420atatggacat tgtctttttg gctgacccta tggtacattt tcgttatttc gtcaacaaag480tccaaagata aaccctctaa cctttccacc aatatacttg tcgcccttgt tgctgtccta540tcatcgaggc tttcgaccac aatcgacgta ttctgttttc ttttaatttg tattcagttg600aatatcattc tacccactta tttatcggtg acgaataagg tagtaccaat aatttcaaat660attattgtat actcattttt gaatgttgct ctaggttgga tttatatgct gttgattttc720tttgcttcag tattttatat tactgtttta cctaagtggt tcatctactg gaaaatcaat780tatcataaac gggataacga tctactaagt acatgggatg caagaacacc aatattggat840tag 843<210>23<211>280<212>PRT<213>酿酒酵母(Saccharomyces cerevisiae)<400>23Met Thr Arg Ser Pro Trp Lys Arg Leu Leu Trp Leu Lys Gln Glu Tyr1 510 15Pro Asp Asn Tyr Thr Asp Pro Ser Phe Ile Glu Leu Arg Ala Arg Gln20 25 30Lys Ala Glu Ser Asn Gln Lys Ser Asp Arg Lys Leu Ser Glu Ala Ala35 40 45Arg Ala Gln Ile Arg Leu Asp Phe Ile Ser Phe Tyr Gln Thr Ile Leu50 55 60Asn Thr Ser Phe Ile Tyr Ile Thr Phe Thr Tyr Ile Tyr Tyr Tyr Gly65 70 75 80Phe Asp Pro Ile Pro Pro Thr Ile Phe Leu Ser Phe Ile Thr Leu Ile85 90 95Ile Ser Arg Thr Lys Val Asp Pro Leu Leu Ser Ser Phe Met Asp Val100 105 110Lys Ser Ser Leu Ile Ile Thr Phe Ala Met Leu Thr Leu Ser Pro Val115 120 125
Leu Lys Ser Leu Ser Lys Thr Thr A1a Ser Asp Ser Ile Trp Thr Leu130 135 140Ser Phe Trp Leu Thr Leu Trp Tyr Ile Phe Val Ile Ser Set Thr Lys145 150 155 160Ser Lys Asp Lys Pro Ser Asn Leu Ser Thr Asn Ile Leu Val Ala Leu165 170 175Val Ala Val Leu Ser Ser Arg Leu Ser Thr Thr Ile Asp Val Phe Cys180 185 190Phe Leu Leu Ile Cys Ile Gln Leu Asn Ile Ile Leu Pro Thr Tyr Leu195 200 205Ser Val Thr Asn Lys Val Val Pro Ile Ile Ser Asn Ile Ile Val Tyr210 215 220Ser Phe Leu Asn Val Ala Leu Gly Trp Ile Tyr Met Leu Leu Ile Phe225 230 235 240Phe Ala Ser Val Phe Tyr Ile Thr Val Leu Pro Lys Trp Phe Ile Tyr245 250 255Trp Lys Ile Asn Tyr His Lys Arg Asp Asn Asp Leu Leu Ser Thr Trp260 265 270Asp Ala Arg Thr Pro Ile Leu Asp275 280<210>24<211>764<212>DNA<213>酿酒酵母(Saccharomyces cerevisiae)<400>24atgattagta aagagtatga atttggtaag actagtatac tgaatagaaa gaagtataca 60ttagttatcg atgaagacaa gaatggcaat tttataagat ttaccgtttt acctgtatct 120aaccgaaagt tcaaaaaagt caagcaaaat gggagggtag agattaacat gggcatacga 180tatcaccaaa ttgtacttat tttactactg aatattttgt tctatgtaat ttgcctaaga 240teaagatttc tcgaacatat taatagaact tttgaagtga caatcgcgcg aagtttccag 300atcttaatta taatgggatt gtttgcctta ggtacaatta tacttgtgag gggacctagt 360gtggaaactg taacaatttt caaagaaagt ggactacagc tgtccagagt gaagggtatg 420gttatatttc ctcaacaatg gaatcggaag ttctttgaac aagtagagtt tatatcgaat 480gaaagaatta ttgatgtagt gatcaatgaa ggattctgtc ggggatttcg agtgatattc 540tatcttgcag caattgtacg taaatcgtct acgcttaagc tattatttcc agtatgtatt 600
caagcgaatt taagattgtt tcttacttca acgatctgat tagaaatact aacacaacaa660tgcagtcaaa tttgcccagt atcgatgacc aacgtctaat atacaacata tctagaaaat 720atctcagtaa gcaagaaaaa cccctgagca gaccaaaaga ttga 764<210>25<211>229<212>PRT<213>酿酒酵母(Saccharomyces cerevisiae)<400>25Met Ile Ser Lys Glu Tyr Glu Phe Gly Lys Thr Ser Ile Leu Asn Arg1 5 10 15Lys Lys Tyr Thr Leu Val Ile Asp Glu Asp Lys Asn Gly Asn Phe Ile20 25 30Arg Phe Thr Val Leu Pro Val Ser Asn Arg Lys Phe Lys Lys Val Lys35 40 45Gln Asn Gly Arg Val Glu Ile Asn Met Gly Ile Gln Tyr His Gln Ile50 55 60Val Leu Ile Leu Leu Leu Asn Ile Leu Phe Tyr Val Ile Cys Leu Arg65 70 75 80Ser Arg Phe Leu Glu His Ile Asn Arg Thr Phe Glu Val Thr Ile Ala85 90 95Arg Ser Phe Gln Ile Leu Ile Ile Met Gly Leu Phe Ala Leu Gly Thr100 105 110Ile Ile Leu Val Arg Gly Pro Ser Val Glu Thr Val Thr Ile Phe Lys115 120 125Glu Ser Gly Leu Gln Leu Ser Arg Val Lys Gly Met Val Ile Phe Pro130 135 140Gln Gln Trp Asn Arg Lys Phe Phe Glu Gln Val Glu Phe Ile Ser Asn145 150 155 160Glu Arg Ile Ile Asp Val Val Ile Asn Glu Gly Phe Cys Arg Gly Phe165 170 175Arg Val Ile Phe Tyr Leu Ala Ala Ile Val Arg Lys Ser Ser Thr Leu180 185 190Lys Leu Leu Phe Pro Ser Asn Leu Pro Ser Ile Asp Asp Gln Arg Leu195 200 205Ile Tyr Asn Ile Ser Arg Lys Tyr Leu Ser Lys Gln Glu Lys Pro Leu210 215 220Ser Arg Pro Lys Asp225
<210>26<211>423<212>DNA<213>酿酒酵母(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>酿酒酵母(Saccharomyces cerevisiae)<400>27Met Tyr Thr Lys Glu Tyr Tyr Trp Phe Ser Gln Tyr Met Ile Ile Thr1 5 10 15Ser Thr Leu Val Leu Thr Ile Ile Trp Ser Ile Leu Pro Ser Ser Leu20 25 30Gly Glu Ala Ala Pro Lys Gln Phe Ile Asn Thr Leu Leu Asp Ile Phe35 40 45Pro Gln Arg Arg Trp Ile Ile Thr Leu Glu Ser Ile Met Leu Met Gly50 55 60Met Leu Cys Thr Tyr Ile Gly Leu Leu Met Tyr Asn Glu Asp Thr Leu65 70 75 80Thr Pro Pro Leu Asp Ser Leu Ser Thr Val Thr Asp Ala Gly Gly Gln85 90 95Leu Val Ile Glu Asp Asp Pro Asp Val Phe Val Lys Lys Trp Ala Phe100 105 110Lys Glu Thr Ser Gly Ile Tyr Asp Leu Ser Leu Met Asp Ala Cys Gln115 120 125Leu Leu Tyr Leu Tyr Asp Asn Asp His Thr Ser Thr130 135 140<210>28
<211>915<212>DNA<213>酿酒酵母(Saccharomyces cerevisiae)<400>28atgaagatgt tgaggcgtac aaaggtaaat ttttcaaaat tgctttacaa gattactaaa 60ctagcaatag tactgacgat cctatatatt tattttacgc ceaaaatcgt ctcccgaaac120aatgcatcat tgcagcatat ttttcctcat aaatatggcg attatgaaat caatttggtc180atagcgcacc ctgacgacga agttatgttt ttttccccea taatttctca actgaattcg240tactttccga gaaccgtccc atttaacata atctgcttat caaagggcaa cgccgaaggt300cttggcgaaa ccagggtaag agaattaaat gagtcggccg ctttattgct acacaatgaa360agagcagtct ccgtacaggt gatggatttc caggatggta tggacgaaat atgggatatt420gattctataa cttcttctct ttcacaaaag atagatataa agaatcataa cttgaaccag480attatcgtta cctttgattc atatggtgta tcaaatcata tcaaccacaa aagctgttat540gctgccgtta aaaagttggt ggatgattat gctcaaccta agaceaaaag aaatgaacaa600ccacctcatg tcactgcgct ttatttgaga agctacaaga acaacatcgt tttaaagtac660aactccttta tttgggaaat cctaaaaata ctttacgacc tgatttctcc attccgtaga720ataattcagg cgcttccgcc taacacagcc gccgaaaaag acaagctttc acttatgaat780acacatgcac aatacgtact agcgtttgcc actatgctaa atgctcacga atcccaagtt840gtgtggttta gatacggatg gtggatattt tccagatttg tcttcgttaa tgaatttgat900gtttatacat attag915<210>29<211>304<212>PRT<213>酿酒酵母(Saccharomyces cerevisiae)<400>29Met Lys Met Leu Arg Arg Thr Lys Val Asn Phe Ser Lys Leu Leu Tyr1 5 10 15Lys Ile Thr Lys Leu Ala Ile Val Leu Thr Ile Leu Tyr Ile Tyr Phe20 25 30Thr Pro Lys Ile Val Ser Arg Asn Asn Ala Ser Leu Gln His Ile Phe35 40 45Pro His Lys Tyr Gly Asp Tyr Glu Ile Asn Leu Val Ile Ala His Pro50 55 60Asp Asp Glu Val Met Phe Phe Ser Pro Ile Ile Ser Gln Leu Asn Ser65 70 75 80
Tyr Phe Pro Arg Thr Val Pro Phe Asn Ile Ile Cys Leu Ser Lys Gly85 90 95Asn Ala Glu Gly Leu Gly Glu Thr Arg Val Arg Glu Leu Asn Glu Ser100 105 110Ala Ala Leu Leu Leu His Asn Glu Arg Ala Val Ser Val Gln Val Met115 120 125Asp Phe Gln Asp Gly Met Asp Glu Ile Trp Asp Ile Asp Ser Ile Thr130 135 140Ser Ser Leu Ser Gln Lys Ile Asp Ile Lys Asn His Asn Leu Asn Gln145 150 155 160Ile Ile Val Thr Phe Asp Ser Tyr Gly Val Ser Asn His Ile Asn His165 170 175Lys Ser Cys Tyr Ala Ala Val Lys Lys Leu Val Asp Asp Tyr Ala Gln180 185 190Pro Lys Thr Lys Arg Asn Glu Gln Pro Pro His Val Thr Ala Leu Tyr195 200 205Leu Arg Ser Tyr Lys Asn Asn Ile Val Leu Lys Tyr Asn Ser Phe Ile210 215 220Trp Glu Ile Leu Lys Ile Leu Tyr Asp Leu Ile Ser Pro Phe Arg Arg225 230 235 240Ile Ile Gln Ala Leu Pro Pro Asn Thr Ala Ala Glu Lys Asp Lys Leu245 250 255Ser Leu Met Asn Thr His Ala Gln Tyr Val Leu Ala Phe Ala Thr Met260 265 270Leu Asn Ala His Glu Ser Gln Val Val Trp Phe Arg Tyr Gly Trp Trp275 280 285Ile Phe Ser Arg Phe Val Phe Val Asn Glu Phe Asp Val Tyr Thr Tyr290 295 300<210>30<211>2760<212>DNA<213>酿酒酵母(Saccharomyces cerevi siae)<400>30atgtggaaca aaaccagaac gacgcttctg gctgttggtg tcttatttca tttattttac 60ctatggtcta tttttgatat ctatttcatt tcaccgctcg ttcatggtat gagcccatat120caaagtactc caacccctcc tgcaaagaga ttgtttttga ttgtcggtga tggtttacgt180gcagatacca cttttgataa agtcactcat ccagtatccg gaaaaacaga atttctggca240
ccttttatta gatctttggt aatgaataat gccacctacg gtatatcaca taccagaatg300ccaactgaat cccgtcctgg tcatgttgct atgattgctg ggttttacga agatgttagt360gccgtcacaa aaggttggaa gtcaaaccct gtcaatttcg atagtttttt caaccaatct420actcatactt attcattcgg ttcacctgac attttaccta tgttcaaaga tggcgcttct480gacccaaata aagttgacac ttggatgtat gatcatactt tcgaggattt tacgcaatct540tccatcgagc tggatgcttt tgtctttaga cacttggatc aattattcca caattccaca600ctgaactcaa cattggatta tgaaattagg caagacggta atgtattctt tctacatcta660ctaggttgcg atactgccgg acattcttat agaccatatt ctgccgagta ttatgacaat720gtcaaatata ttgatgatca aatccctatc cttatagaca aagtcaacaa gttttttgcg780gacgacaaaa ccgcatttat ttttacagca gatcatggta tgagtgcatt tggatcacat840ggtgacggtc atcctaacaa cacaaggacc cctcttgttg cttggggtgc gggtttgaat900aaaccagtac ataatccttt tccggtatcc gacaactata ctgaaaattg ggagctttcg960agcattaaaa gaaatgatgt caagcaagca gatattgctt ctttaatgtc atacttgatt1020ggtgtgaact atcctaaaaa ttcagttggt gagttaccaa tagcatatat cgatggaaaa1080gaaagtgaca agcttgccgc attgtacaac aacgcaagaa gcattttaga gcagtactta1140gtcaagcaag atgaggtaat agactctcaa tttttttata aggaatactt caagtttgtt1200gaaaagtctc attcacatta cttagaagag atagaaacct taattcagcg tatatctgaa1260ggagaaaact atttggaaca agaagcaatc acccttacag aggaattaat gcagataaca1320ttggaaggtt tacattattt gacaacctat aattggagat tcattagaac tattgttaca1380tttgggtttg ttgggtggat ctttttttct tttataatat ttttgaaatc attcatatta1440gagaatgtaa ttgatgacca aaaagcgtca ccattaagcc atgcagtatt tggttccata1500ggaattttac taaattggat tttgttctac caacattctc cgttcaattt ttacatgtac1560cttcttttcc cattatactt ttggagctat atttttacaa atagatccgt actacgttca1620ggtatcaagg aattcttcaa aggtacctct ccttggaaaa gagttttaat aacaatctct1680attatatcag tttatgaggg aattgtatat ggatttttcc atagatggac gtttacgcta1740attacaaata tattggcgtt ttacccgttt atttgtgggg tgagagagct atccgtgaat1800atattgtgga tcataactag tgttctttta tctacattta ccttatttga cgctgttaaa1860attgaggact tgaaccagat acatctagca gggttattaa tcattctcag tgccttttat1920gctctttaca aaatacattc caggataaat tcctacacgc gtgctatatt tgccattcaa1980
atttccttgg tggctgccat gttggcggtt actcatcgtt cagttatctc tttacagcta2040agacaagggt taccaagaga gtcacaggtc gctggatgga taattttttt tgtatctctt2100tttgtaatgc caattttaca ttataggaag cccaacaatg attacaaagt gagattattg2160atcatttatt taaccttcgc accatccttt atcattttga ctatatcatt cgaatccctt2220ttctacttct tgttcactag ttacatggta caatggattg aaattgagaa caaaatcaaa2280gaaatgaaga cccaaaaaga tgaaaattgg ttacaagtgc taagagtttc agtaatcggg2340ttctttttac ttcaagtcgc attctttgga actggtaacg tcgcttcaat ctcttcattt2400tcattggagt ctgtttgtag attgttgcca atttttgatc ctttcctgat gggcgcatta2460ttgatgttga aattgataat tccctacggg ctattgtcca catgcctagg tatactgaat2520ttaaaactta acttcaagga ctacacaatc tcatcattaa ttatttccat gagtgatatt2580ctgtcgttga attttttcta ccttttaaga acggaggggt cgtggttgga tattggcata2640accatttcca actattgttt ggcgatccta tcatctttgt tcatgcttat tttggaagta2700ctcggtcatg tgttgctaaa aaatgtcatc atacaggata aaaccaaaaa aacacaatag2760<210>31<211>919<212>PRT<213>酿酒酵母(Saccharomyces cerevisiae)<400>31Met Trp Asn Lys Thr Arg Thr Thr Leu Leu Ala Val Gly Val Leu Phe1 5 10 15His Leu Phe Tyr Leu Trp Ser Ile Phe Asp Ile Tyr Phe Ile Ser Pro20 25 30Leu Val His Gly Met Ser Pro Tyr Gln Ser Thr Pro Thr Pro Pro Ala35 40 45Lys Arg Leu Phe Leu Ile Val Gly Asp Gly Leu Arg Ala Asp Thr Thr50 55 60Phe Asp Lys Val Thr His Pro Val Ser Gly Lys Thr Glu Phe Leu Ala65 70 75 80Pro Phe Ile Arg Ser Leu Val Met Asn Asn Ala Thr Tyr Gly Ile Ser85 90 95His Thr Arg Met pro Thr Glu Ser Arg Pro Gly His Val Ala Met Ile100 105 110Ala Gly Phe Tyr Glu Asp Val Ser Ala Val Thr Lys Gly Trp Lys Ser115 120 125Asn Pro Val Asn Phe Asp Ser Phe Phe Asn Gln Ser Thr His Thr Tyr
130 135 140Ser Phe Gly Ser Pro Asp Ile Leu Pro Met Phe Lys Asp Gly Ala Ser145 150 155 160Asp Pro Asn Lys Val Asp Thr Trp Met Tyr Asp His Thr Phe Glu Asp165 170 175Phe Thr Gln Ser Ser Ile Glu Leu Asp Ala Phe Val Phe Arg His Leu180 185 190Asp Gln Leu Phe His Asn Ser Thr Leu Asn Ser Thr Leu Asp Tyr Glu195 200 205Ile Arg Gln Asp Gly Asn Val Phe Phe Leu His Leu Leu Gly Cys Asp210 215 220Thr Ala Gly His Ser Tyr Arg Pro Tyr Ser Ala Glu Tyr Tyr Asp Asn225 230 235 240Val Lys Tyr Ile Asp Asp Gln Ile Pro Ile Leu Ile Asp Lys Val Asn245 250 255Lys Phe Phe Ala Asp Asp Lys Thr Ala Phe Ile Phe Thr Ala Asp His260 265 270Gly Met Ser Ala Phe Gly Ser His Gly Asp Gly His Pro Asn Asn Thr275 280 285Arg Thr Pro Leu Val Ala Trp Gly Ala Gly Leu Asn Lys Pro Val His290 295 300Asn Pro Phe Pro Val Ser Asp Asn Tyr Thr Glu Asn Trp Glu Leu Ser305 310 315 320Ser Ile Lys Arg Asn Asp Val Lys Gln Ala Asp Ile Ala Ser Leu Met325 330 335Ser Tyr Leu Ile Gly Val Asn Tyr Pro Lys Asn Ser Val Gly Glu Leu340 345 350Pro Ile Ala Tyr Ile Asp Gly Lys Glu Ser Asp Lys Leu Ala Ala Leu355 360 365Tyr Asn Asn Ala Arg Ser Ile Leu Glu Gln Tyr Leu Val Lys Gln Asp370 375 380Glu Val Ile Asp Ser Gln Phe Phe Tyr Lys Glu Tyr Phe Lys Phe Val385 390 395 400Glu Lys Ser His Ser His Tyr Leu Glu Glu Ile Glu Thr Leu Ile Gln405 410 415Arg Ile Ser Glu Gly Glu Asn Tyr Leu Glu Gln Glu Ala Ile Thr Leu420 425 430Thr Glu Glu Leu Met Gln Ile Thr Leu Glu Gly Leu His Tyr Leu Thr435 440 445
Thr Tyr Asn Trp Arg Phe Ile Arg Thr Ile Val Thr Phe Gly Phe Val450 455 460Gly Trp Ile Phe Phe Ser Phe Ile Ile Phe Leu Lys Ser Phe Ile Leu465 470 475 480Glu Asn Val Ile Asp Asp Gln Lys Ala Ser Pro Leu Ser His Ala Val485 490 495Phe Gly Ser Ile Gly Ile Leu Leu Asn Trp Ile Leu Phe Tyr Gln His500 505 510Ser Pro Phe Asn Phe Tyr Met Tyr Leu Leu Phe Pro Leu Tyr Phe Trp515 520 525Ser Tyr Ile Phe Thr Asn Arg Ser Val Leu Arg Ser Gly Ile Lys Glu530 535 540Phe Phe Lys Gly Thr Ser Pro Trp Lys Arg Val Leu Ile Thr Ile Ser545 550 555 560Ile Ile Ser Val Tyr Glu Gly Ile Val Tyr Gly Phe Phe His Arg Trp565 570 575Thr Phe Thr Leu Ile Thr Asn Ile Leu Ala Phe Tyr Pro Phe Ile Cys580 585 590Gly Val Arg Glu Leu Ser Val Asn Ile Leu TrpIle Ile Thr Ser Val595 600605Leu Leu Ser Thr Phe Thr Leu Phe Asp Ala Val Lys Ile Glu Asp Leu610 615 620Asn Gln Ile His Leu Ala Gly Leu Leu Ile Ile Leu Ser Ala Phe Tyr625 630 635 640Ala Leu Tyr Lys Ile His Ser Arg Ile Asn Ser Tyr Thr Arg Ala Ile645 650 655Phe Ala Ile Gln Ile Ser Leu Val Ala Ala Met Leu Ala Val Thr His660 665 670Arg Ser Val Ile Ser Leu Gln Leu Arg Gln Gly Leu Pro Arg Glu Ser675 680 685Gln Val Ala Gly Trp Ile Ile Phe Phe Val Ser Leu Phe Val Met Pro690 695 700Ile Leu His Tyr Arg Lys Pro Asn Asn Asp Tyr Lys Val Arg Leu Leu705 710 715 720Ile Ile Tyr Leu Thr Phe Ala Pro Ser Phe Ile Ile Leu Thr Ile Ser725 730 735Phe Glu Ser Leu Phe Tyr Phe Leu Phe Thr Ser Tyr Met Val Gln Trp740 745 750Ile Glu Ile Glu Asn Lys Ile Lys GIu Met Lys Thr Gln Lys Asp Glu
755 760 765Asn Trp Leu Gln Val Leu Arg Val Ser Val Ile Gly Phe Phe Leu Leu770 775 780Gln Val Ala Phe Phe Gly Thr Gly Asn Val Ala Ser Ile Ser Ser Phe785 790 795 800Ser Leu Glu Ser Val Cys Arg Leu Leu Pro Ile Phe Asp Pro Phe Leu805 810 815Met Gly Ala Leu Leu Met Leu Lys Leu Ile Ile Pro Tyr Gly Leu Leu820 825 830Ser Thr Cys Leu Gly Ile Leu Asn Leu Lys Leu Asn Phe Lys Asp Tyr835 840 845Thr Ile Ser Ser Leu Ile Ile Ser Met Ser Asp Ile Leu Ser Leu Asn850 855 860Phe Phe Tyr Leu Leu Arg Thr Glu Gly Ser Trp Leu Asp Ile Gly Ile865 870 875 880Thr Ile Ser Asn Tyr Cys Leu Ala Ile Leu Ser Ser Leu Phe Met Leu885 890 895Ile Leu Glu Val Leu Gly His Val Leu Leu Lys Asn Val Ile Ile Gln900 905 910Asp Lys Thr Lys Lys Thr Gln915<210>32<211>660<212>DNA<213>酿酒酵母(Saccharomyces cerevisiae)<400>32atgccagcta aaaaaaggac tagaaagaca gtgaaaaaaa ccgtatcatt ctccgatgac 60acaacattaa caacgcacca aaatcgtgag aaaaagaacg tagatcatga tcgtccacct120gtgtatgtga ggaaaacccc tctgatgaca tttccatacc atttagtagc actactttat180tactacgttt ttgtatcttc aaatttcaat acggtgaagt tgctaagttt tttgattcct240acacaagttg cttatttagt tttacaattc aataaatgca cagtttacgg taacaaaatc300attaagatca attactcatt gaccattatt tgtctaggtg ttacattttt gttgagcttt360cccacaatgt tattaactat attatttggt gcgccattaa tggacttatt gtgggaaacc420tggctgttgt cactgcattt tgcattttta gcataccctg cagtttattc tgtatttaat480tgtgatttca aagtgggatt atggaagaag tattttatct ttatcgttgt agggggttgg540attagttgtg ttgtcattcc tttggattgg gatagagatt ggcagaattg gccaattcct600
attgttgttg gaggttattt gggcgctttg gtgggctata ctatcggtgc ctatatataa660<210>33<211>219<212>PRT<213>酿酒酵母(Saccharomyces cerevisiae)<400>33Met Pro Ala Lys Lys Arg Thr Arg Lys Thr Val Lys Lys Thr Val Ser1 510 15Phe Ser Asp Asp Thr Thr Leu Thr Thr His Gln Asn Arg Glu Lys Lys20 25 30Asn Val Asp His Asp Arg Pro Pro Val Tyr Val Arg Lys Thr Pro Leu35 40 45Met Thr Phe Pro Tyr His Leu Val Ala Leu Leu Tyr Tyr Tyr Val Phe50 55 60Val Ser Ser Asn Phe Asn Thr Val Lys Leu Leu Ser Phe Leu Ile Pro65 70 75 80Thr Gln Val Ala Tyr Leu Val Leu Gln Phe Asn Lys Cys Thr Val Tyr85 90 95Gly Asn Lys Ile Ile Lys Ile Asn Tyr Ser Leu Thr Ile Ile Cys Leu100 105 110Gly Val Thr Phe Leu Leu Ser Phe Pro Thr Met Leu Leu Thr Ile Leu115 120 125Phe Gly Ala Pro Leu Met Asp Leu Leu Trp Glu Thr Trp Leu Leu Ser130 135 140Leu His Phe Ala Phe Leu Ala Tyr Pro Ala Val Tyr Ser Val Phe Asn145 150 155 160Cys Asp Phe Lys Val Gly Leu Trp Lys Lys Tyr Phe Ile Phe Ile Val165 170 175Val Gly Gly Trp Ile Ser Cys Val Val Ile Pro Leu Asp Trp Asp Arg180 185 190A sp Trp Gln Asn Trp Pro Ile Pro Ile Val Val Gly Gly Tyr Leu Gly195 200 2 05Ala Leu Val Gly Tyr Thr Ile Gly Ala Tyr Ile210 215<210> 34<211> 2493<212>DNA<213>酿酒酵母(Saccharomyces cerevisiae)
<400> 34atgaacttga agcagttcac gtgcctatca tgcgctcaat tactcgctat tctgctcttt60atctttgctt ttttccctag aaaaatcgtg ctgacaggta tatcaaagca agatccggat120caagaccgtg atctccagcg cgataggccc ttccagaaat tggtgtttgt gatcattgat180gctctcagat cagactttct ttttgattcg cagatttccc acttcaacaa cgtgcaccaa240tggctcaata cgggcgaagc atggggttac acgtcatttg ctaatccgcc taccgtgacg300ctgcctagac tcaaaagtat tactacggga tctacaccta gcttcattga cttgctgctg360aatgtagccc aggacataga ttccaacgat ctttcggagc acgattcctg gctgcagcag420ttcatccaac ataataacac gattcgtttc atgggcgatg acacctggct gaaactgttc480ceacagcaat ggtttgactt cgctgacccg acacactcgt tctttgtcag tgatttcact540caagtcgata ataatgtgac gaggaacttg cccgggaaat tatttcagga atgggcccag600tgggacgtgg ctatcctgca ttacttgggt cttgaccata tcgggcataa agatggcccg660cattcaaagt ttatggctgc taaacatcaa gaaatggaca gcattctgaa gtcaatatat720gatgaagtgt tggaacatga agatgacgat gatacactga tttgtgttct tggcgaccat780ggaatgaacg aactgggcaa ccatggtggc tcttcagccg gcgaaacatc agcaggattg840ttgtttttgt cacctaagct ggcgcaattt gctaggccag aatcgcaagt aaactacaca900ttgcccatca acgctagtcc ggactggaat ttccagtatt tagagactgt tcaacaaatt960gatatcgtcc ccaccatagc agcactgttt ggtatgccaa tccccatgaa cagtgttggg1020ataataatac ctgacttttt acaactgttg cccaataagt tggcaagtat gaaagaaaat1080tttatgcatt tgtggaaatt atcagaccat cacggcgagg ttgctcttga cgatttcact1140gccgaagata tttatacaaa gatgtacact attcaagaaa cgttaaccaa gtctgcaaca1200aattataatt atcctctttt gacactggct tttgttggtt tcctcataat aacaatcatc1260gccatttatg tattattacg ttattctggg cctgattttt ggcagttgcg cgtttcttcc1320ctgtctgttc tgttagtttc cattatacta ggcgtttcca catttgcaag tagtttcatt1380gaagaggagc accaactgtg gtggtggata gtaactgcat tctcggcggt ccctctgttc1440gtataccgat tgaatgtgct cataatcgtg cgctggttta taatgatggc atgcgtacgc1500tcaatcaagt tttggaataa cagtggccag aaattcattt attctaacgt tatgtccaat1560ctacttaatc agaatccttc ctggaagtgg tgcttaaata tgttgacatt tctagtgctg1620ataatggcat ctgctggttt tcaagtacta cattttattg tcactactat tttggtgggg1680ttgtgtttca cgtacaaaat ctcgtgggaa atcgtcaatg gtaaccaggc agaaataccg1740
ctctttatgc atgatttact ggctaagata gactttgcac caactgaaag taacttgatt1800gtacttgcgc gcgttttctt ccaagcttgg gctattgttg tcatttcaag gttggtcctg1860acgaaattga aagtacttaa caagaactac ctcattaaag atatgaaagt ttatataaca1920attcttttga tgttccaaac ttcttctcag aacataggtc aatttctcgt tttccaaata1980ttagagtccc aaatttttta ctttttccaa aatattccaa ccgcctcatt aacatcaaca2040agtaagattt atttttcgaa tttggtgtcc ttaattttac aaaattttac atttttccaa2100ttcggtggca caaattccat ttctactata gaccttggaa acgcatacca tggtgtttcc2160tcagactaca acatctacgt agtggggata ttaatgtccg ttgccaattt cgcgccggca 2220atatactggt ccatgctacc gtggtcaata aactacgcct ctattccagc acaagttaag2280ttgcaaacgt tcatcagaag taagttacct gccttcacct atcattgtat atttggaact2340tgtttgatga cggcatgcgt cgttttgaga tttcatctet ttatttggtc cgttttcagt 2400ccaaaattat gttattttct tgggtggaat tttgtgatgg gattgctgaa tggctggtta2460cctgaattgg ccctcctttg cgctcttgat taa 2493<210>35<211> 830<212>PRT<213>酿酒酵母(Saccharomyces cerevisiae)<400> 35Met Asn Leu Lys Gln Phe Thr Cys Leu Ser Cys Ala Gln Leu Leu Ala1 5 10 15Ile Leu Leu Phe Ile Phe Ala Phe Phe Pro Arg Lys Ile Val Leu Thr20 25 30Gly Ile Ser Lys Gln Asp Pro Asp Gln Asp Arg Asp Leu Gln Arg Asp35 40 45Arg Pro Phe Gln Lys Leu Val Phe Val Ile Ile Asp Ala Leu Arg Ser50 55 60Asp Phe Leu Phe Asp Ser Gln Ile Ser His Phe Asn Asn Val His Gln65 70 75 80Trp Leu A sn Thr Gly Glu Ala Trp Gly Tyr Thr Ser Phe Ala Asn Pro85 90 95Pro Thr Val Thr Leu Pro Arg Leu Lys Ser Ile Thr Thr Gly Ser Thr100 105 110Pro Ser Phe Ile Asp Leu Leu Leu Asn Val Ala Gln Asp Ile Asp Ser115 120 125
Asn Asp Leu Ser Glu His Asp Ser Trp Leu Gln Gln Phe Ile Gln His130 135 140Asn Asn Thr Ile Arg Phe Met Gly Asp Asp Thr Trp Leu Lys Leu Phe145 150 155 160Pro Gln Gln Trp Phe Asp Phe Ala Asp Pro Thr His Ser Phe Phe Val165 170 175Ser Asp Phe Thr Gln Val Asp Asn Asn Val Thr Arg Asn Leu Pro Gly180 185 190Lys Leu Phe Gln Glu Trp Ala Gln Trp Asp Val Ala Ile Leu His Tyr195 200 205Leu Gly Leu Asp His Ile Gly His Lys Asp Gly Pro Hi s Ser Lys Phe210 215 220Met Ala Ala Lys His Gln Glu Met Asp Ser Ile Leu Lys Ser Ile Tyr225 230 235 240Asp Glu Val Leu Glu Hi s Glu Asp Asp Asp Asp Thr Leu Ile Cys Val245 250 255Leu Gly Asp His Gly Met Asn Glu Leu Gly Asn Hi s Gly Gly Ser Ser260 265 270Ala Gly Glu Thr Ser Ala Gly Leu Leu Phe Leu Ser Pro Lys Leu Ala275 280 285Gln Phe Ala Arg Pro Glu Ser Gln Val Asn Tyr Thr Leu Pro Ile Asn290 295 300Ala Ser Pro Asp Trp Asn Phe Gln Tyr Leu Glu Thr Val Gln Gln Ile305 310 315 320Asp Ile Val Pro Thr Ile Ala Ala Leu Phe Gly Met Pro Ile Pro Met325 330 335Asn Ser Val Gly Ile Ile Ile Pro Asp Phe Leu Gln Leu Leu Pro Asn340 345 350Lys Leu Ala Ser Met Lys Glu Asn Phe Met Hi s Leu Trp Lys Leu Ser355 360 365Asp His His Gly Glu Val Ala Leu Asp Asp Phe Thr Ala Glu Asp Ile370 375 380Tyr Thr Lys Met Tyr Thr Ile Gln Glu Thr Leu Thr Lys Ser Ala Thr385 390 395 400Asn Tyr Asn Tyr Pro Leu Leu Thr Leu Ala Phe Val Gly Phe Leu Ile405 410 415Ile Thr Ile Ile Ala Ile Tyr Val Leu Leu Arg Tyr Ser Gly Pro Asp420 425 430Phe Trp Gln Leu Arg Val Ser Ser Leu Ser Val Leu Leu Val Ser Ile
435 440 445Ile Leu Gly Val Ser Thr Phe Ala Sel Ser Phe Ile Glu Glu Glu His450 455 460Gln Leu Trp Trp Trp Ile Val Thr Ala Phe Ser Ala Val Pro Leu Phe465 470 475 480Val Tyr Arg Leu Asn Val Leu Ile Ile Val Arg Trp Phe Ile Met Met485 490 495Ala Cys Val Arg Ser Ile Lys Phe Trp Asn Asn Ser Gly Gln Lys Phe500 505 510Ile Tyr Ser Asn Val Met Ser Asn Leu Leu Asn Gln Asn Pro Ser Trp515 520 525Lys Trp Cys Leu Asn Met Leu Thr Phe Leu Val Leu Ile Met Ala Ser530 535 540Ala Gly Phe Gln Val Leu His Phe Ile Val Thr Thr Ile Leu Val Gly545 550 555 560Leu Cys Phe Thr Tyr Lys Ile Ser Trp Glu Ile Val A sn Gly Asn Gln565 570 575Ala Glu Ile Pro Leu Phe Met His Asp Leu Leu Ala Lys Ile Asp Phe580 585 590Ala Pro Thr Glu Ser Asn Leu Ile Val Leu Ala Arg Val Phe Phe Gln595 600 605Ala Trp Ala Ile Val Val Ile Ser Arg Leu Val Leu Thr Lys Leu Lys610 615 620Val Leu Asn Lys Asn Tyr Leu Ile Lys Asp Met Lys Val Tyr Ile Thr625 630 635 640Ile Leu Leu Met Phe Gln Thr Ser Ser Gln Asn Ile Gly Gln Phe Leu645 650 655Val Phe Gln Ile Leu Glu Ser Gln Ile Phe Tyr Phe Phe Gln Asn Ile660 665 670Pro Thr Ala Ser Leu Thr Ser Thr Ser Lys Ile Tyr Phe Ser Asn Leu675 680 685Val Ser Leu Ile Leu Gln Asn Phe Thr Phe Phe Gln Phe Gly Gly Thr690 695 700Asn Ser Ile Ser Thr Ile Asp Leu Gly Asn Ala Tyr Hi s Gly Val Ser705 710 715 720Ser Asp Tyr Asn Ile Tyr Val Val Gly Ile Leu Met Ser Val Ala Asn725 730 735Phe Ala Pro Ala Ile Tyr Trp Ser Met Leu Pro Trp Ser Ile Asn Tyr740 745 750
Ala Ser Ile Pro Ala Gln Val Lys Leu Gln Thr Phe Ile Arg Ser Lys755 760 76 5Leu Pro Ala Phe Thr Tyr Hi s Cys Ile Phe Gly Thr Cys Leu Met Thr770 775 780Ala Cys Val Val Leu Arg Phe Hi s Leu Phe Ile Trp Ser Val Phe Ser785 790 795 800Pro Lys Leu Cys Tyr Phe Leu Gly Trp Asn Phe Val Met Gly Leu Leu805 810 815Asn Gly Trp Leu Pro Glu Leu Ala Leu Leu Cys Ala Leu Asp820 825 830<210>36<211> 1605<212>DNA<213>酿酒酵母(Saccharomyces cerevisiae)<400>36atgtccaatg caaatctaag aaaatgggtt ggtttttgct ttgttgccat ttatctcttt 60ttaggtgttc cactgtggta caagctaact acagtttata gagcatcact accaataaat120tacattgagt cacttcaaaa taacaaattc caagatattc atctcgtaat accggtgtat180gttaagtcag atacttacag atttcctgac gttcatgacg ctatccaagt acaagttaac240catttattga attctcagga gcaacgggtc ccttggtctt tacaagttct tccatataat300gagactattg agcagatgga aagtgaaggc aaccagtttc atgtcgttac tttgaagtta360gacgaattta ttggttactc atcagcttac gacaccaaag aaacactagt atattacgac420gatgctgccg ttttaagtaa tgatctaccg ttttttgttg ctcaaacatt ggtagagcac480actttccaat tggaatggac gcatttgaat aaaacgtgtg aaggcgtttc tacaaacaac540gatgtcgcaa tatcttatga tccaaacatt catttaagtg taactttatt gtcaggtgat600gggaatcctg ttgcatggga aattgagcct acattaactg actacttttc accttttagg660aagttcttat caccactggt aaattttaca gtagattcat ccattgttta tcataatgat720ttgaatttgc attcattaaa tggatcatgt acaagcgtta cgtggtttga tctctctcat780actattgatc tttctgaact ttcttcaatg gcctattacc cagaagattc tgcactgaat840ttagccatag tctttcctag tgcttcttea agtcccgatg gtctggcgtt cattaatggc900actcggattt cagacgaaat aaccacatta gattggaata gttatctagt tcctcaatgg960ggggttataa taataaataa aatgccgttg aagccaaatt cagtcattag cgaagattat1020ttagaaccta tgatgtaccg ttttgcgaca gatatttttc aactattggg attaacggag1080
ggctcgcaag atttgttatc accttatatt accatagatt cattcaaaag gttgacaatt 1140ttacagaatc tagataaagc tacggaaaca ttatggtcgt tagtgaaatt aactcaacaa 1200tttcagggca tgtctatccc acgcgaagta tcggataatg ttatcgaagc tttagactta 1260aggctacaga ttattgattt attaaatgat cctggaaagg gtggagatat cgtctggaac 1320aatgccctgc atctaagtaa tgaattggtt aaactatgcg aaaaggcatt tttcaatgga 1380gaaatggttc aacaaaattt cttcccacaa gagcacatga tagctgtgta tttaccttta 1440ttaggcccaa tatcggcagt catgttcttt ggtttctaca acgtgatgaa ggaaaagaat 1500caaaagagta aaaagaatgg aaccgagaga gaagttgcta aagaaaaatt agagttgaaa 1560gaggctcaaa aattacatgc tattgatggt gaagatgaat tatga 1605<210> 37<211>534<212>PRT<213>酿酒酵母(Saccharomyces cerevisiae)<400> 37Met Ser Asn Ala Asn Leu Arg Lys Trp Val Gly Phe Cys Phe Val Ala15 10 15Ile Tyr Leu Phe Leu Gly Val Pro Leu Trp Tyr Lys Leu Thr Thr Val20 25 30Tyr Arg Ala Ser Leu Pro Ile Asn Tyr Ile Glu Ser Leu Gln Asn Asn35 40 45Lys Phe Gln Asp Ile His Leu Val Ile Pro Val Tyr Val Lys Ser Asp50 55 60Thr Tyr Arg Phe Pro Asp Val Hi s Asp Ala Ile Gln Val Gln Val Asn65 70 75 80His Leu Leu Asn Set Gln Glu Gln Arg Val Pro Trp Ser Leu Gln Val85 90 95Leu Pro Tyr Asn Glu Thr Ile Glu Gln Met Glu Ser Glu Gly Asn Gln100 l05 110Phe His Val Val Thr Leu Lys Leu Asp Glu Phe Ile Gly Tyr Ser Ser115 120 125Ala Tyr Asp Thr Lys Glu Thr Leu Val Tyr Tyr Asp Asp Ala Ala Val130 135 140Leu Ser Asn Asp Leu Pro Phe Phe Val Ala Gln Thr Leu Val Glu His145 150 155 160Thr Phe Gln Leu Glu Trp Thr Hi s Leu Asn Lys Thr Cys Glu Gly Val165 170 175
Ser Thr Asn Asn Asp Val Ala Ile Ser Tyr Asp Pro Asn Ile His Leu180 185 190Ser Val Thr Leu Leu Ser Gly Asp Gly Asn Pro Val Ala Trp Glu Ile195 200 205Glu Pro Thr Leu Thr Asp Tyr Phe Ser Pro Phe Arg Lys Phe Leu Ser210 215 220Pro Leu Val Asn Phe Thr Val Asp Ser Ser Ile Val Tyr His Asn Asp225 230 235 240Leu Asn Leu His Ser Leu Asn Gly Ser Cys Thr Ser Val Thr Trp Phe245 250 255Asp Leu Ser His Thr Ile Asp Leu Ser Glu Leu Ser Ser Met Ala Tyr260 265 270Tyr Pro Glu Asp Ser Ala Leu Asn Leu Ala Ile Val Phe Pro Ser Ala275 2 80 285Ser Ser Ser Pro Asp Gly Leu Ala Phe Ile Asn Gly Thr Arg Ile Ser290 295 300Asp Glu Ile Thr Thr Leu Asp Trp Asn Ser Tyr Leu Val Pro Gln Trp305 310 3l5 320Gly Val Ile Ile Ile Asn Lys Met Pro Leu Lys Pro Asn Ser Val Ile325 330 335Ser Glu Asp Tyr Leu Glu Pro Met Met Tyr Arg Phe Ala Thr Asp Ile340 345 350Phe Gln Leu Leu Gly Leu Thr Glu Gly Ser Gln Asp Leu Leu Ser Pro355 360 365Tyr Ile Thr Ile Asp Ser Phe Lys Arg Leu Thr Ile Leu Gln Asn Leu370 375 380Asp Lys Ala Thr Glu Thr Leu Trp Ser Leu Val Lys Leu Thr Gln Gln385 390 395 400Phe Gln Gly Met Ser Ile Pro Arg Glu Val Ser Asp Asn Val Ile Glu405 410 415Ala Leu Asp Leu Arg Leu Gln Ile Ile Asp Leu Leu Asn Asp Pro Gly420 425 430Lys Gly Gly Asp Ile Val Trp Asn Asn Ala Leu Hi s Leu Ser Asn Glu435 440 445Leu Val Lys Leu Cys Glu Ly s Ala Phe Phe Asn Gly Glu Met Val Gln450 455 460Gln Asn Phe Phe Pro Gln Glu Hi s MetIle Ala Val Tyr Leu Pro Leu465 470 475 480
Leu Gly Pro Ile Ser Ala Val Met Phc Phe Gly Phe Tyr Asn Val Met485 490 495Lys Glu Lys Asn Gln Lys Ser Lys Lys Asn Gly Thr Glu Arg Glu Val500 505 51 0Ala Lys Glu Lys Leu Glu Leu Lys Glu Ala Gln Lys Leu His Ala Ile515 520 525Asp Gly Glu Asp Glu Leu530<210> 38<211> 1833<212>DNA<213>酿酒酵母(Saccharomyces cerevisiae)<400> 38atgatcctca cactggccta tttcatgctg ggcactctac tactgggtgt gttcgcagaa 60gacacggtgt cgcagattgg tataaatgac agtttatggt atccgtacga cgaagcactg120gtgttgaagc cgctgcccaa caatgatctg ctactctcat ttgcattcca actgcaatcg180gaaccgtttg acccggccgt atcatccatg tcatatgatg cgtatgagca ctacacgact240ttcccacggg ccatcccacc attgttggaa tctactgcca cgcgtcagtt tcatttaaga300tttaccagag gattctggga tgccctgtcg tggggacagt tgccacatgc tggaaaagag360gcaggtgcct caggtgtgga attgtggtcg caagtgcagg ccatggatca ggaacaggcg420ttccataatt ggaaaaaact gtccaattca ttgageggat tgttttgttc ttctttaaat480tttatcgacg agtcaaggac gacctttccc cggcggtcat atgcttctga tataggagct540cctcttttca atagcaccga gaaactgtac ctgatgcgag catcgttgcc caatgaaccc600atctgtaccg agaacttgac gccgttcata aaactattgc ctactagggg caaatccggt660ttgacatctc tcttggatgg tcataaattg ttcgactctc tatggaatag tatttccttg720gatattgcca ctatttgttc tgaagatgaa gatgctcttt gtcactacga gatggacgca780cgcatagaaa tggtaacaca cgttccctcc gccttggcaa gaggtgagag acctatcccc840aaacctttgg atgggaacac attgcgttgt gacacggata aaccctttga ttcttaccaa900tgcttccctc taccggaacc ttcgcagact cacttcaagc tgtctcagct gtttgccaga960ccaataaaca atggcaacct gtttgctaat aggcccacaa gaatttgtgc agaagttgac1020cgttctacct ggactgcgtt tttgtcagtt gacgatacta ttttcagcac acatgataat1080tgctttgact tatcaaacga tcaaaatgag ggtggttcgg gctacgactt tattttagaa1140tcgacggaca ctactaaagt tactcccata gttcctgtcc caattcacgt aagcagatct1200
ctgactggta atggacaaga tcgtggtgga atgcgtattg ttttccataa cgacaatgat 1260acccctgtga agttgattta tttcgaatca ttgccatggt tcatgagagt ttacctatcc 1320tctcttcaaa ttacttctac tacctctccg caattgcaag aaaacgatat catcttagat 1380aaatactatt tacaagcggc cgatagaaaa agacctggcc acttggagtt cacgatgtta 1440attccagcta atacggacat tgtaatgact tatcaattcg ataaagctct tctgcaattt 1500gccgagtatc caccagatgc aaaccatggt tttgaaatcg atgcagctgt aatcaccgtg 1560ctatctttgg agtcctcatc gtctctttat gaaatgagaa cctctaccct attgttatcc 1620ctgtctacac cggattttag tatgccgtat aacgtcatca ttctaacatc tacaatcatg 1680ggactcatat tcggtatgct atacaatttg atggtgaaga gaatggtcac cgtcgaagag 1740gccgataaga ttacgttgca atctggctta aaatacaaat tgctaaagct aaaggaaaag 1800ttcctaggga aaaaaaagac taaaacagac taa 1833<210> 39<211> 610<212>PRT<213>酿酒酵母(Saccharomyces cerevisiae)<400> 39Met Ile Leu Thr Leu Ala Tyr Phe Met Leu Gly Thr Leu Leu Leu Gly1 5 10 l5Val Phe Ala Glu Asp Thr Val Ser Gln Ile Gly Ile Asn Asp Ser Leu20 25 30Trp Tyr Pro Tyr Asp Glu Ala Leu Val Leu Lys Pro Leu Pro Asn Asn35 40 45Asp Leu Leu Leu Ser Phe Ala Phe Gln Leu Gln Ser Glu Pro Phe Asp50 55 60Pro Ala Val Ser Ser Met Ser Tyr Asp Ala Tyr Glu Hi s Tyr Thr Thr65 70 7580Phe Pro Arg Ala Ile Pro Pro Leu Leu Glu Ser Thr Ala Thr Arg Gln85 90 95Phe His Leu Arg Phe Thr Arg Gly Phe Trp Asp Ala Leu Ser Trp Gly100 105 110Gln Leu Pro Hi s Ala Gly Lys Glu Ala Gly Ala Ser Gly Val Glu Leu115 120 125Trp Ser Gln Val Gln Ala Met Asp Gln Glu Gln Ala Phe Hi s Asn Trp130 135 140Lys Lys Leu Ser Asn Ser Leu Ser Gly Leu Phe Cys Ser Ser Leu Asn
145 150 155 160Phe Ile Asp Glu Ser Arg Thr Thr Phe Pro Arg Arg Ser Tyr Al a Ser165 170 175Asp Ile Gly Ala Pro Leu Phe Asn Ser Thr Glu Lys Leu Tyr Leu Met180 185 190Arg Ala Ser Leu Pro Asn Glu Pro Ile Cy s Thr Glu Asn Leu Thr Pro195 2 00 205Phe Ile Lys Leu Leu Pro Thr Arg Gly Lys Ser Gly Leu Thr Ser Leu210 215 220Leu Asp Gly His Lys Leu Phe Asp Ser Leu Trp Asn Ser Ile Ser Leu225 230 235 240Asp Ile Ala Thr Ile Cys Ser Glu Asp Glu Asp Ala Leu Cys His Tyr245 250 255Glu Met Asp Ala Arg Ile Glu Met Val Thr Hi s Val Pro Ser Ala Leu260 265 270Ala Arg Gly Glu Arg Pro Ile Pro Lys Pro Leu Asp Gly Asn Thr Leu275 280 285Arg Cys Asp Thr Asp Lys Pro Phe Asp Ser Tyr Gln Cy s Phe Pro Leu290 295 300Pro Glu Pro Ser Gln Thr Hi s Phe Lys Leu Ser Gln Leu Phe Ala Arg305 310 315 320Pro Ile Asn Asn Gly Asn Leu Phe Ala Asn Arg Pro Thr Arg Ile Cys325 33 0 335Ala Glu Val Asp Arg Ser Thr Trp Thr Ala Phe Leu Ser Val Asp Asp340 345 350Thr Il e Phe Ser Thr His Asp Asn Cys Phe Asp Leu Ser Asn Asp Gln355 360 365Asn Glu Gly Gly Ser Gly Tyr Asp Phe Ile Leu Glu Ser Thr A sp Thr370 375 380Thr Lys Val Thr Pro Ile Val Pro Val Pro Ile Hi s Val Ser Arg Ser385 390 395 400Leu Thr Gly Asn Gly Gln Asp Arg Gly Gly Met Arg Ile Val Phe His405 41 0 415Asn Asp Asn Asp Thr Pro Val Lys Leu Ile Tyr Phe Glu Ser Leu Pro420 425 430Trp Phe Met Arg Val Tyr Leu Ser Ser Leu Gln Ile Thr Ser Thr Thr435 440 445Ser Pro Gln Leu GlnGlu Asn Asp Ile Ile Leu Asp Lys Tyr Tyr Leu450 455 460
Gln Ala Ala Asp Arg Lys Arg Pro Gly Hi s Leu Glu Phe Thr Met Leu465 470 475 480Ile Pro Ala Asn Thr Asp Ile Val Met Thr Tyr Gln Phe Asp Lys Ala485 490 495Leu Leu Gln Phe Ala Glu Tyr Pro Pro Asp Ala Asn His Gly Phe Glu500 505 510Ile Asp Ala Ala Val Ile Thr Val Leu Ser Leu Glu Ser Ser Ser Ser515 520 525Leu Tyr Glu Met Arg Thr Ser Thr Leu Leu Leu Ser Leu Ser Thr Pro530 535 540Asp Phe Ser Met Pro Tyr Asn Val Ile Ile Leu Thr Ser Thr Ile Met545 550 555 560Gly Leu Ile Phe Gly Met Leu Tyr Asn Leu Met Val Lys Arg Met Val565 570 575Thr Val Glu Glu Ala Asp Lys Ile Thr Leu Gln Ser Gly Leu Lys Tyr580 585 590Lys Leu Leu Lys Leu Lys Glu Lys Phe Leu Gly Lys Lys Lys Thr Lys595 600 605Thr Asp610<210> 40<211>1185<212>DNA<213>酿酒酵母(Saccharomyces cerevisiae)<400>40atggattcca cagcacttaa ggtagctcta ggctgtattg caattcgttt ggctgtgaac 60agcctttttc cctctctaca acaacaactg gaccagtctg tagaattctc aactcccgta120acttcattta ggtcactaca ggaaggtata tacctactgc ggaacaacat ccaagtatat180aatcatgggg ttgttcacca tcctccaatt ttgatttttt ttctttccct ctttaattcc240gacaggttaa tttccctcat atacgcttta attgatggat taattgcgta tcagctgaca300gaggtaacaa aggctttcaa aaacttgaaa ctgaaagttt ggctacctgg acttctttat360gccgtgaatc ctttgaccct tttatcgtgc attagtcggt catcaatcat attcacaaat420tttgctattt catcgtcatt gtattgcata ttagctgaag gaaacgttct tttgtcctct480gttatgattt ctatatctgg atatttgtca gtatacccta ttctcctctt aattccgcta540ttaggtatgc tgaaaagttg gaggcaaaga atattatctg ccattgtttc cataetatct600ttattaattc tgctattatt cagctacagt atattaggca gccaaagttg gtcatttttg660
acacaggttt atggatctat tataaccttt gagaaggttt ttccaaatct gggtttgtgg720tggtacttct tcattgaaat gtttgacacc ttcataccgt tcttcaaggc tgtattcaac780atttttattg cagtattcat tacaccattt actttgcgct atcataagca gccattctac840gcattcattt tatgcattgg gtggattgtc cttacaaagc catatccctc actaggtgac900gctggttttt tcttcagctt cctacctttc ttcacgccac tatttggata tttaagatac960cccatcatat cagcattact gtttttacac gcaattgttt tggcgccaat tttctatcat1020ctttgggttg ttttaggttc agggaatagt aattttttct atgctatttc cctagtttat1080gctctggcta tagcatctat attagttgac ttgaactggg cgatgctgag aattgaatac1140gataacggta tcccaaattt caaattgaag gtaacacaaa tttaa1185<210>41<211>394<212>PRT<213>酿酒酵母(Saccharomyces cerevisiae)<400>41Met Asp Ser Thr Ala Leu Lys Val Ala Leu Gly Cys Ile Ala Ile Arg1 5 10 15Leu Ala Val Asn Ser Leu Phe Pro Ser Leu Gln Gln Gln Leu Asp Gln20 25 30Ser Val Glu Phe Ser Thr Pro Val Thr Ser Phe Arg Ser Leu Gln Glu35 40 45Gly Ile Tyr Leu Leu Arg Asn Asn Ile Gln Val Tyr Asn His Gly Val50 55 60Val His His Pro Pro Ile Leu Ile Phe Phe Leu Ser Leu Phe Asn Ser65 70 75 80Asp Arg Leu Ile Ser Leu Ile Tyr Ala Leu Ile Asp Gly Leu Ile Ala85 90 95Tyr Gln Leu Thr Glu Val Thr Lys Ala Phe Lys Asn Leu Lys Leu Lys100 105 110Val Trp Leu Pro Gly Leu Leu Tyr Ala Val Asn Pro Leu Thr Leu Leu115 120 125Ser Cys Ile Ser Arg Ser Ser Ile Ile Phe Thr Asn Phe Ala Ile Ser130 135 140Ser Ser Leu Tyr Cys Ile Leu Ala Glu Gly Asn Val Leu Leu Ser Ser145 150 155 160Val Met Ile Ser Ile Ser Gly Tyr Leu Ser Val Tyr Pro Ile Leu Leu165 170 175
Leu Ile Pro Leu Leu Gly Met Leu Lys Ser Trp Arg Gln Arg Ile Leu180 185 190Ser Ala Ile Val Ser Ile Leu Ser Leu Leu Ile Leu Leu Leu Phe Ser195 200 205Tyr Ser Ile Leu Gly Ser Gln Ser Trp Ser Phe Leu Thr Gln Val Tyr210 215 220Gly Ser Ile Ile Thr Phe Glu Lys Val Phe Pro Asn Leu Gly Leu Trp225 230 235 240Trp Tyr Phe Phe Ile Glu Met Phe Asp Thr Phe Ile Pro Phe Phe Lys245 250 255Ala Val Phe Asn Ile Phe Ile Ala Val Phe Ile Thr Pro Phe Thr Leu260 265 270Arg Tyr His Lys Gln Pro Phe Tyr Ala Phe Ile Leu Cys Ile Gly Trp275 280 285Ile Val Leu Thr Lys Pro Tyr Pro Ser Leu Gly Asp Ala Gly Phe Phe290 295 300Phe Ser Phe Leu Pro Phe Phe Thr Pro Leu Phe Gly Tyr Leu Arg Tyr305 310 315 320Pro Ile Ile Ser Ala Leu Leu Phe Leu His Ala Ile Val Leu Ala Pro325 330 335Ile Phe Tyr His Leu Trp Val Val Leu Gly Ser Gly Asn Ser Asn Phe340 345 350Phe Tyr Ala Ile Ser Leu Val Tyr Ala Leu Ala Ile Ala Ser Ile Leu355 360 365Val Asp Leu Asn Trp Ala Met Leu Arg Ile Glu Tyr Asp Asn Gly Ile370 375 380Pro Asn Phe Lys Leu Lys Val Thr Gln Ile385 390<210>42<211>255<212>DNA<213>人(Homo sapiens)<400>42atggccacgg ggacagacca ggtggtggga ctcggcctcg tcgccgttag cctgatcatc 60ttcacctact acaccgcctg ggtgattctc ttgccattca tcgacagtca gcatgtcatc120cacaagtatt tcctgccccg agcctatgct gtcgccatcc cactggctgc aggcctcctg180ctgctcctgt ttgtgggact gttcatctcc tatgtgatgc tgaagaccaa gagagtgacc240
aagaaggctc agtga255<210>43<211>84<212>PRT<213>人(Homo sapiens)<400>43Met Ala Thr Gly Thr Asp Gln Val yal Gly Leu Gly Leu Val Ala Val1 5 10 15Ser Leu Ile Ile Phe Thr Tyr Tyr Thr Ala Trp Val Ile Leu Leu Pro20 25 30Phe Ile Asp Ser Gln His Val Ile His Lys Tyr Phe Leu Pro Arg Ala35 40 45Tyr Ala Val Ala Ile Pro Leu Ala Ala Gly Leu Leu Leu Leu Leu Phe50 55 60Val Gly Leu Phe Ile Ser Tyr Val Met Leu Lys Thr Lys Arg Val Thr65 70 75 80Lys Lys Ala Gln<210>44<211>369<212>DNA<213>人(Homo sapiens)<400>44atgctctccg tgggcgggct tcggttgagt ttggtccgct tttcctttct gctcctcagg60ggagcattgc ttccttctct cgcagtgacc atgacgaaat tagcgcagtg gctttgggga120ctagcgatcc tgggctccac ctgggtggcc ctgaccacgg gagccttggg cctggagctg180cccttgtcct gccaggaagt cctgtggcca ctgcccgcct acttgctggt gtccgccggc240tgctatgccc tgggcactgt gggctatcgt gtggccactt ttcatgactg cgaggacgcc300gcacgcgagc tgcagagcca gatacaggag gcccgagccg acttagcccg cagggggctg360cgct tctga 369<210>45<211>122<212>PRT<213>人(Homo sapiens)<400>45Met Leu Ser Val Gly Gly Leu Arg Leu Ser Leu Val Arg Phe Ser Phe1 5 10 15
Leu Leu Leu Arg Gly Ala Leu Leu Pro Ser Leu Ala Val Thr Met Thr20 25 30Lys Leu Ala Gln Trp Leu Trp Gly Leu Ala Ile Leu Gly Ser Thr Trp35 40 45Val Ala Leu Thr Thr Gly Ala Leu Gly Leu Glu Leu Pro Leu Ser Cys50 55 60Gln Glu Val Leu Trp Pro Leu Pro Ala Tyr Leu Leu Val Ser Ala Gly65 70 75 80Cys Tyr Ala Leu Gly Thr Val Gly Tyr Arg Val Ala Thr Phe His Asp85 90 95Cys Glu Asp Ala Ala Arg Glu Leu Gln Ser Gln Ile Gln Glu Ala Arg100 105 110Ala Asp Leu Ala Arg Arg Gly Leu Arg Phe115 120<210>46<211>744<212>DNA<213>人(Homo sapiens)<400>46atggcggccg aggcggacgg accgcttaaa cggctgctcg tgccgattct tttacctgag60aaatgctacg accaactttt cgttcagtgg gacttgcttc acgtcccctg cctcaagatt120ctcctcagca aaggcctggg gctgggcatt gtggctggct cacttctagt aaagctgccc180caggtgttta aaatccgggg agccaagagt gctgaagggt tgagtctcca gtctgtaatg240ctggagctag tggcattgac tgggaccatg gtctacagca tcactaacaa cttcccattc300agctcttggg gtgaagcctt attcctgatg ctccagacga tcaccatctg cttcctggtc360atgcactaca gaggacagac tgtgaaaggt gtcgctttcc tcgcttgcta cggcctggtc420ctgctggtgc ttctctcacc tctgacgccc ttgactgtag tcaccctgct ccaggcctcc480aatgtgcctg ctgtggtggt ggggaggctt ctccaggcag ccaccaacta ccacaacggg540tacacaggcc agctctcagc catcacagtc ttcctgctgt ttgggggctc cctggcccga600atcttcactt ccattcagga aaccggagat cccctgatgg ctgggacctt tgtggtctcc660tctctctgca acggcctcat cgccgcccag ctgctcttct actggaatgc aaagcctccc720cacaagcaga aaaaggcgca gtag 744<210>47<211>247<212>PRT
<213>人(Homo sapiens)<400>47Met Ala Ala Glu Ala Asp Gly Pro Leu Lys Arg Leu Leu Val Pro Ile1 5 10 15Leu Leu Pro Glu Lys Cys Tyr Asp Gln Leu Phe Val Gln Trp Asp Leu20 25 30Leu His Val Pro Cys Leu Lys Ile Leu Leu Ser Lys Gly Leu Gly Leu35 40 45Gly Ile Val Ala Gly Ser Leu Leu Val Lys Leu Pro Gln Val Phe Lys50 55 60Ile Arg Gly Ala Lys Ser Ala Glu Gly Leu Ser Leu Gln Ser Val Met65 70 75 80Leu Glu Leu Val Ala Leu Thr Gly Thr Met Val Tyr Ser Ile Thr Asn85 90 95Asn Phe Pro Phe Ser Ser Trp Gly Glu Ala Leu Phe Leu Met Leu Gln100 105 110Thr Ile Thr Ile Cys Phe Leu Val Met His Tyr Arg Gly Gln Thr Val115 120 125Lys Gly Val Ala Phe Leu Ala Cys Tyr Gly Leu Val Leu Leu Val Leu130 135 140Leu Ser Pro Leu Thr Pro Leu Thr Val Val Thr Leu Leu Gln Ala Ser145 150 155 160Asn Val Pro Ala Val Val Val Gly Arg Leu Leu Gln Ala Ala Thr Asn165 170 175Tyr His Asn Gly Tyr Thr Gly Gln Leu Ser Ala Ile Thr Val Phe Leu180 185 190Leu Phe Gly Gly Ser Leu Ala Arg Ile Phe Thr Ser Ile Gln Glu Thr195 200 205Gly Asp Pro Leu Met Ala Gly Thr Phe Val Val Ser Ser Leu Cys Asn210 215 220Gly Leu Ile Ala Ala Gln Leu Leu Phe Tyr Trp Asn Ala Lys Pro Pro225 230 235 240His Lys Gln Lys Lys Ala Gln245<210>48<211>25<212>DNA<213>人工的<220>
<223>人工合成的引物序列<400>48atgacaatgt ggggaagtca acggg25<210>49<211>30<212>DNA<213>人工的<220>
<223>人工合成的引物序列<400>49tgtgtggtta ccgttctttg aatacataga30<210>50<211> 29<212>DNA<213>人工的<220>
<223>人工合成的引物序列<400>50atagaaaatg atttatggta cagctcaaa 29<210>51<211>30<212>DNA<213>人工的<220>
<223>人工合成的引物序列<400>51agaccaaatt aattatgcct ttacatgtac 30<210>52<211>40<212>DNA<213>人工的<220>
<223>人工合成的引物序列<400>52agaattcacc atgagcaaca tgaatatact tgcgtatctt40<210>53<211>30<212>DNA<213>人工的
<220>
<223>人工合成的引物序列<400>53gaaattccaa tgtattccat attcacttat30<210>54<211>42<212>DNA<213>人工的<220>
<223>人工合成的引物序列<400>54aagatctaat acattaaaac attttagatt aatgaatatg tg42<210>55<211>34<212>DNA<213>人工的<220>
<223>人工合成的引物序列<400>55aggtaccgta cactccactc tatgatgatc attc 3权利要求
1.由核苷酸序列SEQ ID NO5组成的DNA。
2.载体，其中插入权利要求1的DNA。
3.转化体，保留可表达状态的权利要求1所述DNA或者权利要求2所述载体。
4.权利要求3所述的转化体，其为GPI合酶基因-缺陷型真菌。
5.权利要求3所述的转化体，其为GPI合酶基因-缺陷型酵母。
6.一种制备由权利要求1所述DNA编码的蛋白的方法，该方法包括培养权利要求3-5中的任一转化体，并从该转化体或其培养上清液收集表达的蛋白的步骤。
7.一种筛选具有抗疟活性的化合物的方法，该方法包括以下步骤(1)将试样与表达权利要求1所述DNA的GPI合酶基因-缺陷型酵母接触；(2)分析该酵母的生长，和(3)选出抑制该酵母生长的被试化合物。
8.一种筛选具有抗疟活性的化合物的方法，该方法包括以下步骤(1)将试样与表达权利要求1所述DNA的GPI合酶基因-缺陷型酵母接触，(2)检测GPI-锚定蛋白转运到酵母细胞壁的量，和(3)选出使步骤(2)中所检测的GPI-锚定蛋白转运到酵母细胞壁的量减少的试样。
9.一种筛选具有抗疟活性的化合物的方法，该方法包括以下步骤(1)将权利要求1所述DNA导入GPI合酶基因-缺陷型酵母，并使该DNA编码的蛋白表达，(2)获得步骤(1)中表达的蛋白，(3)使所得蛋白与试样和具有与该蛋白结合的活性的标记化合物接触，(4)检测与该蛋白结合的标记化合物，和(5)选出减少与该蛋白结合的标记化合物的量的试样。
10.一种筛选具有抗疟活性的化合物的方法，该方法包括以下步骤(1)将(i)权利要求1的DNA，或者(ii)插入了权利要求1的DNA的载体，导入GWT1-缺陷型酵母，且表达由该DNA编码的蛋白(2)获得步骤(1)中表达的蛋白，(3)加入UDP-GlcNAc或酰基辅酶A，(4)使所得蛋白与试样接触，(5)检测GlcN-(酰基)PI，和(6)选出减少GlcN-(酰基)PI水平的被试化合物。
全文摘要
本发明人成功地分离出了GWT1(PfGWT1)，其是一种在恶性疟原虫中参与GPI生物合成的酶。此外，本发明人揭示比编码PfGWT1蛋白的DNA具有更低AT含量的简并突变体DNA，能补偿GWT1－缺陷型酵母的表型。在此发现的基础上，本发明提供了疟原虫的GWT1蛋白，以及该蛋白在筛选抗疟药物的方法中的用途。本发明还提供了编码参有GPI生物合成的蛋白的简并突变体DNA，该DNA比天然DNA具有更低的AT含量。本发明还提供了利用该简并突变体DNA筛选抗疟药物的方法。
文档编号G01N33/569GK101092628SQ20071010428
公开日2007年12月26日 申请日期2003年11月21日 优先权日2002年11月22日
发明者畑桂, 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 申请人:卫材R&D管理有限公司