筛选抑制gwt1基因产物酶活性的化合物的方法

专利名称：筛选抑制gwt1基因产物酶活性的化合物的方法
技术领域：
本发明涉及筛选具有抑制GPI合酶活性的抗真菌剂的方法，所述GPI合酶参与真菌细胞壁的合成。
背景技术：
本发明人注意到，粘附宿主细胞对于真菌发挥其病原性是非常重要的，而且参与真菌细胞粘附的粘附因子在糖基磷脂酰肌醇(GPI)锚定到细胞膜上以后，就被转运到细胞壁表层(非专利文献1Hamada K等，Mol.Gen.Genet.，25853-59，1998)。因此，本发明人认为通过抑制锚定GPI的蛋白(GPI-锚定蛋白)向细胞壁转运的过程，可以制得新抗真菌剂，该抗真菌剂能抑制真菌细胞壁的合成，并且还能抑制真菌细胞粘附宿主细胞。
本发明的公开本发明的目的是通过抑制真菌细胞壁的合成，通过抑制真菌细胞粘附宿主细胞，并抑制GPI-锚定蛋白转运到真菌细胞壁的过程，来开发出防止病原性真菌发挥病原性的抗真菌剂。
在WO 02/04626中，本发明人发现了下述参与GPI-锚定蛋白到细胞壁转运过程的蛋白由含有SEQ ID NO1核苷酸序列的DNA编码的酿酒酵母(Saccharomyces cerevisiae)蛋白；由含有SEQ ID NO3和5核苷酸序列的DNA编码的白色念珠菌(Candida albicans)蛋白；由含有SEQ ID NO7核苷酸序列的DNA编码的粟酒裂殖酵母(Schizosaccharomyces pombe)蛋白；由含有SEQ ID NO9和11核苷酸序列的DNA编码的烟曲霉(Aspergillusfumigatus)蛋白；和由含有SEQ ID NO12和13核苷酸序列的DNA编码的新生隐球菌(Cryptococcus neoformans)蛋白。这些核苷酸序列称为GWT1基因。此外，本发明人发现GWT1基因-缺陷型真菌不能合成细胞壁。另外，本发明人发现式(Ia)所示化合物与上述蛋白结合能抑制GPI-锚定蛋白到细胞壁的转运，从而抑制真菌细胞壁的合成。
然后，本发明人发现在GPI合成途径中GWT1基因产物(下文中称作“GWT1蛋白”)具有将酰基转移至G1cN-PI而合成GlcN-(酰基)PI的活性(

图1；Kinoshita和Inoue，Curr Opin Chem Biol 2000Dec；4(6)632-8；Ferguson等，Biochim Biophys Acta 1999 Oct 8；1455(2-3)327-40)。本发明人认为抑制真菌细胞壁合成的化合物可以通过对抑制此活性的化合物进行筛选而得到，以此完成本发明。
具体地，本发明提供了下述[1]-[4]。
筛选具有抗真菌活性的化合物的方法，其中该方法包括下述步骤(1)将试样与由GWT1基因编码且过表达的蛋白接触；(2)检测GlcN-(酰基)PI；和(3)选出减少GlcN-(酰基)PI的试样。
“GWT1”基因是指参与真菌细胞壁合成的基因，该基因公开于WO02/04626中。术语“过表达”并不是指天然基因的表达，而是指外源导入基因的表达。
“GlcN-(酰基)PI”是指在GPI生物合成途径中，酰基与葡糖胺基-磷脂酰肌醇(GleN-PI)的肌醇连接的葡糖胺基-酰基磷脂酰肌醇(图1；Kinoshita和Inoue，Curr Opin Chem Biol 2000 Dec；4(6)632-8；Ferguson等，BiochimBiophys Acta 1999 Oct 8；1455(2-3)327-40)。
项[1]所述的方法，其中所述GWT1基因是下述任一种(a)编码包含氨基酸序列SEQ ID NO2，4，6，8，10或14的蛋白的DNA；(b)包含核苷酸序列SEQ ID NO1，3，5，7，9，11，12或13的DNA；(c)在严谨条件下，与包含核苷酸序列SEQ ID NO1，3，5，7，9，11，12或13的DNA杂交的DNA；和(d)编码包含氨基酸序列SEQ ID NO2，4，6，8，10或14的蛋白的DNA，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。
术语“严谨条件”意思是，例如在65℃于4xSSC中进行杂交，然后在65℃用0.1xSSC洗涤1小时。可选的严谨条件是指在42℃，于含50％甲酰胺的4xSSC中进行杂交。其它可接受的条件是在65℃于PerfectHybTM溶液(TOYOBO)中杂交2.5小时，然后(1)在25℃，用2xSSC，0.05％SDS洗涤5分钟；(2)在25℃，用2xSSC，0.05％SDS洗涤15分钟；和(3)在50℃，用0.1xSSC，0.1％SDS洗涤20分钟。
“包含其中添加，缺失，取代和/或插入一个或多个氨基酸的氨基酸序列的蛋白”可采用本领域技术人员所熟知的方法来进行制备，例如定点诱变(Sambrook，J.，Fritsch，E.F.和Maniatis，T.(1989)Molecular CloningALaboratory Manual，Cold Spring Harbor Laboratory，Cold Spring Harbor，NY)。所述突变也可以是自然发生的。对于突变的氨基酸数量并无限制，只要所得蛋白保持将酰基转移至GlcN-PI的活性。突变的氨基酸数量通常为30个或更少，优选10个或更少，更优选3个或更少。对于突变氨基酸的位置并无限制，只要蛋白保持上述活性。
采用上述杂交技术制得的蛋白和蛋白突变体通常在氨基酸水平上，与由SEQ ID NO2，4，6，8，10或14氨基酸序列组成的蛋白具有很高的同源性(例如60％或更高，70％或更高，80％或更高，90％或更高，或95％或更高的同源性)。氨基酸序列同源性可采用BLASTx程序(以氨基酸水平；Altschul等，J Mol.Biol.215403-410，1990)来进行测定。该程序以Karlin和Altschul的BLAST算法为基础(Proc.Natl.Acad.Sci.USA 872264-2268，1990；Proc.Natl.Acad.Sci.USA 905873-5877，1993)。当使用BLASTX分析氨基酸序列时，使用的参数是如得分＝50，字长＝3。作为选择，当使用Gapped BLAST程序时，可以利用Altschul等所描述的方法(Nucleic.Acids.Res.253389-3402，1997)对氨基酸序列进行分析。当使用BLAST和Gapped BLAST程序时，使用各程序的默认参数值。这些分析的详细步骤是本领域已知的(http//www.ncbi.nim.nih.gov)。
权利要求1或2所述的方法，其中检测酰基化GPI的步骤是薄层层析。
项[1]-[3]中任一项所述的方法，其中该方法进一步包含步骤(4)，测定选出的试样是否抑制GPI-锚定蛋白转运到真菌细胞壁的过程，试样是否抑制GPI-锚定蛋白在真菌细胞表面上的表达，或试样是否抑制真菌的增殖。
本发明制备GWT1蛋白的方法[1]，和测定转酰基活性的方法[2]将在下面公开。
1.制备GWT1蛋白的方法GWT1蛋白是从真菌膜级分，优选酿酒酵母(S.Cerevisiae)，白色念珠菌(C.Albicans)，粟酒裂殖酵母(S.pombe)，烟曲霉(A.Fumigatus)，或新生隐球菌(C.Neoformans)，更优选酿酒酵母的膜级分制得的。转酰基活性可以采用直接制备膜级分或在纯化后制备膜级分来进行测定。通过将SEQ ID NO1，3，5，7，9，11，12或13核苷酸序列的DNA导入真菌细胞来过表达GWT1蛋白可以很容易地对转酰基活性进行测定。利用酿酒酵母的方法将在下面进行详细地描述(1)GWT1基因的导入用真菌DNA作模板，和在SEQ ID NO1，3，5，7，9，11，12或13核苷酸序列基础上设计的引物，通过PCR制备GWT1基因。
通过将适合的启动子和终止子，如衍生自pKT10的GAPDH启动子和GAPDH终止子(Tanaka等，Mol.Cell Biol.，104303-4313，1990)插到在酿酒酵母中起作用的表达载体如YEp 352的多克隆位点内，并将GWT1基因插到该表达载体内来制备GWT1表达质粒。将酿酒酵母如G2-10菌株，在适宜的温度如30℃，合适的培养基如酵母提取物-聚蛋白胨-葡萄糖(YPD)培养基中振荡孵育。在对数生长晚期收获真菌细胞。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。所述乙酸锂法描述于YEASTMAKERTM酵母转化系统所附的使用手册(Clontech)中。通过将细胞在30℃，SD(ura-)培养基中培养两天而得到GWT1-过表达菌株和导入空载体的菌株。
引入GWT1基因的真菌菌株优选是缺失了其天然GWT1基因的缺陷型菌株。酿酒酵母GWT1基因-缺陷型细胞采用下述方法获得。
利用标记基因，优选粟酒裂殖酵母his5基因作模板，采用引物来进行PCR扩增，引物被设计成使得PCR产物包含将要缺失的GWT1基因(如SEQ ID NO1序列)的30个碱基对，更优选40个碱基对或更多。将获得的PCR产物提纯并导入真菌细胞中。通过进行对标记基因合适的筛选，如当标记为his5时就将细胞培养在his-培养基中，得到缺陷型菌株。
除酿酒酵母之外真菌的表达载体和基因导入法描述于Igarashi等，Nature 35380-83，1991，粟酒裂殖酵母表达载体pcL等和导入该载体的方法；Pla J等，Yeast，121677-1702，1996，白色念珠菌表达载体pRM10等和导入该载体的方法；Punt PJ等，GENE，56117-124，1987，烟曲霉表达载体pAN7-1等和导入该载体的方法；Monden P等，FEMS Microbiol.Lett.，18741-45，2000，新生隐球菌表达载体pPM8等和导入该载体的方法。
制备白色念珠菌缺陷菌株的方法描述于Fonzi WA等，Genetics 134717-728，1993中。
(2)制备膜级分的方法在适宜的温度如24℃，适合的培养基如SD(ura-)液体培养基中对导入了GWT1基因的酿酒酵母细胞进行振荡培养。在对数生长中期时收获真菌细胞。用TM缓冲液(50mM Tris-HCl(pH7.5)和2mM MgCl2)洗涤后，用合适量(如2ml)的TM缓冲液+蛋白酶抑制剂(CompleteTM；Roche)对真菌细胞进行悬浮。将合适量(如1.5ml)的玻璃珠加到悬浮液中。涡旋样品，并置于冰上，重复此步骤(如涡旋30秒，置于冰上30秒，循环10次)，使真菌细胞破碎。
将样品离心，如1000g离心5分钟，使玻璃珠和未破碎的真菌细胞沉淀。将所得上清液移到另一试管内，然后离心，如13000g离心20分钟，使含细胞器的膜级分(总膜级分)沉淀。如果需要，用1ml合适的试验缓冲液进一步悬浮该沉淀物并离心，如1000g离心1分钟，除去那些未悬浮成分。再离心上清液，如13 000g离心20分钟，用合适的试验缓冲液重悬浮所得的沉淀物，得到膜级分。
采用Yoko-o等，Eur.J.Biochem.257630-637，1998，用于粟酒裂殖酵母；Sentandreu M等，J.Bacteriol.，180282-289，1998，用于白色念珠菌；Mouyna I等，J.Biol.Chem.，27514882-14889，2000，用于烟曲霉；ThompsonJR等J.Bacteriol.，181444-453，1999，用于新生隐球菌的所述方法从除酿酒酵母之外的真菌细胞中制得膜级分。
作为选择，可通过让GWT1蛋白在真菌之外的细胞，如哺乳动物细胞，昆虫细胞和大肠杆菌细胞中表达来制备GWT1蛋白。
当使用哺乳动物细胞时，可以通过将GWT1插到如含有CMV启动子的过表达载体内；将该载体导入哺乳动物细胞；然后，进行Petaja-Repo等J.Biol.Chem.，2764416-23，2001中所述的方法来制备膜级分。
当使用昆虫细胞时，可以利用杆状病毒表达试剂盒，如BAC-TO-BAC杆状病毒表达系统(Invitrogen)制备GWT1-表达昆虫细胞(如Sf9细胞)；然后，利用该细胞进行Okamoto等，J.Biol.Chem.，276742-751，2001中所述方法来制备膜级分。
当使用大肠杆菌时，GWT1蛋白可通过将GWT1插到大肠杆菌表达载体，如pGEX(Amersham Biosciences)内；然后将该载体导入大肠杆菌细胞如BL21中来加以制备。
2.测定转酰基活性的方法酰基移至GPI的反应可通过Costello和Orlean，J.Biol.Chem.(1992)2678599-8603中所述方法，或Franzot和Doering，Biochem.J.(1999)34025-32中所述方法来进行检测。该方法的具体实例将在下面进行说明，但下述实验条件优选是根据所用的GWT1基因产物进行了优化的将上述部分1中制得的GWT1基因产物，优选含有GWT1基因产物的膜级分与待测化合物一起加到缓冲液中，所述缓冲液含有合适的金属离子(Mg，Mn)；ATP；和辅酶A；和优选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-连接的糖链合成抑制剂的衣霉素(tunicamycin)。将该混合物在适宜的温度下孵育适当长的时间(如在24℃孵育15分钟)。
然后，将用合适标记标记的，优选用放射性同位素标记的GlcN-(酰基)PI前体(如UDP-GlcNAc，或酰基-辅酶A，优选UDP-[14C]GlcNAc)加到混合物中。将所得混合物进一步孵育适当长的时间(如在24℃孵育1小时)。将氯仿∶甲醇的1∶2混合物加到该混合物中，搅拌停止反应。然后从混合物中抽提脂质。抽提的反应产物用合适的溶剂优选丁醇溶解，然后进行HPLC，薄层层析(TLC)等，优选TLC，将反应中产生的GlcN-(酰基)PI分离出来。可对TLC展开剂(developing solvent)进行适当地选择，例如可以是CHCl3/CH3OH/H2O(65∶25∶4)，CHCl3/CH3OH/1M NH4OH(10∶10∶3)，或CHCl3/吡啶/HCOOH(35∶30∶7)，优选CHCl3/CH3OH/1M NH4OH(10∶10∶3)。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。
当在待测化合物存在的条件下，制得的GlcN-(酰基)PI量减少就判定该待测化合物含有抑制由GWT1蛋白引起的酰基转移的活性。
优选对上述已发现含有抑制酰基转移的活性的试样进行进一步的检测以确定它是否抑制GPI-锚定蛋白转运到细胞壁的过程，它是否抑制GPI-锚定蛋白在真菌细胞表面上的表达，或它是否抑制真菌生长。如果检测结果显示试样抑制GPI-锚定蛋白转运到细胞壁的过程，抑制GPI-锚定蛋白在真菌细胞表面上的表达，或抑制真菌生长，则该样品就可望成为抗真菌剂候选物。
可用(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)检测真菌细胞与动物细胞粘附的方法；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对试样是否抑制GPI-锚定蛋白转运到细胞壁的过程或抑制GPI-锚定蛋白在真菌细胞表面上的表达进行检测。
方法(1)-(4)公开在WO 02/04626中，并在实施例中进行了具体说明。利用(1)-(4)的方法，优选其组合，可以确定试样是抑制GPI-锚定蛋白转运到真菌细胞壁的过程，还是抑制GPI-锚定蛋白在真菌细胞表面上的表达。另外，当由本发明DNA编码的蛋白在真菌细胞中过表达，由试样引起的抑制受损或消失时，可以确定试样对GPI-锚定蛋白转运到细胞壁的过程有影响。
用测定抗真菌活性的常规方法还可以检测试样是否抑制真菌生长(National Committee for Clinical Laboratory Standards.1992.Reference methodfor broth dilution antifungal susceptibility testing for yeasts.Proposed standardM27-P.National Committee for Clinical Laboratory Standards，Villanova，Pa.)。
附图的简单说明图1示GPT生物合成途径。
图2照片显示在从野生型菌株(WT)，GWT1基因遭破坏的Δgwt1菌株(Δ)，和GWT1基因-导入的Δgwt1菌株(Δ/G)制得的膜级分中测定的GPI酰基化结果。
图3照片显示在酰基化GPI检测系统中，化合物1-(4-丁基苄基)异喹啉和化合物N-(3-(4-(1-异喹啉基甲基)苯基)-2-丙炔基)乙酰胺的GPI酰基化抑制活性的检测结果。在公开了GWT1基因的WO02/04626中，这两种化合物列于WO02/04626的表1，并分别描述于WO02/04626的实施例B2和实施例B60中。
图4照片显示在酰基化GPI检测系统中，化合物N-(3-(4-(1-异喹啉基甲基)苯基)丙基)-N-甲基乙酰胺和化合物5-丁基-2-(1-异喹啉基甲基)苯酚的GPI酰基化抑制活性的检测结果。在公开了GWT1基因的WO02/04626中，这两种化合物列于WO02/04626的表1，并分别描述于WO02/04626的实施例B73和实施例B85中。
进行发明的最佳实施方式下面，将利用实施例详细地描述本发明，但是本发明并不限于此。
制备表达GWT1的膜级分(1)制备GWT1表达质粒通过将GAPDH启动子和GAPDH终止子，其都衍生自pKT10(Tanaka等，Mol.Cell Biol.，104303-4313，1990)插到YEp352的多克隆位点内；并用pUC 18的多克隆位点代替该多克隆位点制备用于在酿酒酵母中表达的载体，YEp352GAPII载体。另外，为了促进GWT1基因的插入，用ClaI位点取代多克隆位点中的SalI位点制得YEp352GAPIIClaIΔSal载体。
利用SEQ ID NOs15和16引物扩增包含SEQ ID NO1核苷酸序列的酿酒酵母GWT1基因。将获得的PCR产物插到YEp352GAPIIClaIΔSal载体的多克隆位点，获得GWT1过表达质粒。
(2)制备酿酒酵母GWT1基因-缺陷型菌株Δgwt1利用粟酒裂殖酵母his5基因(Longtine MS等，Yeast，14953-961，1998)作模板，SEQ ID NOs17和18序列作引物PCR扩增两端都包含GWT1序列的his5盒。
培养并收获酿酒酵母细胞，然后用上述PCR产物转化。然后，将细胞在30℃，SD(His-)培养基中培养5-7天，获得GWT1基因-缺陷型菌株Δgwt1。
(3)制备表达GWT1的细胞将Δgwt1菌株细胞在30℃，酵母提取物-聚蛋白胨-葡萄糖(YPD)培养基中振荡培养。对数生长晚期收获细胞，然后洗涤。采用乙酸锂法(YEASTMAKERTM酵母转化系统(Clontech))将GWT1表达质粒导入Δgwt1菌株细胞中。将细胞在30℃，SD(ura-)培养基中培养两天，得到过表达GWT1基因的Δgwt1菌株。
(4)制备膜级分将野生型酿酒酵母菌株，GWT1基因-缺陷型菌株Δgwt1，和导入了GWT1过表达质粒的菌株Δgwt1分别在24℃，100ml的YPD培养基中振荡培养，然后在对数生长中期收获细胞(OD600＝1～3)。用TM缓冲液(50mMTris-HCl(pH7.5)和2mM MgCl2)洗涤真菌细胞，然后重悬浮在2ml TM缓冲液+蛋白酶抑制剂(1片CompleteTM(Roche)/25ml)中。将1.5ml玻璃珠加到悬浮液中。涡旋混合物30秒，然后置于冰上30秒。重复此步骤10次，破坏真菌细胞。将细胞匀浆移至一新试管内，4℃，1000g离心5分钟，使玻璃珠和未破坏的真菌细胞沉淀。将上清液移至另一试管内，4℃，13000g离心20分钟，使含细胞器的膜级分(总膜级分)沉淀。获得的沉淀物作为膜级分。
(5)检测酰基化的GPI已知在GPI生物合成反应路径中，N-乙酰基-葡糖胺基-磷脂酰肌醇(GlcNAc-PI)脱乙酰基化形成葡糖胺基-磷脂酰肌醇(GlcN-PI)，其在添加酰基后生成葡糖胺基-酰基磷脂酰肌醇(GlcN-(酰基)PI)(图1)。因此，利用下述方法，本发明人可以对Gwt1蛋白是否参与该转酰基反应进行检测。
用缓冲液稀释膜级分制剂(300μg蛋白)，所述缓冲液由50mM Tris-HCl(pH7.5)，2mM MgCl2，2mM MnCl2，1mMATP，1mM辅酶A，21μg/ml衣霉素，10μM尼可霉素Z和0.5mM二硫苏糖醇组成。将溶液调成总量为140μl，作为反应液。24℃孵育溶液15分钟后，将15μCi UDP-[14C]GlcNAc加到试管内，然后在24℃孵育1小时。将1ml氯仿∶甲醇(1∶2)加到该溶液中，搅拌终止反应。然后，从该溶液中抽提脂质，干燥，经丁醇抽提脱盐。用薄层层析(HCl3/CH3OH/1M NH4OH(10∶10∶3))将酰基化GPI(GlcN-(酰基)PI)，非酰基化GPI(GlcN-PI)，以及GPI分开，其中GPI既不酰基化也不去酰基化(GlcNAc-PI)。各斑点用放射自显影进行检测。
结果，如图2所示，在GWT1基因-缺陷型菌株(Δgwt1)中未检测到酰基化GPI斑点，在野生型菌株中检测到酰基化GPI斑点。在引入GWT1基因的Δgwt1菌株中也检测到了酰基化GPI斑点，这说明该菌株已经恢复了酰基化能力。此发现表明Gwt1蛋白是一种能催化酰基转移到GPI的酶。
上述结果说明当具有抑制GWT1基因产物活性的化合物存在于用于分析GPI合酶活性的系统中时，酰基化GlcN-(酰基)PI的斑点强度(intensity)就会降低或消失。这样，利用GlcN-(酰基)PI的斑点强度作为指示剂，可以对抑制GWT1基因产物的酶活性的化合物，以及抑制真菌细胞壁合成的化合物进行筛选。
(6)筛选抑制酰基化反应的化合物将下述化合物加到(5)所述的酰基化GPI检测系统中，测定抑制GPI酰基化的活性。这些化合物描述于WO 02/04626的实施例B2，实施例B60，实施例B73和实施例B85中，在所述WO 02/04626中公开了GWT1基因。在WO 02/04626中，这些化合物还在表1中列出，该表显示了这些化合物在反映GWT1基因产物活性的报告系统中的抑制活性。这些化合物的结构如下所示实施例B2中所述化合物1-(4-丁基苄基)异喹啉 实施例B60中所述化合物N-(3-(4-(1-异喹啉基甲基)苯基)-2-丙炔基)乙酰胺 实施例B73中所述化合物N-(3-(4-(1-异喹啉基甲基)苯基)丙基)-N-甲基乙酰胺
实施例B85中所述化合物5-丁基-2-(1-异喹啉基甲基)苯酚 实验结果示于图3和4。在WO 02/04626表1所列的化合物中，实施例B2和实施例B85中的所述化合物在酰基化GPI的斑点强度中显示出剂量依赖性的减少，所述化合物具有IC50为1μg/ml或更低的抑制活性。实施例B73中所述化合物的斑点强度未观察到减少，该化合物IC50为50μg/ml。
此结果说明利用GPI酰基化实验系统可以对抑制GWT1基因产物的酶活性的化合物进行筛选。
工业实用性利用简单的转酰基活性实验，本发明可以对抑制GPI-锚定蛋白到真菌细胞壁的转运过程的化合物进行筛选。
序列表<110>卫材株式会社(Eisai Co.，Ltd.)独立行政法人产业技术总合研究所(National Institute of Advanced Industrial Scienceand Technology)<120>筛选抑制GWT1基因产物酶活性的化合物的方法<130>E1-A0209P<150>JP 2002-339418<151>2002-11-22<160>18<170>PatentIn Ver.2.0<210>1<211>1497<212>DNA<213>酿酒酵母(Saccharomyces cerevisiae)<220>
<221>CDS<222>(1)..(1494)<400>1atg gca aca gta cat cag aag aat atg tcg act tta aaa cag aga aaa 48Met Ala Thr Val His Gln Lys Asn Met Ser Thr Leu Lys Gln Arg Lys1 5 10 15gag gac ttt gtg aca ggg ctc aat ggc ggt tct ata aca gaa att aac 96Glu Asp Phe Val Thr Gly Leu Asn Gly Gly Ser Ile Thr Glu Ile Asn20 25 30gca gtg aca tca att gct ttg gta act tac ata tca tgg aac tta ttg 144Ala Val Thr Ser Ile Ala Leu Val Thr Tyr Ile Ser Trp Asn Leu Leu35 40 45aaa aat tcc aac ctt atg cct cct ggc att tcc agc gtg caa tac ata 192Lys Asn Ser Asn Leu Met Pro Pro Gly Ile Ser Ser Val Gln Tyr Ile50 55 60
att gat ttt gca ttg aac tgg gtt gct ttg ctt cta tct att act att240Ile Asp Phe Ala Leu Asn Trp Val Ala Leu Leu Leu Ser Ile Thr Ile65 70 75 80tat gct agt gaa cca tac ctt cta aac acg cta ata ctg tta cct tgt288Tyr Ala Ser Glu Pro Tyr Leu Leu Asn Thr Leu Ile Leu Leu Pro Cys85 90 95ttg ctc gca ttc ata tat gga aaa ttt act agc tcg agt aaa cct tct336Leu Leu Ala Phe Ile Tyr Gly Lys Phe Thr Ser Ser Ser Lys Pro Ser100 105 110aat cca ata tac aat aaa aaa aaa atg att aca cag cgg ttc caa cta384Asn Pro Ile Tyr Asn Lys Lys Lys Met Ile Thr Gln Arg Phe Gln Leu115 120 125gaa aaa aag ccg tat att act gcg tat cgt ggt ggg atg ctt att ctg432Glu Lys Lys Pro Tyr Ile Thr Ala Tyr Arg Gly Gly Met Leu Ile Leu130 135 140act gct att gcc atc ttg gct gta gat ttt cca att ttc cca agg agg480Thr Ala Ile Ala Ile Leu Ala Val Asp Phe Pro Ile Phe Pro Arg Arg145 150 155 160ttt gcc aag gtg gaa act tgg ggg aca tcc ctg atg gat ctt ggt gta528Phe Ala Lys Val Glu Thr Trp Gly Thr Ser Leu Met Asp Leu Gly Val165 170 175gga tca ttc gtt ttc agt aac ggt att gtt tct tct agg gca ctg ttg576Gly Ser Phe Val Phe Ser Asn Gly Ile Val Ser Ser Arg Ala Leu Leu180 185 190aaa aac cta agc ttg aag agt aaa ccc agc ttc tta aaa aat gca ttt624Lys Asn Leu Ser Leu Lys Ser Lys Pro Ser Phe Leu Lys Asn Ala Phe195 200 205aat gcc tta aaa tca gga gga act cta ttg ttc cta gga ttg ctg agg 672Asn Ala Leu Lys Ser Gly Gly Thr Leu Leu Phe Leu Gly Leu Leu Arg210 215 220ttg ttt ttt gta aaa aat ttg gaa tat caa gaa cat gtc aca gaa tat 720Leu Phe Phe Val Lys Asn Leu Glu Tyr Gln Glu His Val Thr Glu Tyr225 230 235 240
ggg gtt cat tgg aat ttt ttt atc acc cta tca ttg ttg cca ctt gta768Gly Val His Trp Asn Phe Phe Ile Thr Leu Ser Leu Leu Pro Leu Val245 250 255ttg acc ttt att gat ccc gtc aca aga atg gtt cca cgc tgc tca att816Leu Thr Phe Ile Asp Pro Val Thr Arg Met Val Pro Arg Cys Ser Ile260 265 270gca ata ttc att tca tgc att tat gaa tgg cta ctt tta aag gac gat864Ala Ile Phe Ile Ser Cys Ile Tyr Glu Trp Leu Leu Leu Lys Asp Asp275 280 285cgc act tta aac ttt tta att ttg gct gat aga aat tgt ttc ttc agt912Arg Thr Leu Asn Phe Leu Ile Leu Ala Asp Arg Asn Cys Phe Phe Ser290 295 300gct aat aga gaa ggc atc ttc tca ttt cta ggt tat tgc tcg att ttt960Ala Asn Arg Glu Gly Ile Phe Ser Phe Leu Gly Tyr Cys Ser Ile Phe305 310 315 320ctt tgg ggc caa aac acg gga ttt tac ttg ttg gga aat aaa cca act1008Leu Trp Gly Gln Asn Thr Gly Phe Tyr Leu Leu Gly Asn Lys Pro Thr325 330 335tta aac aat ctt tat aag cct tct acg caa gac gta gtt gca gca tca1056Leu Asn Asn Leu Tyr Lys Pro Ser Thr Gln Asp Val Val Ala Ala Ser340 345 350aag aag tct tcg act tgg gac tat tgg act tca gta acc cca tta agt1104Lys Lys Ser Ser Thr Trp Asp Tyr Trp Thr Ser Val Thr Pro Leu Ser355 360 365ggc ctc tgt ata tgg agt aca att ttt ctt gtt atc agc cag ttg gtt1152Gly Leu Cys Ile Trp Ser Thr Ile Phe Leu Val Ile Ser Gln Leu Val370 375 380ttt caa tac cat cct tat agt gtt tca aga agg ttt gct aac tta cca1200Phe Gln Tyr His Pro Tyr Ser Val Ser Arg Arg Phe Ala Asn Leu Pro385 390 395 400tat act ttg tgg gtc att act tat aat tta cta ttt ttg act ggg tac1248Tyr Thr Leu Trp Val Ile Thr Tyr Asn Leu Leu Phe Leu Thr Gly Tyr405 410 415
tgc ttg act gac aaa att ttc ggt aat tct tcg gaa tat tat aaa gtt 1296Cys Leu Thr Asp Lys Ile Phe Gly Asn Ser Ser Glu Tyr Tyr Lys Val420 425 430gcc gaa tgc ttg gaa tca atc aac tcc aat ggg ttg ttt tta ttt ttg 1344Ala Glu Cys Leu Glu Ser Ile Asn Ser Asn Gly Leu Phe Leu Phe Leu435 440 445ttg gca aat gtc tct act ggt tta gtc aat atg tct atg gtc acg ata 1392Leu Ala Asn Val Ser Thr Gly Leu Val Asn Met Ser Met Val Thr Ile450 455 460gat tct tca ccc tta aaa tca ttc ctg gtt ttg ttg gca tac tgc tca 1440Asp Ser Ser Pro Leu Lys Ser Phe Leu Val Leu Leu Ala Tyr Cys Ser465 470 475 480ttc ata gct gtc ata tcg gtt ttc ttg tat aga aaa aga ata ttc att 1488Phe Ile Ala Val Ile Ser Val Phe Leu Tyr Arg Lys Arg Ile Phe Ile485 490 495aag cta taa 1497Lys Leu<210>2<211>498<212>PRT<213>酿酒酵母(Saccharomyces cerevisiae)<400>2Met Ala Thr Val His Gln Lys Asn Met Ser Thr Leu Lys Gln Arg Lys1 5 10 15Glu Asp Phe Val Thr Gly Leu Asn Gly Gly Ser Ile Thr Glu Ile Asn20 25 30Ala Val Thr Ser Ile Ala Leu Val Thr Tyr Ile Ser Trp Asn Leu Leu35 40 45Lys Asn Ser Asn Leu Met Pro Pro Gly Ile Ser Ser Val Gln Tyr Ile50 55 60Ile Asp Phe Ala Leu Asn Trp Val Ala Leu Leu Leu Ser Ile Thr Ile65 70 75 80
Tyr Ala Ser Glu Pro Tyr Leu Leu Asn Thr Leu Ile Leu Leu Pro Cys85 90 95Leu Leu Ala Phe Ile Tyr Gly Lys Phe Thr Ser Ser Ser Lys Pro Ser100 105 110Asn Pro Ile Tyr Asn Lys Lys Lys Met Ile Thr Gln Arg Phe Gln Leu115 120 125Glu Lys Lys Pro Tyr Ile Thr Ala Tyr Arg Gly Gly Met Leu Ile Leu130 135 140Thr Ala Ile Ala Ile Leu Ala Val Asp Phe Pro Ile Phe Pro Arg Arg145 150 155 160Phe Ala Lys Val Glu Thr Trp Gly Thr Ser Leu Met Asp Leu Gly Val165 170 175Gly Ser Phe Val Phe Ser Asn Gly Ile Val Ser Ser Arg Ala Leu Leu180 185 190Lys Asn Leu Ser Leu Lys Ser Lys Pro Ser Phe Leu Lys Asn Ala Phe195 200 205Asn Ala Leu Lys Ser Gly Gly Thr Leu Leu Phe Leu Gly Leu Leu Arg210 215 220Leu Phe Phe Val Lys Asn Leu Glu Tyr Gln Glu His Val Thr Glu Tyr225 230 235 240Gly Val His Trp Asn Phe Phe Ile Thr Leu Ser Leu Leu Pro Leu Val245 250 255Leu Thr Phe Ile Asp Pro Val Thr Arg Met Val Pro Arg Cys Ser Ile260 265 270Ala Ile Phe Ile Ser Cys Ile Tyr Glu Trp Leu Leu Leu Lys Asp Asp275 280 285Arg Thr Leu Asn Phe Leu Ile Leu Ala Asp Arg Asn Cys Phe Phe Ser290 295 300
Ala Asn Arg Glu Gly Ile Phe Ser Phe Leu Gly Tyr Cys Ser Ile Phe305 310 315 320Leu Trp Gly Gln Asn Thr Gly Phe Tyr Leu Leu Gly Asn Lys Pro Thr325 330 335Leu Asn Asn Leu Tyr Lys Pro Ser Thr Gln Asp Val Val Ala Ala Ser340 345 350Lys Lys Ser Ser Thr Trp Asp Tyr Trp Thr Ser Val Thr Pro Leu Ser355 360 365Gly Leu Cys Ile Trp Ser Thr Ile Phe Leu Val Ile Ser Gln Leu Val370 375 380Phe Gln Tyr His Pro Tyr Ser Val Ser Arg Arg Phe Ala Asn Leu Pro385 390 395 400Tyr Thr Leu Trp Val Ile Thr Tyr Asn Leu Leu Phe Leu Thr Gly Tyr405 410 415Cys Leu Thr Asp Lys Ile Phe Gly Asn Ser Ser Glu Tyr Tyr Lys Val420 425 430Ala Glu Cys Leu Glu Ser Ile Asn Ser Asn Gly Leu Phe Leu Phe Leu435 440 445Leu Ala Asn Val Ser Thr Gly Leu Val Asn Met Ser Met Val Thr Ile450 455 460Asp Ser Ser Pro Leu Lys Ser Phe Leu Val Leu Leu Ala Tyr Cys Ser465 470 475 480Phe Ile Ala Val Ile Ser Val Phe Leu Tyr Arg Lys Arg Ile Phe Ile485 490 495Lys Leu<210>3<211>1458<212>DNA<213>白色念珠菌(Candida albicans)
<220>
<221>CDS<222>(1)..(1455)<400>3atg tca tcg tct tta aaa caa ttg aaa gaa caa ttt gtc tca gat ttg48Met Ser Ser Ser Leu Lys Gln Leu Lys Glu Gln Phe Val Ser Asp Leu1 5 10 15act ggt ggc aca att gaa gaa att tat gct gta acc agt ata gca tta96Thr Gly Gly Thr Ile Glu Glu Ile Tyr Ala Val Thr Ser Ile Ala Leu20 25 30tca tct tat ttg tcc ttt aga ttg ttg aaa aag tct ctt ggt gat tta144Ser Ser Tyr Leu Ser Phe Arg Leu Leu Lys Lys Ser Leu Gly Asp Leu35 40 45gct ttg att tac gac tac att ctt aat gtg ttg aca att cta gca tcc192Ala Leu Ile Tyr Asp Tyr Ile Leu Asn Val Leu Thr Ile Leu Ala Ser50 55 60att act gtt tat agc aac agc cct tct tat ttg cat tat ttt att gtt240Ile Thr Val Tyr Ser Asn Ser Pro Ser Tyr Leu His Tyr Phe Ile Val65 70 75 80att cca tca tta gtt ata tat cta gtg aat tac cat gtt gag aaa cca288Ile Pro Ser Leu Val Ile Tyr Leu Val Asn Tyr His Val Glu Lys Pro85 90 95tct tca ccc cat aga caa aat gat aca aaa gaa gat aaa tcg gac gaa336Ser Ser Pro His Arg Gln Asn Asp Thr Lys Glu Asp Lys Ser Asp Glu100 105 110cta ttg ccg aga aaa caa ttt ata aca gcc tat cgt tct caa atg ttg384Leu Leu Pro Arg Lys Gln Phe Ile Thr Ala Tyr Arg Ser Gln Met Leu115 120 125ata att act aat cta gct ata tta gct gtt gat ttt cct att ttc cca432Ile Ile Thr Asn Leu Ala Ile Leu Ala Val Asp Phe Pro Ile Phe Pro130 135 140aga aga ttt gcc aaa gtg gaa aca tgg ggc acg tca atg atg gat tta480Arg Arg Phe Ala Lys Val Glu Thr Trp Gly Thr Ser Met Met Asp Leu145 150 155 160
gga gtt ggg tcg ttt gtg ttc tcc atg ggg ttg gct aat tct cga caa528Gly Val Gly Ser Phe Val Phe Ser Met Gly Leu Ala Asn Ser Arg Gln165 170 175ttg atc aag aac cac acc gac aac tac aaa ttt agt tgg aag agt tat576Leu Ile Lys Asn His Thr Asp Asn Tyr Lys Phe Ser Trp Lys Ser Tyr180 185 190ttg aaa aca atc aag cag aac ttt atc aag tca gtg cct ata ctt gtt624Leu Lys Thr Ile Lys Gln Asn Phe Ile Lys Ser Val Pro Ile Leu Val195 200 205tta gga gct att cgt ttt gtt agt gtt aag caa ttg gac tat cag gaa672Leu Gly Ala Ile Arg Phe Val Ser Val Lys Gln Leu Asp Tyr Gln Glu210 215 220cac gaa aca gag tat gga atc cat tgg aat ttt ttc ttc aca tta ggg720His Glu Thr Glu Tyr Gly Ile His Trp Asn Phe Phe Phe Thr Leu Gly225 230 235 240ttc ttg cca att gta ttg gga ata tta gac ccg gtg ttg aat ttg gtt768Phe Leu Pro Ile Val Leu Gly Ile Leu Asp Pro Val Leu Asn Leu Val245 250 255cca cgc ttc ata ata gga att ggt atc tca att gct tat gag gta gcg816Pro Arg Phe Ile Ile Gly Ile Gly Ile Ser Ile Ala Tyr Glu Val Ala260 265 270ttg aat aag act ggt ttg ttg aag ttc att ttg agc agc gaa aac aga864Leu Asn Lys Thr Gly Leu Leu Lys Phe Ile Leu Ser Ser Glu Asn Arg275 280 285ctt gaa tct ctc atc acc atg aat aaa gaa ggt att ttt tcg ttt att912Leu Glu Ser Leu Ile Thr Met Asn Lys Glu Gly Ile Phe Ser Phe Ile290 295 300gga tat ctt tgt att ttt ata att ggt cag tct ttt ggg tca ttt gtt960Gly Tyr Leu Cys Ile Phe Ile Ile Gly Gln Ser Phe Gly Ser Phe Val305 310 315 320tta aca ggc tac aaa aca aag aac aac tta ata acc att agc aaa att1008Leu Thr Gly Tyr Lys Thr Lys Asn Asn Leu Ile Thr Ile Ser Lys Ile325 330 335
cgt att tca aaa aaa caa cac aag aaa gag ctg ctg ctg ttt ttc tca1056Arg Ile Ser Lys Lys Gln His Lys Lys Glu Leu Leu Leu Phe Phe Ser340 345 350gtc gcc act act cag gga tta tat ttg gca tgt atc ttc tat cac tta1104Val Ala Thr Thr Gln Gly Leu Tyr Leu Ala Cys Ile Phe Tyr His Leu355 360 365gct ttc agt ttg ttc atc agc aac tta tca ttc ttg caa cca att tca1152Ala Phe Ser Leu Phe Ile Ser Asn Leu Ser Phe Leu Gln Pro Ile Ser370 375 380aga cga ttg gcc aat ttc ccc tac gtc atg tgg gtc gtt tcg tac aat1200Arg Arg Leu Ala Asn Phe Pro Tyr Val Met Trp Val Val Ser Tyr Asn385 390 395 400gct acg ttt tta tta tgt tat gac tta att gaa aaa ttt atc ccg ggg1248Ala Thr Phe Leu Leu Cys Tyr Asp Leu Ile Glu Lys Phe Ile Pro Gly405 410 415aac ctt act tct act gta ttg gac tct att aat aac aat ggt tta ttt1296Asn Leu Thr Ser Thr Val Leu Asp Ser Ile Asn Asn Asn Gly Leu Phe420 425 430atc ttc ttg gtc agc aat tta tta aca ggg ttt att aac atg tcc atc1344Ile Phe Leu Val Ser Asn Leu Leu Thr Gly Phe Ile Asn Met Ser Ile435 440 445aac act ttg gaa act agc aat aaa atg gca gtg att atc ttg att ggc1392Asn Thr Leu Glu Thr Ser Asn Lys Met Ala Val Ile Ile Leu Ile Gly450 455 460tat agt ctt act tgg aca ttg ctc gcc tta tat ttg gat aag agg aag1440Tyr Ser Leu Thr Trp Thr Leu Leu Ala Leu Tyr Leu Asp Lys Arg Lys465 470 475 480atc tac atc aag ctt tag1458Ile Tyr Ile Lys Leu485<210>4<211>485
<212>PRT<213>白色念珠菌(Candida albicans)<400>4Met Ser Ser Ser Leu Lys Gln Leu Lys Glu Gln Phe Val Ser Asp Leu1 5 10 15Thr Gly Gly Thr Ile Glu Glu Ile Tyr Ala Val Thr Ser Ile Ala Leu20 25 30Ser Ser Tyr Leu Ser Phe Arg Leu Leu Lys Lys Ser Leu Gly Asp Leu35 40 45Ala Leu Ile Tyr Asp Tyr Ile Leu Asn Val Leu Thr Ile Leu Ala Ser50 55 60Ile Thr Val Tyr Ser Asn Ser Pro Ser Tyr Leu His Tyr Phe Ile Val65 70 75 80Ile Pro Ser Leu Val Ile Tyr Leu Val Asn Tyr His Val Glu Lys Pro85 90 95Ser Ser Pro His Arg Gln Asn Asp Thr Lys Glu Asp Lys Ser Asp Glu100 105 110Leu Leu Pro Arg Lys Gln Phe Ile Thr Ala Tyr Arg Ser Gln Met Leu115 120 125Ile Ile Thr Asn Leu Ala Ile Leu Ala Val Asp Phe Pro Ile Phe Pro130 135 140Arg Arg Phe Ala Lys Val Glu Thr Trp Gly Thr Ser Met Met Asp Leu145 150 155 160Gly Val Gly Ser Phe Val Phe Ser Met Gly Leu Ala Asn Ser Arg Gln165 170 175Leu Ile Lys Asn His Thr Asp Asn Tyr Lys Phe Ser Trp Lys Ser Tyr180 185 190Leu Lys Thr Ile Lys Gln Asn Phe Ile Lys Ser Val Pro Ile Leu Val195 200 205
Leu Gly Ala Ile Arg Phe Val Ser Val Lys Gln Leu Asp Tyr Gln Glu210 215 220His Glu Thr Glu Tyr Gly Ile His Trp Asn Phe Phe Phe Thr Leu Gly225 230 235 240Phe Leu Pro Ile Val Leu Gly Ile Leu Asp Pro Val Leu Asn Leu Val245 250 255Pro Arg Phe Ile Ile Gly Ile Gly Ile Ser Ile Ala Tyr Glu Val Ala260 265 270Leu Asn Lys Thr Gly Leu Leu Lys Phe Ile Leu Ser Ser Glu Asn Arg275 280 285Leu Glu Ser Leu Ile Thr Met Asn Lys Glu Gly Ile Phe Ser Phe Ile290 295 300Gly Tyr Leu Cys Ile Phe Ile Ile Gly Gln Ser Phe Gly Ser Phe Val305 310 315 320Leu Thr Gly Tyr Lys Thr Lys Asn Asn Leu Ile Thr Ile Ser Lys Ile325 330 335Arg Ile Ser Lys Lys Gln His Lys Lys Glu Leu Leu Leu Phe Phe Ser340 345 350Val Ala Thr Thr Gln Gly Leu Tyr Leu Ala Cys Ile Phe Tyr His Leu355 360 365Ala Phe Ser Leu Phe Ile Ser Asn Leu Ser Phe Leu Gln Pro Ile Ser370 375 380Arg Arg Leu Ala Asn Phe Pro Tyr Val Met Trp Val Val Ser Tyr Asn385 390 395 400Ala Thr Phe Leu Leu Cys Tyr Asp Leu Ile Glu Lys Phe Ile Pro Gly405 410 415Asn Leu Thr Ser Thr Val Leu Asp Ser Ile Asn Asn Asn Gly Leu Phe420 425 430Ile Phe Leu Val Ser Asn Leu Leu Thr Gly Phe Ile Asn Met Ser Ile
435 440 445Asn Thr Leu Glu Thr Ser Asn Lys Met Ala Val Ile Ile Leu Ile Gly450 455 460Tyr Ser Leu Thr Trp Thr Leu Leu Ala Leu Tyr Leu Asp Lys Arg Lys465 470 475 480Ile Tyr Ile Lys Leu485<210>5<211>1458<212>DNA<213>白色念珠菌(Candida albicans)<220>
<221>CDS<222>(1)..(1455)<400>5atg tca tcg tct tta aaa caa ttg aaa gaa caa ttt gtc tca gat ttg48Met Ser Ser Ser Leu Lys Gln Leu Lys Glu Gln Phe Val Ser Asp Leu1 5 10 15act ggt ggc aca att gaa gaa att tat gct gta acc agt ata gca tta96Thr Gly Gly Thr Ile Glu Glu Ile Tyr Ala Val Thr Ser Ile Ala Leu20 25 30tca tct tat ttg tcc ttt aga ttg ttg aaa aag tct ctt ggt gat tta144Ser Ser Tyr Leu Ser Phe Arg Leu Leu Lys Lys Ser Leu Gly Asp Leu35 40 45gct ttg att tac gac tac att ctt aat gtg ttg aca att cta gca tcc192Ala Leu Ile Tyr Asp Tyr Ile Leu Asn Val Leu Thr Ile Leu Ala Ser50 55 60att act gtt tat agc aac agc cct tct tat ttg cat tat ttt att gtt240Ile Thr Val Tyr Ser Asn Ser Pro Ser Tyr Leu His Tyr Phe Ile Val65 70 75 80att cca tca tta gtt ata tat cta gtg aat tac cat gtt gag aaa cca288Ile Pro Ser Leu Val Ile Tyr Leu Val Asn Tyr His Val Glu Lys Pro
85 90 95tct tca ccc cat aga caa aat gat aca aaa gaa gat aaa tcg gac gaa336Ser Ser Pro His Arg Gln Asn Asp Thr Lys Glu Asp Lys Ser Asp Glu100 105 110cta ttg ccg aga aaa caa ttt ata aca gcc tat cgt tct caa atg ttg384Leu Leu Pro Arg Lys Gln Phe Ile Thr Ala Tyr Arg Ser Gln Met Leu115 120 125ata att act aat cta gct ata tta gct gtt gat ttt cct att ttc cca432Ile Ile Thr Asn Leu Ala Ile Leu Ala Val Asp Phe Pro Ile Phe Pro130 135 140aga aga ttt gcc aaa gtg gaa aca tgg ggc acg tca atg atg gat tta480Arg Arg Phe Ala Lys Val Glu Thr Trp Gly Thr Ser Met Met Asp Leu145 150 155 160gga gtt ggg tcg ttt gtg ttc tcc atg ggg ttg gct aat tct cga caa528Gly Val Gly Ser Phe Val Phe Ser Met Gly Leu Ala Asn Ser Arg Gln165 170 175ttg atc aag aac cac acc gac aat tac aaa ttt agt tgg aag agt tat576Leu Ile Lys Asn His Thr Asp Asn Tyr Lys Phe Ser Trp Lys Ser Tyr180 185 190ttg aaa aca atc aag cag aac ttt atc aag tca gtg cct ata ctt gtt624Leu Lys Thr Ile Lys Gln Asn Phe Ile Lys Ser Val Pro Ile Leu Val195 200 205tta gga gct att cgt ttt gtt agt gtt aag caa ttg gac tat cag gaa672Leu Gly Ala Ile Arg Phe Val Ser Val Lys Gln Leu Asp Tyr Gln Glu210 215 220cac gaa aca gag tat gga atc cat tgg aat ttt ttc ttc aca tta ggg720His Glu Thr Glu Tyr Gly Ile His Trp Asn Phe Phe Phe Thr Leu Gly225 230 235 240ttc ttg cca att gta ttg gga ata tta gac ccg gtg ttg aat ttg gtt768Phe Leu Pro Ile Val Leu Gly Ile Leu Asp Pro Val Leu Asn Leu Val245 250 255cca cgc ttc ata ata gga att ggt atc tca att ggt tat gag gta gcg816Pro Arg Phe Ile Ile Gly Ile Gly Ile Ser Ile Gly Tyr Glu Val Ala
260 265 270ttg aat aag act ggt ttg ttg aag ttc att ttg agc agc gaa aac aga864Leu Asn Lys Thr Gly Leu Leu Lys Phe Ile Leu Ser Ser Glu Asn Arg275 280 285ctt gaa tct ctc atc gcc atg aat aaa gaa ggt att ttt tcg ttt att912Leu Glu Ser Leu Ile Ala Met Asn Lys Glu Gly Ile Phe Ser Phe Ile290 295 300gga tat ctt tgt att ttt ata att ggt cag tct ttt ggg tca ttt gtt960Gly Tyr Leu Cys Ile Phe Ile Ile Gly Gln Ser Phe Gly Ser Phe Val305 310 315 320tta aca ggc tac aaa aca aag aac aac tta ata acc att agc aaa att1008Leu Thr Gly Tyr Lys Thr Lys Asn Asn Leu Ile Thr Ile Ser Lys Ile325 330 335cgt att tca aaa aaa caa cac aag aaa gag ctg ctg ctg ttt ttc tca1056Arg Ile Ser Lys Lys Gln His Lys Lys Glu Leu Leu Leu Phe Phe Ser340 345 350gtc gcc act act cag gga tta tat ttg gca tgt atc ttc tat cac tta1104Val Ala Thr Thr Gln Gly Leu Tyr Leu Ala Cys Ile Phe Tyr His Leu355 360 365gct ttc agt ttg ttc atc agc aac tta tca ttc ttg caa cca att tca1152Ala Phe Ser Leu Phe Ile Ser Asn Leu Ser Phe Leu Gln Pro Ile Ser370 375 380aga cga ttg gcc aat ttc ccc tac gtc atg tgg gtc gtt tcg tac aat1200Arg Arg Leu Ala Asn Phe Pro Tyr Val Met Trp Val Val Ser Tyr Asn385 390 395 400gct acg ttt tta tta tgt tat gac tta att gaa aaa ttt atc ccg ggg1248Ala Thr Phe Leu Leu Cys Tyr Asp Leu Ile Glu Lys Phe Ile Pro Gly405 410 415aac ctt act tct act gta ttg gac tct att aat aac aat ggt tta ttt1296Asn Leu Thr Ser Thr Val Leu Asp Ser Ile Asn Asn Asn Gly Leu Phe420 425 430atc ttc ttg gtc agc aat tta tta aca ggg ttt att aac atg tcc atc1344Ile Phe Leu Val Ser Asn Leu Leu Thr Gly Phe Ile Asn Met Ser Ile
435 440 445aac act ttg gaa act agc aat aaa atg gca gtg att atc ttg att ggc 1392Asn Thr Leu Glu Thr Ser Asn Lys Met Ala Val Ile Ile Leu Ile Gly450 455 460tat agt ctt act tgg aca ttg ctc gcc tta tat ttg gat aag agg aag 1440Tyr Ser Leu Thr Trp Thr Leu Leu Ala Leu Tyr Leu Asp Lys Arg Lys465 470 475 480atc tac atc aag ctt tag 1458Ile Tyr Ile Lys Leu485<210>6<211>485<212>PRT<213>白色念珠菌(Candida albicans)<400>6Met Ser Ser Ser Leu Lys Gln Leu Lys Glu Gln Phe Val Ser Asp Leu1 5 10 15Thr Gly Gly Thr Ile Glu Glu Ile Tyr Ala Val Thr Ser Ile Ala Leu20 25 30Ser Ser Tyr Leu Ser Phe Arg Leu Leu Lys Lys Ser Leu Gly Asp Leu35 40 45Ala Leu Ile Tyr Asp Tyr Ile Leu Asn Val Leu Thr Ile Leu Ala Ser50 55 60Ile Thr Val Tyr Ser Asn Ser Pro Ser Tyr Leu His Tyr Phe Ile Val65 70 75 80Ile Pro Ser Leu Val Ile Tyr Leu Val Asn Tyr His Val Glu Lys Pro85 90 95Ser Ser Pro His Arg Gln Asn Asp Thr Lys Glu Asp Lys Ser Asp Glu100 105 110Leu Leu Pro Arg Lys Gln Phe Ile Thr Ala Tyr Arg Ser Gln Met Leu115 120 125
Ile Ile Thr Asn Leu Ala Ile Leu Ala Val Asp Phe Pro Ile Phe Pro130 135 140Arg Arg Phe Ala Lys Val Glu Thr Trp Gly Thr Ser Met Met Asp Leu145 150 155 160Gly Val Gly Ser Phe Val Phe Ser Met Gly Leu Ala Asn Ser Arg Gln165 170 175Leu Ile Lys Asn His Thr Asp Asn Tyr Lys Phe Ser Trp Lys Ser Tyr180 185 190Leu Lys Thr Ile Lys Gln Asn Phe Ile Lys Ser Val Pro Ile Leu Val195 200 205Leu Gly Ala Ile Arg Phe Val Ser Val Lys Gln Leu Asp Tyr Gln Glu210 215 220His Glu Thr Glu Tyr Gly Ile His Trp Asn Phe Phe Phe Thr Leu Gly225 230 235 240Phe Leu Pro Ile Val Leu Gly Ile Leu Asp Pro Val Leu Asn Leu Val245 250 255Pro Arg Phe Ile Ile Gly Ile Gly Ile Ser Ile Gly Tyr Glu Val Ala260 265 270Leu Asn Lys Thr Gly Leu Leu Lys Phe Ile Leu Ser Ser Glu Asn Arg275 280 285Leu Glu Ser Leu Ile Ala Met Asn Lys Glu Gly Ile Phe Ser Phe Ile290 295 300Gly Tyr Leu Cys Ile Phe Ile Ile Gly Gln Ser Phe Gly Ser Phe Val305 310 315 320Leu Thr Gly Tyr Lys Thr Lys Asn Asn Leu Ile Thr Ile Ser Lys Ile325 330 335Arg Ile Ser Lys Lys Gln His Lys Lys Glu Leu Leu Leu Phe Phe Ser340 345 350Val Ala Thr Thr Gln Gly Leu Tyr Leu Ala Cys Ile Phe Tyr His Leu
355 360 365Ala Phe Ser Leu Phe Ile Ser Asn Leu Ser Phe Leu Gln Pro Ile Ser370 375 380Arg Arg Leu Ala Asn Phe Pro Tyr Val Met Trp Val Val Ser Tyr Asn385 390 395 400Ala Thr Phe Leu Leu Cys Tyr Asp Leu Ile Glu Lys Phe Ile Pro Gly405 410 415Asn Leu Thr Ser Thr Val Leu Asp Ser Ile Asn Asn Asn Gly Leu Phe420 425 430Ile Phe Leu Val Ser Asn Leu Leu Thr Gly Phe Ile Asn Met Ser Ile435 440 445Asn Thr Leu Glu Thr Ser Asn Lys Met Ala Val Ile Ile Leu Ile Gly450 455 460Tyr Ser Leu Thr Trp Thr Leu Leu Ala Leu Tyr Leu Asp Lys Arg Lys465 470 475 480Ile Tyr Ile Lys Leu485<210>7<211>1380<212>DNA<213>粟酒裂殖酵母(Schizosaccharomyces pombe)<220>
<221>CDS<222>(1)..(1380)<400>7atg tca tac aaa ttg gaa aaa gaa gca ttt gtc tca aac ctg acg ggt48Met Ser Tyr Lys Leu Glu Lys Glu Ala Phe Val Ser Asn Leu Thr Gly1 5 10 15tca agt tcc att gag aca tgt ggc ttg tta tta ata gga att gct tgc96Ser Ser Ser Ile Glu Thr Cys Gly Leu Leu Leu Ile Gly Ile Ala Cys
20 25 30aac gtt ttg tgg gta aac atg act gcg aga aac atc tta ccc aaa ggg144Asn Val Leu Trp Val Asn Met Thr Ala Arg Asn Ile Leu Pro Lys Gly35 40 45aat ctt ggg ttt ctt gtt gag ttt ttc atc ttt tgc tta att cca tta192Asn Leu Gly Phe Leu Val Glu Phe Phe Ile Phe Cys Leu Ile Pro Leu50 55 60ttt gtc att tac gtt tca tcg aaa gtt ggc gtt ttc act ctt tgc ata240Phe Val Ile Tyr Val Ser Ser Lys Val Gly Val Phe Thr Leu Cys Ile65 70 75 80gcc tct ttt ttg cct tcc ttc gtc ctt cat gtt ata agt cca att aat288Ala Ser Phe Leu Pro Ser Phe Val Leu His Val Ile Ser Pro Ile Asn85 90 95tgg gat gtg ctg aga aga aaa cct ggt tgt tgt ctt act aaa aaa aat336Trp Asp Val Leu Arg Arg Lys Pro Gly Cys Cys Leu Thr Lys Lys Asn100 105 110gaa aat act ttt gat cga cga att gct gga gtc aca ttt tat cgt tct384Glu Asn Thr Phe Asp Arg Arg Ile Ala Gly Val Thr Phe Tyr Arg Ser115 120 125caa atg atg ttg gtt act gtc act tgc atc ctg gcc gtt gac ttt acc432Gln Met Met Leu Val Thr Val Thr Cys Ile Leu Ala Val Asp Phe Thr130 135 140ctt ttc ccg agg aga tat gcc aaa gtt gaa acc tgg gga aca tca ctg480Leu Phe Pro Arg Arg Tyr Ala Lys Val Glu Thr Trp Gly Thr Ser Leu145 150 155 160atg gat ctt ggt gtt gga tct ttc atg ttt tct tca ggt act gtg gct528Met Asp Leu Gly Val Gly Ser Phe Met Phe Ser Ser Gly Thr Val Ala165 170 175gga cgg aaa aat gac att aaa aaa cca aat gcg ttt aaa aat gta ttg576Gly Arg Lys Asn Asp Ile Lys Lys Pro Asn Ala Phe Lys Asn Val Leu180 185 190tgg aat tct ttc atc ctt ttg att tta gga ttt gcg cgc atg ttt tta624
Trp Asn Ser Phe Ile Leu Leu Ile Leu Gly Phe Ala Arg Met Phe Leu195 200 205acg aaa agc atc aat tac caa gaa cat gta agc gaa tat ggc atg cat672Thr Lys Ser Ile Asn Tyr Gln Glu His Val Ser Glu Tyr Gly Met His210 215 220tgg aac ttt ttt ttc acc cta ggt ttc atg gct ctt ggc gta ttt ttt720Trp Asn Phe Phe Phe Thr Leu Gly Phe Met Ala Leu Gly Val Phe Phe225 230 235 240ttt cgt cgt tct tta aaa aaa gtc tcc tat ttt aat tta gca acc ttc768Phe Arg Arg Ser Leu Lys Lys Val Ser Tyr Phe Asn Leu Ala Thr Phe245 250 255att act ctt ctt cat cat tgt ttg ctt gtt tta acc cct ttc caa aaa816Ile Thr Leu Leu His His Cys Leu Leu Val Leu Thr Pro Phe Gln Lys260 265 270tgg gca cta tcc gcc ccc aga aca aat att ttg gct cag aat aga gag864Trp Ala Leu Ser Ala Pro Arg Thr Asn Ile Leu Ala Gln Asn Arg Glu275 280 285ggt att gct tct ctt ccc gga tac att gct att tac ttt tat gga atg912Gly Ile Ala Ser Leu Pro Gly Tyr Ile Ala Ile Tyr Phe Tyr Gly Met290 295 300tat acc ggt agt gta gtt ttg gct gat cga cct cta atg tat act aga960Tyr Thr Gly Ser Val Val Leu Ala Asp Arg Pro Leu Met Tyr Thr Arg305 310 315 320gct gag tcg tgg aag cgc ttt caa cgt cta tta ttc ccg cta tgc att1008Ala Glu Ser Trp Lys Arg Phe Gln Arg Leu Leu Phe Pro Leu Cys Ile325 330 335ttg tta gtg ttg tat ctt gtg tct aac ttt ttg tca gtt ggt gtt tct1056Leu Leu Val Leu Tyr Leu Val Ser Asn Phe Leu Ser Val Gly Val Ser340 345 350cgc cga ctt gct aat acg cct tat gtt gcg aat gtt gcc ttt atc aat1104Arg Arg Leu Ala Asn Thr Pro Tyr Val Ala Asn Val Ala Phe Ile Asn355 360 365atg ttt ttt ctt act ata tac ata ctt att gat gcc tat tta ttc cca1152
Met Phe Phe Leu Thr Ile Tyr Ile Leu Ile Asp Ala Tyr Leu Phe Pro370 375 380tct tct gtg cca tat gga agt cgc gtc ccc aaa ctg ctt gaa gat gcc 1200Ser Ser Val Pro Tyr Gly Ser Arg Val Pro Lys Leu Leu Glu Asp Ala385 390 395 400aat aat aat ggc ttg ttg gtg ttt ttg att gct aac gtt tta aca gga 1248Asn Asn Asn Gly Leu Leu Val Phe Leu Ile Ala Asn Val Leu Thr Gly405 410 415gta gtt aat tta tcg ttc gac acc ctt cat tct agc aat gca aaa ggc 1296Val Val Asn Leu Ser Phe Asp Thr Leu His Ser Ser Asn Ala Lys Gly420 425 430ttg aca atc atg act atg tat ctt ttt att att tgc tat atg gca cat 1344Leu Thr Ile Met Thr Met Tyr Leu Phe Ile Ile Cys Tyr Met Ala His435 440 445tgg ctt gct caa cac gga att cgt ttt cgc ctt tag 1380Trp Leu Ala Gln His Gly Ile Arg Phe Arg Leu450 455 460<210>8<211>459<212>PRT<213>粟酒裂殖酵母(Schi zosaccharomyces pombe)<400>8Met Ser Tyr Lys Leu Glu Lys Glu Ala Phe Val Ser Asn Leu Thr Gly1 5 10 15Ser Ser Ser Ile Glu Thr Cys Gly Leu Leu Leu Ile Gly Ile Ala Cys20 25 30Asn Val Leu Trp Val Asn Met Thr Ala Arg Asn Ile Leu Pro Lys Gly35 40 45Asn Leu Gly Phe Leu Val Glu Phe Phe Ile Phe Cys Leu Ile Pro Leu50 55 60Phe Val Ile Tyr Val Ser Ser Lys Val Gly Val Phe Thr Leu Cys Ile65 70 75 80
Ala Ser Phe Leu Pro Ser Phe Val Leu His Val Ile Ser Pro Ile Asn85 90 95Trp Asp Val Leu Arg Arg Lys Pro Gly Cys Cys Leu Thr Lys Lys Asn100 105 110Glu Asn Thr Phe Asp Arg Arg Ile Ala Gly Val Thr Phe Tyr Arg Ser115 120 125Gln Met Met Leu Val Thr Val Thr Cys Ile Leu Ala Val Asp Phe Thr130 135 140Leu Phe Pro Arg Arg Tyr Ala Lys Val Glu Thr Trp Gly Thr Ser Leu145 150 155 160Met Asp Leu Gly Val Gly Ser Phe Met Phe Ser Ser Gly Thr Val Ala165 170 175Gly Arg Lys Asn Asp Ile Lys Lys Pro Asn Ala Phe Lys Asn Val Leu180 185 190Trp Asn Ser Phe Ile Leu Leu Ile Leu Gly Phe Ala Arg Met Phe Leu195 200 205Thr Lys Ser Ile Asn Tyr Gln Glu His Val Ser Glu Tyr Gly Met His210 215 220Trp Asn Phe Phe Phe Thr Leu Gly Phe Met Ala Leu Gly Val Phe Phe225 230 235 240Phe Arg Arg Ser Leu Lys Lys Val Ser Tyr Phe Asn Leu Ala Thr Phe245 250 255Ile Thr Leu Leu His His Cys Leu Leu Val Leu Thr Pro Phe Gln Lys260 265 270Trp Ala Leu Ser Ala Pro Arg Thr Asn Ile Leu Ala Gln Asn Arg Glu275 280 285Gly Ile Ala Ser Leu Pro Gly Tyr Ile Ala Ile Tyr Phe Tyr Gly Met290 295 300Tyr Thr Gly Ser Val Val Leu Ala Asp Arg Pro Leu Met Tyr Thr Arg305 310 315 320
Ala Glu Ser Trp Lys Arg Phe Gln Arg Leu Leu Phe Pro Leu Cys Ile325 330 335Leu Leu Val Leu Tyr Leu Val Ser Asn Phe Leu Ser Val Gly Val Ser340 345 350Arg Arg Leu Ala Asn Thr Pro Tyr Val Ala Asn Val Ala Phe Ile Asn355 360 365Met Phe Phe Leu Thr Ile Tyr Ile Leu Ile Asp Ala Tyr Leu Phe Pro370 375 380Ser Ser Val Pro Tyr Gly Ser Arg Val Pro Lys Leu Leu Glu Asp Ala385 390 395 400Asn Asn Asn Gly Leu Leu Val Phe Leu Ile Ala Asn Val Leu Thr Gly405 410 415Val Val Asn Leu Ser Phe Asp Thr Leu His Ser Ser Asn Ala Lys Gly420 425 430Leu Thr Ile Met Thr Met Tyr Leu Phe Ile Ile Cys Tyr Met Ala His435 440 445Trp Leu Ala Gln His Gly Ile Arg Phe Arg Leu450 455<210>9<211>1576<212>DNA<213>烟曲霉(Aspergillus fumigatus)<220>
<221>CDS<222>(31)..(1536)<400>9aaggtgcaaa tcccgcggca ttgagtcaag atg gat cca gat tat aaa gct cgc 54Met Asp Pro Asp Tyr Lys Ala Arg1 5aaa gag gcc ttt gtc tca ggt ctt gca gga gga agc atc ctg gaa atc 102
Lys Glu Ala Phe Val Ser Gly Leu Ala Gly Gly Ser Ile Leu Glu Ile10 15 20aac gcc gtc acc ttg gtt gct tcg gta tcc gtt ttt ctg tgg tca att150Asn Ala Val Thr Leu Val Ala Ser Val Ser Val Phe Leu Trp Ser Ile25 30 35 40cta caa tct cgc cta tcc ttt ttc aca ccc tac agc gcc gct gcc ctt198Leu Gln Ser Arg Leu Ser Phe Phe Thr Pro Tyr Ser Ala Ala Ala Leu45 50 55ctc gtt gat ttc ctg ctc aat gta cta gct atc ttg ttc gca acc act246Leu Val Asp Phe Leu Leu Asn Val Leu Ala Ile Leu Phe Ala Thr Thr60 65 70tta tac tct tcg gcg cct ctt ctt ctc aat ctc ctt cta ata tct ccc294Leu Tyr Ser Ser Ala Pro Leu Leu Leu Asn Leu Leu Leu Ile Ser Pro75 80 85gct ctg ctg ata ctc ctc tct acg aaa cgt cct cgg acc ccc gtc aaa342Ala Leu Leu Ile Leu Leu Ser Thr Lys Arg Pro Arg Thr Pro Val Lys90 95 100gcg aaa cct cct cgc cag tcc gct aga gct ggg aaa gat gac tcg aaa390Ala Lys Pro Pro Arg Gln Ser Ala Arg Ala Gly Lys Asp Asp Ser Lys105 110 115 120cat gcg aca gcc ttg cca gag tct cta ccc att cat cca ttt ctc acg438His Ala Thr Ala Leu Pro Glu Ser Leu Pro Ile His Pro Phe Leu Thr125 130 135aca tat cgc gcc gcc atg atg gtt atc acg tgc atc gct atc ttg gct486Thr Tyr Arg Ala Ala Met Met Val Ile Thr Cys Ile Ala Ile Leu Ala140 145 150gtg gat ttt cgc att ttt cct cgc cga ttc gcc aag gta gaa aac tgg534Val Asp Phe Arg Ile Phe Pro Arg Arg Phe Ala Lys Val Glu Asn Trp155 160 165ggt aca tca ctc atg gat ctg ggc gtt gga tcg ttt gtc ttt tcg ggc582Gly Thr Ser Leu Met Asp Leu Gly Val Gly Ser Phe Val Phe Ser Gly170 175 180gga gta gta tcc gct cgc tca cta ctc aag agc agg acc aat ggc tct630
Gly Val Val Ser Ala Arg Ser Leu Leu Lys Ser Arg Thr Asn Gly Ser185 190 195 200aaa agg ttg cct ctt gcc aag agg ttg att gcg tcg acg cga cac tct678Lys Arg Leu Pro Leu Ala Lys Arg Leu Ile Ala Ser Thr Arg His Ser205 210 215att cct ctg ctc gtc ctc ggc ctg att cgg cta tac agc gtc aaa ggc726Ile Pro Leu Leu Val Leu Gly Leu Ile Arg Leu Tyr Ser Val Lys Gly220 225 230ttg gac tat gcg gag cac gtc acc gag tac ggc gta cat tgg aac ttc774Leu Asp Tyr Ala Glu His Val Thr Glu Tyr Gly Val His Trp Asn Phe235 240 245ttc ttt aca ttg ggt ctt ttg cct ccg ttc gtg gag gtc ttc gac gcc822Phe Phe Thr Leu Gly Leu Leu Pro Pro Phe Val Glu Val Phe Asp Ala250 255 260ttg gct acg atc att ccg tca tac gag gtt ctc tcc gtg ggg atc gcc870Leu Ala Thr Ile Ile Pro Ser Tyr Glu Val Leu Ser Val Gly Ile Ala265 270 275 280gtc ttg tat caa gtt gcc cta gag tca aca gac ttg aaa agc tac atc918Val Leu Tyr Gln Val Ala Leu Glu Ser Thr Asp Leu Lys Ser Tyr Ile285 290 295ctc gtc tcc cct cgt ggg cca agc tta ctg tcc aag aat cgt gaa ggc966Leu Val Ser Pro Arg Gly Pro Ser Leu Leu Ser Lys Asn Arg Glu Gly300 305 310gtc ttc tcc ttc tca ggt tat ctc gcg att ttt ctt gct ggt cgt gcg1014Val Phe Ser Phe Ser Gly Tyr Leu Ala Ile Phe Leu Ala Gly Arg Ala315 320 325atc ggc att cgg ata atc cct cgc gga act tct ttc tca aga agc cca1062Ile Gly Ile Arg Ile Ile Pro Arg Gly Thr Ser Phe Ser Arg Ser Pro330 335 340gaa cag gcc agg aga cgg gtc ctg atc agc ctt ggc gtg caa gcg tta1110Glu Gln Ala Arg Arg Arg Val Leu Ile Ser Leu Gly Val Gln Ala Leu345 350 355 360gtg tgg acc act ctt ttt gtg ttg aac tcc act tat gcg atg gga tac1158
Val Trp Thr Thr Leu Phe Val Leu Asn Ser Thr Tyr Ala Met Gly Tyr365 370 375gga gct aat atc cct gtc tcc cgc cgc ctc gct aac atg ccc tat gtc 1206Gly Ala Asn Ile Pro Val Ser Arg Arg Leu Ala Asn Met Pro Tyr Val380 385 390ctt tgg gtt tcg gcg ttc aac acc gcg caa ctg ttt gtg ttc tgc ctg 1254Leu Trp Val Ser Ala Phe Asn Thr Ala Gln Leu Phe Val Phe Cys Leu395 400 405atc gaa aca ctc tgc ttt cct gca gtt cat cgg aca acg act caa gag 1302Ile Glu Thr Leu Cys Phe Pro Ala Val His Arg Thr Thr Thr Gln Glu410 415 420agc gaa tct gag cga gtc gat ttt gct acg agc cga atc atg tcg gcc 1350Ser Glu Ser Glu Arg Val Asp Phe Ala Thr Ser Arg Ile Met Ser Ala425 430 435 440ttc aat aag aac agt ctc gcg atc ttt ctt ttg gcc aat ctt ctg act 1398Phe Asn Lys Asn Ser Leu Ala Ile Phe Leu Leu Ala Asn Leu Leu Thr445 450 455gga gct gtg aat ctg agc atc tcc aca att gat gct aat aca gcg cag 1446Gly Ala Val Asn Leu Ser Ile Ser Thr Ile Asp Ala Asn Thr Ala Gln460 465 470gcc atc gct gtt ctc att gga tat tca tcc att atc aca ggg gtt gct 1494Ala Ile Ala Val Leu Ile Gly Tyr Ser Ser Ile Ile Thr Gly Val Ala475 480 485cta gca ttg cat cat gcc aat atc aaa gta ctt cct ttc tag 1536Leu Ala Leu His His Ala Asn Ile Lys Val Leu Pro Phe490 495 500ggtatttacg agcaattggt ggtgtgttga agatatatag 1576<210>10<211>501<212>PRT<213>烟曲霉(Aspergillus fumigatus)<400>10
Met Asp Pro Asp Tyr Lys Ala Arg Lys Glu Ala Phe Val Ser Gly Leu1 5 10 15Ala Gly Gly Ser Ile Leu Glu Ile Asn Ala Val Thr Leu Val Ala Ser20 25 30Val Ser Val Phe Leu Trp Ser Ile Leu Gln Ser Arg Leu Ser Phe Phe35 40 45Thr Pro Tyr Ser Ala Ala Ala Leu Leu Val Asp Phe Leu Leu Asn Val50 55 60Leu Ala Ile Leu Phe Ala Thr Thr Leu Tyr Ser Ser Ala Pro Leu Leu65 70 75 80Leu Asn Leu Leu Leu Ile Ser Pro Ala Leu Leu Ile Leu Leu Ser Thr85 90 95Lys Arg Pro Arg Thr Pro Val Lys Ala Lys Pro Pro Arg Gln Ser Ala100 105 110Arg Ala Gly Lys Asp Asp Ser Lys His Ala Thr Ala Leu Pro Glu Ser115 120 125Leu Pro Ile His Pro Phe Leu Thr Thr Tyr Arg Ala Ala Met Met Val130 135 140Ile Thr Cys Ile Ala Ile Leu Ala Val Asp Phe Arg Ile Phe Pro Arg145 150 155 160Arg Phe Ala Lys Val Glu Asn Trp Gly Thr Ser Leu Met Asp Leu Gly165 170 175Val Gly Ser Phe Val Phe Ser Gly Gly Val Val Ser Ala Arg Ser Leu180 185 190Leu Lys Ser Arg Thr Asn Gly Ser Lys Arg Leu Pro Leu Ala Lys Arg195 200 205Leu Ile Ala Ser Thr Arg His Ser Ile Pro Leu Leu Val Leu Gly Leu210 215 220Ile Arg Leu Tyr Ser Val Lys Gly Leu Asp Tyr Ala Glu His Val Thr
225 230 235 240Glu Tyr Gly Val His Trp Asn Phe Phe Phe Thr Leu Gly Leu Leu Pro245 250 255Pro Phe Val Glu Val Phe Asp Ala Leu Ala Thr Ile Ile Pro Ser Tyr260 265 270Glu Val Leu Ser Val Gly Ile Ala Val Leu Tyr Gln Val Ala Leu Glu275 280 285Ser Thr Asp Leu Lys Ser Tyr Ile Leu Val Ser Pro Arg Gly Pro Ser290 295 300Leu Leu Ser Lys Asn Arg Glu Gly Val Phe Ser Phe Ser Gly Tyr Leu305 310 315 320Ala Ile Phe Leu Ala Gly Arg Ala Ile Gly Ile Arg Ile Ile Pro Arg325 330 335Gly Thr Ser Phe Ser Arg Ser Pro Glu Gln Ala Arg Arg Arg Val Leu340 345 350Ile Ser Leu Gly Val Gln Ala Leu Val Trp Thr Thr Leu Phe Val Leu355 360 365Asn Ser Thr Tyr Ala Met Gly Tyr Gly Ala Asn Ile Pro Val Ser Arg370 375 380Arg Leu Ala Asn Met Pro Tyr Val Leu Trp Val Ser Ala Phe Asn Thr385 390 395 400Ala Gln Leu Phe Val Phe Cys Leu Ile Glu Thr Leu Cys Phe Pro Ala405 410 415Val His Arg Thr Thr Thr Gln Glu Ser Glu Ser Glu Arg Val Asp Phe420 425 430Ala Thr Ser Arg Ile Met Ser Ala Phe Asn Lys Asn Ser Leu Ala Ile435 440 445Phe Leu Leu Ala Asn Leu Leu Thr Gly Ala Val Asn Leu Ser Ile Ser450 455 460
Thr Ile Asp Ala Asn Thr Ala Gln Ala Ile Ala Val Leu Ile Gly Tyr465 470 475 480Ser Ser Ile Ile Thr Gly Val Ala Leu Ala Leu His His Ala Asn Ile485 490 495Lys Val Leu Pro Phe500<210>11<211>1648<212>DNA<213>烟曲霉(Aspergillus fumigatus)<220>
<221>内含子<222>(122)..(198)<220>
<221>CDS<222>(26)..(121)<220>
<221>CDS<222>(199)..(1608)<400>11gcaaatcccg cggcattgag tcaag atg gat cca gat tat aaa gct cgc aaa 52Met Asp Pro Asp Tyr Lys Ala Arg Lys1 5gag gcc ttt gtc tca ggt ctt gca gga gga agc atc ctg gaa atc aac 100Glu Ala Phe Val Ser Gly Leu Ala Gly Gly Ser Ile Leu Glu Ile Asn10 15 20 25gcc gtc acc ttg gtt gct tcg gttcgtgtta ctatcttatt gtggctactt 151Ala Val Thr Leu Val Ala Ser30cgcctacatt gtttctcgac taaccgagtc tctttgcgat caatcag gta tcc gtt 207Val Ser Val35
ttt ctg tgg tca att cta caa tct cgc cta tcc ttt ttc aca ccc tac255Phe Leu Trp Ser Ile Leu Gln Ser Arg Leu Ser Phe Phe Thr Pro Tyr40 45 50agc gcc gct gcc ctt ctc gtt gat ttc ctg ctc aat gta cta gct atc303Ser Ala Ala Ala Leu Leu Val Asp Phe Leu Leu Asn Val Leu Ala Ile55 60 65ttg ttc gca acc act tta tac tct tcg gcg cct ctt ctt ctc aat ctc351Leu Phe Ala Thr Thr Leu Tyr Ser Ser Ala Pro Leu Leu Leu Asn Leu70 75 80ctt cta ata tct ccc gct ctg ctg ata ctc ctc tct acg aaa cgt cct399Leu Leu Ile Ser Pro Ala Leu Leu Ile Leu Leu Ser Thr Lys Arg Pro85 90 95cgg acc ccc gtc aaa gcg aaa cct cct cgc cag tcc gct aga gct ggg447Arg Thr Pro Val Lys Ala Lys Pro Pro Arg Gln Ser Ala Arg Ala Gly100 105 110 115aaa gat gac tcg aaa cat gcg aca gcc ttg cca gag tct cta ccc att495Lys Asp Asp Ser Lys His Ala Thr Ala Leu Pro Glu Ser Leu Pro Ile120 125 130cat cca ttt ctc acg aca tat cgc gcc gcc atg atg gtt atc acg tgc543His Pro Phe Leu Thr Thr Tyr Arg Ala Ala Met Met Val Ile Thr Cys135 140 145atc gct atc ttg gct gtg gat ttt cgc att ttt cct cgc cga ttc gcc591Ile Ala Ile Leu Ala Val Asp Phe Arg Ile Phe Pro Arg Arg Phe Ala150 155 160aag gta gaa aac tgg ggt aca tca ctc atg gat ctg ggc gtt gga tcg639Lys Val Glu Asn Trp Gly Thr Ser Leu Met Asp Leu Gly Val Gly Ser165 170 175ttt gtc ttt tcg ggc gga gta gta tcc gct cgc tca cta ctc aag agc687Phe Val Phe Ser Gly Gly Val Val Ser Ala Arg Ser Leu Leu Lys Ser180 185 190 195agg acc aat ggc tct aaa agg ttg cct ctt gcc aag agg ttg att gcg735Arg Thr Asn Gly Ser Lys Arg Leu Pro Leu Ala Lys Arg Leu Ile Ala200 205 210tcg acg cga cac tct att cct ctg ctc gtc ctc ggc ctg att cgg cta783
Ser Thr Arg His Ser Ile Pro Leu Leu Val Leu Gly Leu Ile Arg Leu215 220 225tac agc gtc aaa ggc ttg gac tat gcg gag cac gtc acc gag tac ggc831Tyr Ser Val Lys Gly Leu Asp Tyr Ala Glu His Val Thr Glu Tyr Gly230 235 240gta cat tgg aac ttc ttc ttt aca ttg ggt ctt ttg cct ccg ttc gtg879Val His Trp Asn Phe Phe Phe Thr Leu Gly Leu Leu Pro Pro Phe Val245 250 255gag gtc ttc gac gcc ttg gct acg atc att ccg tca tac gag gtt ctc927Glu Val Phe Asp Ala Leu Ala Thr Ile Ile Pro Ser Tyr Glu Val Leu260 265 270 275tcc gtg ggg atc gcc gtc ttg tat caa gtt gcc cta gag tca aca gac975Ser Val Gly Ile Ala Val Leu Tyr Gln Val Ala Leu Glu Ser Thr Asp280 285 290ttg aaa agc tac atc ctc gtc tcc cct cgt ggg cca agc tta ctg tcc1023Leu Lys Ser Tyr Ile Leu Val Ser Pro Arg Gly Pro Ser Leu Leu Ser295 300 305aag aat cgt gaa ggc gtc ttc tcc ttc tca ggt tat ctc gcg att ttt1071Lys Asn Arg Glu Gly Val Phe Ser Phe Ser Gly Tyr Leu Ala Ile Phe310 315 320ctt gct ggt cgt gcg atc ggc att cgg ata atc cct cgc gga act tct1119Leu Ala Gly Arg Ala Ile Gly Ile Arg Ile Ile Pro Arg Gly Thr Ser325 330 335ttc tca aga agc cca gaa cag gcc agg aga cgg gtc ctg atc agc ctt1167Phe Ser Arg Ser Pro Glu Gln Ala Arg Arg Arg Val Leu Ile Ser Leu340 345 350 355ggc gtg caa gcg tta gtg tgg acc act ctt ttt gtg ttg aac tcc act1215Gly Val Gln Ala Leu Val Trp Thr Thr Leu Phe Val Leu Asn Ser Thr360 365 370tat gcg atg gga tac gga gct aat atc cct gtc tcc cgc cgc ctc gct1263Tyr Ala Met Gly Tyr Gly Ala Asn Ile Pro Val Ser Arg Arg Leu Ala375 380 385aac atg ccc tat gtc ctt tgg gtt tcg gcg ttc aac acc gcg caa ctg1311
Asn Met Pro Tyr Val Leu Trp Val Ser Ala Phe Asn Thr Ala Gln Leu390 395 400ttt gtg ttc tgc ctg atc gaa aca ctc tgc ttt cct gca gtt cat cgg 1359Phe Val Phe Cys Leu Ile Glu Thr Leu Cys Phe Pro Ala Val His Arg405 410 415aca acg act caa gag agc gaa tct gag cga gtc gat ttt gct acg agc 1407Thr Thr Thr Gln Glu Ser Glu Ser Glu Arg Val Asp Phe Ala Thr Ser420 425 430 435cga atc atg tcg gcc ttc aat aag aac agt ctc gcg atc ttt ctt ttg 1455Arg Ile Met Ser Ala Phe Asn Lys Asn Ser Leu Ala Ile Phe Leu Leu440 445 450gcc aat ctt ctg act gga gct gtg aat ctg agc atc tcc aca att gat 1503Ala Asn Leu Leu Thr Gly Ala Val Asn Leu Ser Ile Ser Thr Ile Asp455 460 465gct aat aca gcg cag gcc atc gct gtt ctc att gga tat tca tcc att 1551Ala Asn Thr Ala Gln Ala Ile Ala Val Leu Ile Gly Tyr Ser Ser Ile470 475 480atc aca ggg gtt gct cta gca ttg cat cat gcc aat atc aaa gta ctt 1599Ile Thr Gly Val Ala Leu Ala Leu His His Ala Asn Ile Lys Val Leu485 490 495cct ttc tag ggtatttacg agcaattggt ggtgtgttga agatatatag 1648Pro Phe500<210>12<211>2045<212>DNA<213>新生隐球菌(Cryptococcus neoformans)<220>
<221>内含子<222>(137)..(198)<220>
<221>内含子<222>(892)..(942)
<220>
<221>内含子<222>(1636)..(1686)<220>
<221>CDS<222>(44)..(2001)<400>12gtcatagcat taaatccccg ccataataag ctactgaatt gca atg ggg gat tac 55Met Gly Asp Tyr1aag tcg gcc aaa gag gcc ttt gtc tcg gat aac cca ggt gct tct atc 103Lys Ser Ala Lys Glu Ala Phe Val Ser Asp Asn Pro Gly Ala Ser Ile5 10 15 20tgg agt atc aac gct gtc agc ctg gtc gca ctg gtatgtagct cgttctccga 156Trp Ser Ile Asn Ala Val Ser Leu Val Ala Leu25 30ggggttctgt catttggaga cgcttattaa ttgggatcgc ag gcg aca tat gct210Ala Thr Tyr Ala35ctc tgg atc gcc tta tcg ccg tac atc cgt cat gga ctc ctg aac aac 258Leu Trp Ile Ala Leu Ser Pro Tyr Ile Arg His Gly Leu Leu Asn Asn40 45 50tac ctg atc tgt gtt ctt ccc cta tta ttc ggg gtg acc atc ttc tca 306Tyr Leu Ile Cys Val Leu Pro Leu Leu Phe Gly Val Thr Ile Phe Ser55 60 65act tcg cct ctc gta ttt acc tct ttt ttg tcc att att tcc ctc gct 354Thr Ser Pro Leu Val Phe Thr Ser Phe Leu Ser Ile Ile Ser Leu Ala70 75 80ttc atc acg aaa tcc caa aaa tgc ttc aaa tct gtc agt tcg ccc gaa 402Phe Ile Thr Lys Ser Gln Lys Cys Phe Lys Ser Val Ser Ser Pro Glu85 90 95aag cca aaa ggc caa tgg cta gac gaa tca gac tcc gat gag gaa cca 450Lys Pro Lys Gly Gln Trp Leu Asp Glu Ser Asp Ser Asp Glu Glu Pro
100 105 110 115gcg gaa cct gct tct gca gct gga tct gca gca gtc tca cca gta aag498Ala Glu Pro Ala Ser Ala Ala Gly Ser Ala Ala Val Ser Pro Val Lys120 125 130ctt cta cct tcc caa gtg gcg ttc gct tcg gga tcc cta tta tct ccc546Leu Leu Pro Ser Gln Val Ala Phe Ala Ser Gly Ser Leu Leu Ser Pro135 140 145gat ccg aca aca tcc ccc atg tcg cca agt agt tct tca gct tca gga594Asp Pro Thr Thr Ser Pro Met Ser Pro Ser Ser Ser Ser Ala Ser Gly150 155 160cat gaa gac cct ttg ggg att atg ggc gtt aac aga cgg agg tcg cta642His Glu Asp Pro Leu Gly Ile Met Gly Val Asn Arg Arg Arg Ser Leu165 170 175tta gaa gga gtt tcg ctt gat gtt ccg tca cat atc gac tcc aag gtc690Leu Glu Gly Val Ser Leu Asp Val Pro Ser His Ile Asp Ser Lys Val180 185 190 195aga ata tct cct gtt ccc tac ttg agg ctc aaa aag tct agg gca acg738Arg Ile Ser Pro Val Pro Tyr Leu Arg Leu Lys Lys Ser Arg Ala Thr200 205 210aag gcg caa tgg gtg aaa gaa aag gga aga tta cca ttt ttg aca gtg786Lys Ala Gln Trp Val Lys Glu Lys Gly Arg Leu Pro Phe Leu Thr Val215 220 225tac cga gcg cac atg atg ctc atg act gtt atc tgc atc ttg gcg gta834Tyr Arg Ala His Met Met Leu Met Thr Val Ile Cys Ile Leu Ala Val230 235 240gat ttt gaa gtg ttt cct aga tgg cag ggc aag tgc gaa gat ttt ggt882Asp Phe Glu Val Phe Pro Arg Trp Gln Gly Lys Cys Glu Asp Phe Gly245 250 255act agt ctg gtaagctttc cttcagccat ggtccagtgc tcaccgctct931Thr Ser Leu260acttgccgta g atg gac gtg ggt gtc ggg tca ttc gtc ttt tcc ctc ggt 981
Met Asp Val Gly Val Gly Ser Phe Val Phe Ser Leu Gly265 270 275ctc gtc tcc aca aaa tct ctt tct cct cca cct cca act cct acg ccc1029Leu Val Ser Thr Lys Ser Leu Ser Pro Pro Pro Pro Thr Pro Thr Pro280 285 290tcc tcg ccc gct ctc aac tct cac atc att ccc ctc acc ccg tcc ccg1077Ser Ser Pro Ala Leu Asn Ser His Ile Ile Pro Leu Thr Pro Ser Pro295 300 305ttc act tcc atc ctc atc tcg ctc cga aaa tcc atc ccc atc ctc gtc1125Phe Thr Ser Ile Leu Ile Ser Leu Arg Lys Ser Ile Pro Ile Leu Val310 315 320ctc ggc ttt ata cgg ttg att atg gtc aag gga tct gat tat cct gag1173Leu Gly Phe Ile Arg Leu Ile Met Val Lys Gly Ser Asp Tyr Pro Glu325 330 335cat gtg acg gag tac ggc gtg cac tgg aat ttc ttc ttc acc ctc gca1221His Val Thr Glu Tyr Gly Val His Trp Asn Phe Phe Phe Thr Leu Ala340 345 350 355ttg gtt cct gtg ctc gcc gtg ggc att cga cca ttg acg cag tgg ctt1269Leu Val Pro Val Leu Ala Val Gly Ile Arg Pro Leu Thr Gln Trp Leu360 365 370cgc tgg agt gtg ctt ggg gta atc atc tct ttg ctg cat cag ctg tgg1317Arg Trp Ser Val Leu Gly Val Ile Ile Ser Leu Leu His Gln Leu Trp375 380 385tta aca tat tat ctc caa tcc atc gtc ttc tca ttc ggc cgg tca ggt1365Leu Thr Tyr Tyr Leu Gln Ser Ile Val Phe Ser Phe Gly Arg Ser Gly390 395 400atc ttt cta gca aac aag gaa ggc ttc tcc tct ctt cct ggt tat ctt1413Ile Phe Leu Ala Asn Lys Glu Gly Phe Ser Ser Leu Pro Gly Tyr Leu405 410 415tcc ata ttt ttg atc ggc ttg tct att gga gat cat gtt tta agg ctc1461Ser Ile Phe Leu Ile Gly Leu Ser Ile Gly Asp His Val Leu Arg Leu420 425 430 435agt tta cca cca aga aga gag agg gtc gtg tca gaa aca aat gaa gag1509
Ser Leu Pro Pro Arg Arg Glu Arg Val Val Ser Glu Thr Asn Glu Glu440 445 450cat gag cag agt cat ttt gag aga aaa aaa ttg gat ttg att atg gag1557His Glu Gln Ser His Phe Glu Arg Lys Lys Leu Asp Leu Ile Met Glu455 460 465ttg att gga tat agc tta ggc tgg tgg gca ctc tta gga ggc tgg att1605Leu Ile Gly Tyr Ser Leu Gly Trp Trp Ala Leu Leu Gly Gly Trp Ile470 475 480tgg gcc ggc ggg gag gta tcc agg cgt tta gtaagtggac atctttggta 1655Trp Ala Gly Gly Glu Val Ser Arg Arg Leu485 490atattgtacc tatactaatc cctgcataaa g gcc aac gct cct tat gta ttt 1707Ala Asn Ala Pro Tyr Val Phe495 500tgg gta gcg gca tac aat acc acc ttt ctc ctc ggc tac ctc ctc ctt1755Trp Val Ala Ala Tyr Asn Thr Thr Phe Leu Leu Gly Tyr Leu Leu Leu505 510 515acc cac att att cca tct ccc acc tct tcc caa aca tca cca tcg atc1803Thr His Ile Ile Pro Ser Pro Thr Ser Ser Gln Thr Ser Pro Ser Ile520 525 530tta gtg cct ccc ttg ctc gac gct atg aat aaa aac ggt ctc gcg ata1851Leu Val Pro Pro Leu Leu Asp Ala Met Asn Lys Asn Gly Leu Ala Ile535 540 545ttt ttg gcg gcc aac ttg ctt aca gga ctg gtg aat gtg agc atg aag1899Phe Leu Ala Ala Asn Leu Leu Thr Gly Leu Val Asn Val Ser Met Lys550 555 560aca atg tat gcg ccg gcg tgg ttg tca atg ggg gtg tta atg ttg tat1947Thr Met Tyr Ala Pro Ala Trp Leu Ser Met Gly Val Leu Met Leu Tyr565 570 575 580acc ttg aca atc agt tgt gta ggg tgg ata ctg aaa gga cgg agg atc1995Thr Leu Thr Ile Ser Cys Val Gly Trp Ile Leu Lys Gly Arg Arg Ile585 590 595aag ata tagttaaagt gtttaccatg caggatactg agtatctcgg ttca 2045
Lys Ile<210>13<211>1797<212>DNA<213>新生隐球菌(Cryptococcus neoformans)<220>
<221>CDS<222>(1)..(1794)<400>13atg ggg gat tac aag tcg gcc aaa gag gcc ttt gtc tcg gat aac cca 48Met Gly Asp Tyr Lys Ser Ala Lys Glu Ala Phe Val Ser Asp Asn Pro1 5 10 15ggt gct tct atc tgg agt atc aac gct gtc agc ctg gtc gca ctg gcg 96Gly Ala Ser Ile Trp Ser Ile Asn Ala Val Ser Leu Val Ala Leu Ala20 25 30aca tat gct ctc tgg atc gcc tta tcg ccg tac atc cgt cat gga ctc 144Thr Tyr Ala Leu Trp Ile Ala Leu Ser Pro Tyr Ile Arg His Gly Leu35 40 45ctg aac aac tac ctg atc tgt gtt ctt ccc cta tta ttc ggg gtg acc 192Leu Asn Asn Tyr Leu Ile Cys Val Leu Pro Leu Leu Phe Gly Val Thr50 55 60atc ttc tca act tcg cct ctc gta ttt acc tct ttt ttg tcc att att 240Ile Phe Ser Thr Ser Pro Leu Val Phe Thr Ser Phe Leu Ser Ile Ile65 70 75 80tcc ctc gct ttc atc acg aaa tcc caa aaa tgc ttc aaa tct gtc agt 288Ser Leu Ala Phe Ile Thr Lys Ser Gln Lys Cys Phe Lys Ser Val Ser85 90 95tcg ccc gaa aag cca aaa ggc caa tgg cta gac gaa tca gac tcc gat 336Ser Pro Glu Lys Pro Lys Gly Gln Trp Leu Asp Glu Ser Asp Ser Asp100 105 110gag gaa cca gcg gaa cct gct tct gca gct gga tct gca gca gtc tca 384Glu Glu Pro Ala Glu Pro Ala Ser Ala Ala Gly Ser Ala Ala Val Ser115 120 125
cca gta aag ctt cta cct tcc caa gtg gcg ttc gct tcg gga tcc cta432Pro Val Lys Leu Leu Pro Ser Gln Val Ala Phe Ala Ser Gly Ser Leu130 135 140tta tct ccc gat ccg aca aca tcc ccc atg tcg cca agt agt tct tca480Leu Ser Pro Asp Pro Thr Thr Ser Pro Met Ser Pro Ser Ser Ser Ser145 150 155 160gct tca gga cat gaa gac cct ttg ggg att atg ggc gtt aac aga cgg528Ala Ser Gly His Glu Asp Pro Leu Gly Ile Met Gly Val Asn Arg Arg165 170 175agg tcg cta tta gaa gga gtt tcg ctt gat gtt ccg tca cat atc gac576Arg Ser Leu Leu Glu Gly Val Ser Leu Asp Val Pro Ser His Ile Asp180 185 190tcc aag gtc aga ata tct cct gtt ccc tac ttg agg ctc aaa aag tct624Ser Lys Val Arg Ile Ser Pro Val Pro Tyr Leu Arg Leu Lys Lys Ser195 200 205agg gca acg aag gcg caa tgg gtg aaa gaa aag gga aga tta cca ttt672Arg Ala Thr Lys Ala Gln Trp Val Lys Glu Lys Gly Arg Leu Pro Phe210 215 220ttg aca gtg tac cga gcg cac atg atg ctc atg act gtt atc tgc atc720Leu Thr Val Tyr Arg Ala His Met Met Leu Met Thr Val Ile Cys Ile225 230 235 240ttg gcg gta gat ttt gaa gtg ttt cct aga tgg cag ggc aag tgc gaa768Leu Ala Val Asp Phe Glu Val Phe Pro Arg Trp Gln Gly Lys Cys Glu245 250 255gat ttt ggt act agt ctg atg gac gtg ggt gtc ggg tca ttc gtc ttt816Asp Phe Gly Thr Ser Leu Met Asp Val Gly Val Gly Ser Phe Val Phe260 265 270tcc ctc ggt ctc gtc tcc aca aaa tct ctt tct cct cca cct cca act864Ser Leu Gly Leu Val Ser Thr Lys Ser Leu Ser Pro Pro Pro Pro Thr275 280 285cct acg ccc tcc tcg ccc gct ctc aac tct cac atc att ccc ctc acc912Pro Thr Pro Ser Ser Pro Ala Leu Asn Ser His Ile Ile Pro Leu Thr
290 295 300ccg tcc ccg ttc act tcc atc ctc atc tcg ctc cga aaa tcc atc ccc960Pro Ser Pro Phe Thr Ser Ile Leu Ile Ser Leu Arg Lys Ser Ile Pro305 310 315 320atc ctc gtc ctc ggc ttt ata cgg ttg att atg gtc aag gga tct gat1008Ile Leu Val Leu Gly Phe Ile Arg Leu Ile Met Val Lys Gly Ser Asp325 330 335tat cct gag cat gtg acg gag tac ggc gtg cac tgg aat ttc ttc ttc1056Tyr Pro Glu His Val Thr Glu Tyr Gly Val His Trp Asn Phe Phe Phe340 345 350acc ctc gca ttg gtt cct gtg ctc gcc gtg ggc att cga cca ttg acg1104Thr Leu Ala Leu Val Pro Val Leu Ala Val Gly Ile Arg Pro Leu Thr355 360 365cag tgg ctt cgc tgg agt gtg ctt ggg gta atc atc tct ttg ctg cat1152Gln Trp Leu Arg Trp Ser Val Leu Gly Val Ile Ile Ser Leu Leu His370 375 380cag ctg tgg tta aca tat tat ctc caa tcc atc gtc ttc tca ttc ggc1200Gln Leu Trp Leu Thr Tyr Tyr Leu Gln Ser Ile Val Phe Ser Phe Gly385 390 395 400cgg tca ggt atc ttt cta gca aac aag gaa ggc ttc tcc tct ctt cct1248Arg Ser Gly Ile Phe Leu Ala Asn Lys Glu Gly Phe Ser Ser Leu Pro405 410 415ggt tat ctt tcc ata ttt ttg atc ggc ttg tct att gga gat cat gtt1296Gly Tyr Leu Ser Ile Phe Leu Ile Gly Leu Ser Ile Gly Asp His Val420 425 430tta agg ctc agt tta cca cca aga aga gag agg gtc gtg tca gaa aca1344Leu Arg Leu Ser Leu Pro Pro Arg Arg Glu Arg Val Val Ser Glu Thr435 440 445aat gaa gag cat gag cag agt cat ttt gag aga aaa aaa ttg gat ttg1392Asn Glu Glu His Glu Gln Ser His Phe Glu Arg Lys Lys Leu Asp Leu450 455 460att atg gag ttg att gga tat agc tta ggc tgg tgg gca ctc tta gga1440
Ile Met Glu Leu Ile Gly Tyr Ser Leu Gly Trp Trp Ala Leu Leu Gly465 470 475 480ggc tgg att tgg gcc ggc ggg gag gta tcc agg cgt tta gcc aac gct 1488Gly Trp Ile Trp Ala Gly Gly Glu Val Ser Arg Arg Leu Ala Asn Ala485 490 495cct tat gta ttt tgg gta gcg gca tac aat acc acc ttt ctc ctc ggc 1536Pro Tyr Val Phe Trp Val Ala Ala Tyr Asn Thr Thr Phe Leu Leu Gly500 505 510tac ctc ctc ctt acc cac att att cca tct ccc acc tct tcc caa aca 1584Tyr Leu Leu Leu Thr His Ile Ile Pro Ser Pro Thr Ser Ser Gln Thr515 520 525tca cca tcg atc tta gtg cct ccc ttg ctc gac gct atg aat aaa aac 1632Ser Pro Ser Ile Leu Val Pro Pro Leu Leu Asp Ala Met Asn Lys Asn530 535 540ggt ctc gcg ata ttt ttg gcg gcc aac ttg ctt aca gga ctg gtg aat 1680Gly Leu Ala Ile Phe Leu Ala Ala Asn Leu Leu Thr Gly Leu Val Asn545 550 555 560gtg agc atg aag aca atg tat gcg ccg gcg tgg ttg tca atg ggg gtg 1728Val Ser Met Lys Thr Met Tyr Ala Pro Ala Trp Leu Ser Met Gly Val565 570 575tta atg ttg tat acc ttg aca atc agt tgt gta ggg tgg ata ctg aaa 1776Leu Met Leu Tyr Thr Leu Thr Ile Ser Cys Val Gly Trp Ile Leu Lys580 585 590gga cgg agg atc aag ata tag 1797Gly Arg Arg Ile Lys Ile595<210>14<211>598<212>PRT<213>新生隐球菌(Cryptococcus neoformans)<400>14Met Gly Asp Tyr Lys Ser Ala Lys Glu Ala Phe Val Ser Asp Asn Pro1 5 10 15
Gly Ala Ser Ile Trp Ser Ile Asn Ala Val Ser Leu Val Ala Leu Ala20 25 30Thr Tyr Ala Leu Trp Ile Ala Leu Ser Pro Tyr Ile Arg His Gly Leu35 40 45Leu Asn Asn Tyr Leu Ile Cys Val Leu Pro Leu Leu Phe Gly Val Thr50 55 60Ile Phe Ser Thr Ser Pro Leu Val Phe Thr Ser Phe Leu Ser Ile Ile65 70 75 80Ser Leu Ala Phe Ile Thr Lys Ser Gln Lys Cys Phe Lys Ser Val Ser85 90 95Ser Pro Glu Lys Pro Lys Gly Gln Trp Leu Asp Glu Ser Asp Ser Asp100 105 110Glu Glu Pro Ala Glu Pro Ala Ser Ala Ala Gly Ser Ala Ala Val Ser115 120 125Pro Val Lys Leu Leu Pro Ser Gln Val Ala Phe Ala Ser Gly Ser Leu130 135 140Leu Ser Pro Asp Pro Thr Thr Ser Pro Met Ser Pro Ser Ser Ser Ser145 150 155 160Ala Ser Gly His Glu Asp Pro Leu Gly Ile Met Gly Val Asn Arg Arg165 170 175Arg Ser Leu Leu Glu Gly Val Ser Leu Asp Val Pro Ser His Ile Asp180 185 190Ser Lys Val Arg Ile Ser Pro Val Pro Tyr Leu Arg Leu Lys Lys Ser195 200 205Arg Ala Thr Lys Ala Gln Trp Val Lys Glu Lys Gly Arg Leu Pro Phe210 215 220Leu Thr Val Tyr Arg Ala His Met Met Leu Met Thr Val Ile Cys Ile225 230 235 240Leu Ala Val Asp Phe Glu Val Phe Pro Arg Trp Gln Gly Lys Cys Glu245 250 255
Asp Phe Gly Thr Ser Leu Met Asp Val Gly Val Gly Ser Phe Val Phe260 265 270Ser Leu Gly Leu Val Ser Thr Lys Ser Leu Ser Pro Pro Pro Pro Thr275 280 285Pro Thr Pro Ser Ser Pro Ala Leu Asn Ser His Ile Ile Pro Leu Thr290 295 300Pro Ser Pro Phe Thr Ser Ile Leu Ile Ser Leu Arg Lys Ser Ile Pro305 310 315 320Ile Leu Val Leu Gly Phe Ile Arg Leu Ile Met Val Lys Gly Ser Asp325 330 335Tyr Pro Glu His Val Thr Glu Tyr Gly Val His Trp Asn Phe Phe Phe340 345 350Thr Leu Ala Leu Val Pro Val Leu Ala Val Gly Ile Arg Pro Leu Thr355 360 365Gln Trp Leu Arg Trp Ser Val Leu Gly Val Ile Ile Ser Leu Leu His370 375 380Gln Leu Trp Leu Thr Tyr Tyr Leu Gln Ser Ile Val Phe Ser Phe Gly385 390 395 400Arg Ser Gly Ile Phe Leu Ala Asn Lys Glu Gly Phe Ser Ser Leu Pro405 410 415Gly Tyr Leu Ser Ile Phe Leu Ile Gly Leu Ser Ile Gly Asp His Val420 425 430Leu Arg Leu Ser Leu Pro Pro Arg Arg Glu Arg Val Val Ser Glu Thr435 440 445Asn Glu Glu His Glu Gln Ser His Phe Glu Arg Lys Lys Leu Asp Leu450 455 460Ile Met Glu Leu Ile Gly Tyr Ser Leu Gly Trp Trp Ala Leu Leu Gly465 470 475 480Gly Trp Ile Trp Ala Gly Gly Glu Val Ser Arg Arg Leu Ala Asn Ala485 490 495
Pro Tyr Val Phe Trp Val Ala Ala Tyr Asn Thr Thr Phe Leu Leu Gly500 505 510Tyr Leu Leu Leu Thr His Ile Ile Pro Ser Pro Thr Ser Ser Gln Thr515 520 525Ser Pro Ser Ile Leu Val Pro Pro Leu Leu Asp Ala Met Asn Lys Asn530 535 540Gly Leu Ala Ile Phe Leu Ala Ala Asn Leu Leu Thr Gly Leu Val Asn545 550 555 560Val Ser Met Lys Thr Met Tyr Ala Pro Ala Trp Leu Ser Met Gly Val565 570 575Leu Met Leu Tyr Thr Leu Thr Ile Ser Cys Val Gly Trp Ile Leu Lys580 585 590Gly Arg Arg Ile Lys Ile595<210>15<211>35<212>DNA<213>人工序列<220>
<223>人工序列的描述人工合成的引物序列<400>15ggaattcatg tcgactttaa aacagagaaa agagg 35<210>16<211>34<212>DNA<213>人工序列<220>
<223>人工序列的描述人工合成的引物序列
<400>16gcatcgattt atagcttaat gaatattctt tttct atac 34<210>17<211>60<212>DNA<213>酿酒酵母(Saccharomyces cerevisiae)<400>17atggcaacag tacatcagga gaatatgtcg actttaaaac cggatccccg tcgtttaaac 60<210>18<211>60<212>DNA<213>酿酒酵母(Saccharomyces cerevisiae)<400>18ttatagctta atgaatattc tttttctata caagaaaacc gaattcgagc tcgtttaaac 60
权利要求
1.一种筛选具有抗真菌活性的化合物的方法，其中该方法包括下述步骤(1)将试样与由GWT1基因编码且过表达的蛋白接触；(2)检测GlcN-(酰基)PI；和(3)选出减少GlcN-(酰基)PI的试样。
2.权利要求1所述的方法，其中所述GWT1基因是下述任一种(a)编码包含氨基酸序列SEQ ID NO2，4，6，8，10或14的蛋白的DNA；(b)包含核苷酸序列SEQ ID NO1，3，5，7，9，11，12或13的DNA；(c)在严谨条件下，与包含核苷酸序列SEQ ID NO1，3，5，7，9，11，12或13的DNA杂交的DNA；和(d)编码包含氨基酸序列SEQ ID NO2，4，6，8，10或14的蛋白的DNA，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。
3.权利要求1或2所述的方法，其中检测酰基化GPI的步骤是薄层层析。
4.权利要求1-3中任一项所述的方法，其中该方法进一步包含步骤(4)，测定选出的试样是否抑制GPI-锚定蛋白转运到真菌细胞壁的过程，试样是否抑制GPI-锚定蛋白在真菌细胞表面上的表达，或试样是否抑制真菌的增殖。
全文摘要
本发明利用简单的转酰基至GlcN－PI的实验，利用表达GWT1蛋白的膜级分，可以对抑制GPI－锚定蛋白转运到真菌细胞壁的过程的化合物进行筛选。通过抑制GPI－锚定蛋白到真菌细胞壁的转运，可以获得抑制真菌细胞壁合成，并且还抑制与宿主细胞粘附的新抗真菌剂。
文档编号C12N15/55GK1742095SQ20038010911
公开日2006年3月1日 申请日期2003年11月21日 优先权日2002年11月22日
发明者塚原克平, 土谷满美子, 地神芳文, 仲山贤一, 梅村真理子, 冈本美智代 申请人:卫材株式会社, 独立行政法人产业技术总合研究所