專利名稱：篩選抑制gwt1基因產(chǎn)物酶活性的化合物的方法
技術領域：
本發(fā)明涉及篩選具有抑制GPI合酶活性的抗真菌劑的方法，所述GPI合酶參與真菌細胞壁的合成。
背景技術：
本發(fā)明人注意到，粘附宿主細胞對于真菌發(fā)揮其病原性是非常重要的，而且參與真菌細胞粘附的粘附因子在糖基磷脂酰肌醇(GPI)錨定到細胞膜上以后，就被轉運到細胞壁表層(非專利文獻1Hamada K等，Mol.Gen.Genet.，25853-59，1998)。因此，本發(fā)明人認為通過抑制錨定GPI的蛋白(GPI-錨定蛋白)向細胞壁轉運的過程，可以制得新抗真菌劑，該抗真菌劑能抑制真菌細胞壁的合成，并且還能抑制真菌細胞粘附宿主細胞。
本發(fā)明的公開本發(fā)明的目的是通過抑制真菌細胞壁的合成，通過抑制真菌細胞粘附宿主細胞，并抑制GPI-錨定蛋白轉運到真菌細胞壁的過程，來開發(fā)出防止病原性真菌發(fā)揮病原性的抗真菌劑。
在WO 02/04626中，本發(fā)明人發(fā)現(xiàn)了下述參與GPI-錨定蛋白到細胞壁轉運過程的蛋白由含有SEQ ID NO1核苷酸序列的DNA編碼的釀酒酵母(Saccharomyces cerevisiae)蛋白；由含有SEQ ID NO3和5核苷酸序列的DNA編碼的白色念珠菌(Candida albicans)蛋白；由含有SEQ ID NO7核苷酸序列的DNA編碼的粟酒裂殖酵母(Schizosaccharomyces pombe)蛋白；由含有SEQ ID NO9和11核苷酸序列的DNA編碼的煙曲霉(Aspergillusfumigatus)蛋白；和由含有SEQ ID NO12和13核苷酸序列的DNA編碼的新生隱球菌(Cryptococcus neoformans)蛋白。這些核苷酸序列稱為GWT1基因。此外，本發(fā)明人發(fā)現(xiàn)GWT1基因-缺陷型真菌不能合成細胞壁。另外，本發(fā)明人發(fā)現(xiàn)式(Ia)所示化合物與上述蛋白結合能抑制GPI-錨定蛋白到細胞壁的轉運，從而抑制真菌細胞壁的合成。
然后，本發(fā)明人發(fā)現(xiàn)在GPI合成途徑中GWT1基因產(chǎn)物(下文中稱作“GWT1蛋白”)具有將?；D移至G1cN-PI而合成GlcN-(?；?PI的活性(

圖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)。本發(fā)明人認為抑制真菌細胞壁合成的化合物可以通過對抑制此活性的化合物進行篩選而得到，以此完成本發(fā)明。
具體地，本發(fā)明提供了下述[1]-[4]。
篩選具有抗真菌活性的化合物的方法，其中該方法包括下述步驟(1)將試樣與由GWT1基因編碼且過表達的蛋白接觸；(2)檢測GlcN-(?；?PI；和(3)選出減少GlcN-(?；?PI的試樣。
“GWT1”基因是指參與真菌細胞壁合成的基因，該基因公開于WO02/04626中。術語“過表達”并不是指天然基因的表達，而是指外源導入基因的表達。
“GlcN-(?；?PI”是指在GPI生物合成途徑中，酰基與葡糖胺基-磷脂酰肌醇(GleN-PI)的肌醇連接的葡糖胺基-?；字＜〈?圖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)。
項[1]所述的方法，其中所述GWT1基因是下述任一種(a)編碼包含氨基酸序列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)在嚴謹條件下，與包含核苷酸序列SEQ ID NO1，3，5，7，9，11，12或13的DNA雜交的DNA；和(d)編碼包含氨基酸序列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/或插入一個或多個氨基酸。
術語“嚴謹條件”意思是，例如在65℃于4xSSC中進行雜交，然后在65℃用0.1xSSC洗滌1小時。可選的嚴謹條件是指在42℃，于含50％甲酰胺的4xSSC中進行雜交。其它可接受的條件是在65℃于PerfectHybTM溶液(TOYOBO)中雜交2.5小時，然后(1)在25℃，用2xSSC，0.05％SDS洗滌5分鐘；(2)在25℃，用2xSSC，0.05％SDS洗滌15分鐘；和(3)在50℃，用0.1xSSC，0.1％SDS洗滌20分鐘。
“包含其中添加，缺失，取代和/或插入一個或多個氨基酸的氨基酸序列的蛋白”可采用本領域技術人員所熟知的方法來進行制備，例如定點誘變(Sambrook，J.，F(xiàn)ritsch，E.F.和Maniatis，T.(1989)Molecular CloningALaboratory Manual，Cold Spring Harbor Laboratory，Cold Spring Harbor，NY)。所述突變也可以是自然發(fā)生的。對于突變的氨基酸數(shù)量并無限制，只要所得蛋白保持將?；D移至GlcN-PI的活性。突變的氨基酸數(shù)量通常為30個或更少，優(yōu)選10個或更少，更優(yōu)選3個或更少。對于突變氨基酸的位置并無限制，只要蛋白保持上述活性。
采用上述雜交技術制得的蛋白和蛋白突變體通常在氨基酸水平上，與由SEQ ID NO2，4，6，8，10或14氨基酸序列組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)來進行測定。該程序以Karlin和Altschul的BLAST算法為基礎(Proc.Natl.Acad.Sci.USA 872264-2268，1990；Proc.Natl.Acad.Sci.USA 905873-5877，1993)。當使用BLASTX分析氨基酸序列時，使用的參數(shù)是如得分＝50，字長＝3。作為選擇，當使用Gapped BLAST程序時，可以利用Altschul等所描述的方法(Nucleic.Acids.Res.253389-3402，1997)對氨基酸序列進行分析。當使用BLAST和Gapped BLAST程序時，使用各程序的默認參數(shù)值。這些分析的詳細步驟是本領域已知的(http//www.ncbi.nim.nih.gov)。
權利要求1或2所述的方法，其中檢測?；疓PI的步驟是薄層層析。
項[1]-[3]中任一項所述的方法，其中該方法進一步包含步驟(4)，測定選出的試樣是否抑制GPI-錨定蛋白轉運到真菌細胞壁的過程，試樣是否抑制GPI-錨定蛋白在真菌細胞表面上的表達，或試樣是否抑制真菌的增殖。
本發(fā)明制備GWT1蛋白的方法[1]，和測定轉?；钚缘姆椒╗2]將在下面公開。
1.制備GWT1蛋白的方法GWT1蛋白是從真菌膜級分，優(yōu)選釀酒酵母(S.Cerevisiae)，白色念珠菌(C.Albicans)，粟酒裂殖酵母(S.pombe)，煙曲霉(A.Fumigatus)，或新生隱球菌(C.Neoformans)，更優(yōu)選釀酒酵母的膜級分制得的。轉?；钚钥梢圆捎弥苯又苽淠ぜ壏只蛟诩兓笾苽淠ぜ壏謥磉M行測定。通過將SEQ ID NO1，3，5，7，9，11，12或13核苷酸序列的DNA導入真菌細胞來過表達GWT1蛋白可以很容易地對轉酰基活性進行測定。利用釀酒酵母的方法將在下面進行詳細地描述(1)GWT1基因的導入用真菌DNA作模板，和在SEQ ID NO1，3，5，7，9，11，12或13核苷酸序列基礎上設計的引物，通過PCR制備GWT1基因。
通過將適合的啟動子和終止子，如衍生自pKT10的GAPDH啟動子和GAPDH終止子(Tanaka等，Mol.Cell Biol.，104303-4313，1990)插到在釀酒酵母中起作用的表達載體如YEp 352的多克隆位點內，并將GWT1基因插到該表達載體內來制備GWT1表達質粒。將釀酒酵母如G2-10菌株，在適宜的溫度如30℃，合適的培養(yǎng)基如酵母提取物-聚蛋白胨-葡萄糖(YPD)培養(yǎng)基中振蕩孵育。在對數(shù)生長晚期收獲真菌細胞。洗滌后，采用如乙酸鋰法將GWT1表達質粒導入釀酒酵母細胞中。所述乙酸鋰法描述于YEASTMAKERTM酵母轉化系統(tǒng)所附的使用手冊(Clontech)中。通過將細胞在30℃，SD(ura-)培養(yǎng)基中培養(yǎng)兩天而得到GWT1-過表達菌株和導入空載體的菌株。
引入GWT1基因的真菌菌株優(yōu)選是缺失了其天然GWT1基因的缺陷型菌株。釀酒酵母GWT1基因-缺陷型細胞采用下述方法獲得。
利用標記基因，優(yōu)選粟酒裂殖酵母his5基因作模板，采用引物來進行PCR擴增，引物被設計成使得PCR產(chǎn)物包含將要缺失的GWT1基因(如SEQ ID NO1序列)的30個堿基對，更優(yōu)選40個堿基對或更多。將獲得的PCR產(chǎn)物提純并導入真菌細胞中。通過進行對標記基因合適的篩選，如當標記為his5時就將細胞培養(yǎng)在his-培養(yǎng)基中，得到缺陷型菌株。
除釀酒酵母之外真菌的表達載體和基因導入法描述于Igarashi等，Nature 35380-83，1991，粟酒裂殖酵母表達載體pcL等和導入該載體的方法；Pla J等，Yeast，121677-1702，1996，白色念珠菌表達載體pRM10等和導入該載體的方法；Punt PJ等，GENE，56117-124，1987，煙曲霉表達載體pAN7-1等和導入該載體的方法；Monden P等，F(xiàn)EMS Microbiol.Lett.，18741-45，2000，新生隱球菌表達載體pPM8等和導入該載體的方法。
制備白色念珠菌缺陷菌株的方法描述于Fonzi WA等，Genetics 134717-728，1993中。
(2)制備膜級分的方法在適宜的溫度如24℃，適合的培養(yǎng)基如SD(ura-)液體培養(yǎng)基中對導入了GWT1基因的釀酒酵母細胞進行振蕩培養(yǎng)。在對數(shù)生長中期時收獲真菌細胞。用TM緩沖液(50mM Tris-HCl(pH7.5)和2mM MgCl2)洗滌后，用合適量(如2ml)的TM緩沖液+蛋白酶抑制劑(CompleteTM；Roche)對真菌細胞進行懸浮。將合適量(如1.5ml)的玻璃珠加到懸浮液中。渦旋樣品，并置于冰上，重復此步驟(如渦旋30秒，置于冰上30秒，循環(huán)10次)，使真菌細胞破碎。
將樣品離心，如1000g離心5分鐘，使玻璃珠和未破碎的真菌細胞沉淀。將所得上清液移到另一試管內，然后離心，如13000g離心20分鐘，使含細胞器的膜級分(總膜級分)沉淀。如果需要，用1ml合適的試驗緩沖液進一步懸浮該沉淀物并離心，如1000g離心1分鐘，除去那些未懸浮成分。再離心上清液，如13 000g離心20分鐘，用合適的試驗緩沖液重懸浮所得的沉淀物，得到膜級分。
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，用于煙曲霉；ThompsonJR等J.Bacteriol.，181444-453，1999，用于新生隱球菌的所述方法從除釀酒酵母之外的真菌細胞中制得膜級分。
作為選擇，可通過讓GWT1蛋白在真菌之外的細胞，如哺乳動物細胞，昆蟲細胞和大腸桿菌細胞中表達來制備GWT1蛋白。
當使用哺乳動物細胞時，可以通過將GWT1插到如含有CMV啟動子的過表達載體內；將該載體導入哺乳動物細胞；然后，進行Petaja-Repo等J.Biol.Chem.，2764416-23，2001中所述的方法來制備膜級分。
當使用昆蟲細胞時，可以利用桿狀病毒表達試劑盒，如BAC-TO-BAC桿狀病毒表達系統(tǒng)(Invitrogen)制備GWT1-表達昆蟲細胞(如Sf9細胞)；然后，利用該細胞進行Okamoto等，J.Biol.Chem.，276742-751，2001中所述方法來制備膜級分。
當使用大腸桿菌時，GWT1蛋白可通過將GWT1插到大腸桿菌表達載體，如pGEX(Amersham Biosciences)內；然后將該載體導入大腸桿菌細胞如BL21中來加以制備。
2.測定轉?；钚缘姆椒；浦罣PI的反應可通過Costello和Orlean，J.Biol.Chem.(1992)2678599-8603中所述方法，或Franzot和Doering，Biochem.J.(1999)34025-32中所述方法來進行檢測。該方法的具體實例將在下面進行說明，但下述實驗條件優(yōu)選是根據(jù)所用的GWT1基因產(chǎn)物進行了優(yōu)化的將上述部分1中制得的GWT1基因產(chǎn)物，優(yōu)選含有GWT1基因產(chǎn)物的膜級分與待測化合物一起加到緩沖液中，所述緩沖液含有合適的金屬離子(Mg，Mn)；ATP；和輔酶A；和優(yōu)選防止在其它反應中消耗UDP-GlcNAc的抑制劑，如作為幾丁質合成抑制劑的尼可霉素(nikkomycin)Z，或作為天冬酰胺-連接的糖鏈合成抑制劑的衣霉素(tunicamycin)。將該混合物在適宜的溫度下孵育適當長的時間(如在24℃孵育15分鐘)。
然后，將用合適標記標記的，優(yōu)選用放射性同位素標記的GlcN-(?；?PI前體(如UDP-GlcNAc，或?；?輔酶A，優(yōu)選UDP-[14C]GlcNAc)加到混合物中。將所得混合物進一步孵育適當長的時間(如在24℃孵育1小時)。將氯仿∶甲醇的1∶2混合物加到該混合物中，攪拌停止反應。然后從混合物中抽提脂質。抽提的反應產(chǎn)物用合適的溶劑優(yōu)選丁醇溶解，然后進行HPLC，薄層層析(TLC)等，優(yōu)選TLC，將反應中產(chǎn)生的GlcN-(?；?PI分離出來。可對TLC展開劑(developing solvent)進行適當?shù)剡x擇，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)，優(yōu)選CHCl3/CH3OH/1M NH4OH(10∶10∶3)。采用對所用標記而言恰當?shù)姆椒▽Ψ蛛x的GlcN-(?；?PI進行定量。當用放射性同位素標記時，可以基于其放射性對分離的GlcN-(?；?PI進行定量。
當在待測化合物存在的條件下，制得的GlcN-(?；?PI量減少就判定該待測化合物含有抑制由GWT1蛋白引起的酰基轉移的活性。
優(yōu)選對上述已發(fā)現(xiàn)含有抑制?；D移的活性的試樣進行進一步的檢測以確定它是否抑制GPI-錨定蛋白轉運到細胞壁的過程，它是否抑制GPI-錨定蛋白在真菌細胞表面上的表達，或它是否抑制真菌生長。如果檢測結果顯示試樣抑制GPI-錨定蛋白轉運到細胞壁的過程，抑制GPI-錨定蛋白在真菌細胞表面上的表達，或抑制真菌生長，則該樣品就可望成為抗真菌劑候選物。
可用(1)利用報告酶的方法；(2)利用與真菌細胞壁表面糖蛋白反應的抗體的方法；(3)檢測真菌細胞與動物細胞粘附的方法；或者(4)在光學顯微鏡或電子顯微鏡下觀察真菌細胞的方法對試樣是否抑制GPI-錨定蛋白轉運到細胞壁的過程或抑制GPI-錨定蛋白在真菌細胞表面上的表達進行檢測。
方法(1)-(4)公開在WO 02/04626中，并在實施例中進行了具體說明。利用(1)-(4)的方法，優(yōu)選其組合，可以確定試樣是抑制GPI-錨定蛋白轉運到真菌細胞壁的過程，還是抑制GPI-錨定蛋白在真菌細胞表面上的表達。另外，當由本發(fā)明DNA編碼的蛋白在真菌細胞中過表達，由試樣引起的抑制受損或消失時，可以確定試樣對GPI-錨定蛋白轉運到細胞壁的過程有影響。
用測定抗真菌活性的常規(guī)方法還可以檢測試樣是否抑制真菌生長(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.)。
附圖的簡單說明圖1示GPT生物合成途徑。
圖2照片顯示在從野生型菌株(WT)，GWT1基因遭破壞的Δgwt1菌株(Δ)，和GWT1基因-導入的Δgwt1菌株(Δ/G)制得的膜級分中測定的GPI酰基化結果。
圖3照片顯示在?；疓PI檢測系統(tǒng)中，化合物1-(4-丁基芐基)異喹啉和化合物N-(3-(4-(1-異喹啉基甲基)苯基)-2-丙炔基)乙酰胺的GPI?；种苹钚缘臋z測結果。在公開了GWT1基因的WO02/04626中，這兩種化合物列于WO02/04626的表1，并分別描述于WO02/04626的實施例B2和實施例B60中。
圖4照片顯示在?；疓PI檢測系統(tǒng)中，化合物N-(3-(4-(1-異喹啉基甲基)苯基)丙基)-N-甲基乙酰胺和化合物5-丁基-2-(1-異喹啉基甲基)苯酚的GPI?；种苹钚缘臋z測結果。在公開了GWT1基因的WO02/04626中，這兩種化合物列于WO02/04626的表1，并分別描述于WO02/04626的實施例B73和實施例B85中。
進行發(fā)明的最佳實施方式下面，將利用實施例詳細地描述本發(fā)明，但是本發(fā)明并不限于此。
制備表達GWT1的膜級分(1)制備GWT1表達質粒通過將GAPDH啟動子和GAPDH終止子，其都衍生自pKT10(Tanaka等，Mol.Cell Biol.，104303-4313，1990)插到YEp352的多克隆位點內；并用pUC 18的多克隆位點代替該多克隆位點制備用于在釀酒酵母中表達的載體，YEp352GAPII載體。另外，為了促進GWT1基因的插入，用ClaI位點取代多克隆位點中的SalI位點制得YEp352GAPIIClaIΔSal載體。
利用SEQ ID NOs15和16引物擴增包含SEQ ID NO1核苷酸序列的釀酒酵母GWT1基因。將獲得的PCR產(chǎn)物插到YEp352GAPIIClaIΔSal載體的多克隆位點，獲得GWT1過表達質粒。
(2)制備釀酒酵母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擴增兩端都包含GWT1序列的his5盒。
培養(yǎng)并收獲釀酒酵母細胞，然后用上述PCR產(chǎn)物轉化。然后，將細胞在30℃，SD(His-)培養(yǎng)基中培養(yǎng)5-7天，獲得GWT1基因-缺陷型菌株Δgwt1。
(3)制備表達GWT1的細胞將Δgwt1菌株細胞在30℃，酵母提取物-聚蛋白胨-葡萄糖(YPD)培養(yǎng)基中振蕩培養(yǎng)。對數(shù)生長晚期收獲細胞，然后洗滌。采用乙酸鋰法(YEASTMAKERTM酵母轉化系統(tǒng)(Clontech))將GWT1表達質粒導入Δgwt1菌株細胞中。將細胞在30℃，SD(ura-)培養(yǎng)基中培養(yǎng)兩天，得到過表達GWT1基因的Δgwt1菌株。
(4)制備膜級分將野生型釀酒酵母菌株，GWT1基因-缺陷型菌株Δgwt1，和導入了GWT1過表達質粒的菌株Δgwt1分別在24℃，100ml的YPD培養(yǎng)基中振蕩培養(yǎng)，然后在對數(shù)生長中期收獲細胞(OD600＝1～3)。用TM緩沖液(50mMTris-HCl(pH7.5)和2mM MgCl2)洗滌真菌細胞，然后重懸浮在2ml TM緩沖液+蛋白酶抑制劑(1片CompleteTM(Roche)/25ml)中。將1.5ml玻璃珠加到懸浮液中。渦旋混合物30秒，然后置于冰上30秒。重復此步驟10次，破壞真菌細胞。將細胞勻漿移至一新試管內，4℃，1000g離心5分鐘，使玻璃珠和未破壞的真菌細胞沉淀。將上清液移至另一試管內，4℃，13000g離心20分鐘，使含細胞器的膜級分(總膜級分)沉淀。獲得的沉淀物作為膜級分。
(5)檢測?；腉PI已知在GPI生物合成反應路徑中，N-乙?；?葡糖胺基-磷脂酰肌醇(GlcNAc-PI)脫乙?；纬善咸前坊?磷脂酰肌醇(GlcN-PI)，其在添加酰基后生成葡糖胺基-?；字＜〈?GlcN-(?；?PI)(圖1)。因此，利用下述方法，本發(fā)明人可以對Gwt1蛋白是否參與該轉?；磻M行檢測。
用緩沖液稀釋膜級分制劑(300μg蛋白)，所述緩沖液由50mM Tris-HCl(pH7.5)，2mM MgCl2，2mM MnCl2，1mMATP，1mM輔酶A，21μg/ml衣霉素，10μM尼可霉素Z和0.5mM二硫蘇糖醇組成。將溶液調成總量為140μl，作為反應液。24℃孵育溶液15分鐘后，將15μCi UDP-[14C]GlcNAc加到試管內，然后在24℃孵育1小時。將1ml氯仿∶甲醇(1∶2)加到該溶液中，攪拌終止反應。然后，從該溶液中抽提脂質，干燥，經(jīng)丁醇抽提脫鹽。用薄層層析(HCl3/CH3OH/1M NH4OH(10∶10∶3))將酰基化GPI(GlcN-(?；?PI)，非?；疓PI(GlcN-PI)，以及GPI分開，其中GPI既不?；膊蝗ヵ；?GlcNAc-PI)。各斑點用放射自顯影進行檢測。
結果，如圖2所示，在GWT1基因-缺陷型菌株(Δgwt1)中未檢測到?；疓PI斑點，在野生型菌株中檢測到?；疓PI斑點。在引入GWT1基因的Δgwt1菌株中也檢測到了酰基化GPI斑點，這說明該菌株已經(jīng)恢復了酰基化能力。此發(fā)現(xiàn)表明Gwt1蛋白是一種能催化酰基轉移到GPI的酶。
上述結果說明當具有抑制GWT1基因產(chǎn)物活性的化合物存在于用于分析GPI合酶活性的系統(tǒng)中時，?；疓lcN-(?；?PI的斑點強度(intensity)就會降低或消失。這樣，利用GlcN-(酰基)PI的斑點強度作為指示劑，可以對抑制GWT1基因產(chǎn)物的酶活性的化合物，以及抑制真菌細胞壁合成的化合物進行篩選。
(6)篩選抑制?；磻幕衔飳⑾率龌衔锛拥?5)所述的酰基化GPI檢測系統(tǒng)中，測定抑制GPI?；幕钚浴＿@些化合物描述于WO 02/04626的實施例B2，實施例B60，實施例B73和實施例B85中，在所述WO 02/04626中公開了GWT1基因。在WO 02/04626中，這些化合物還在表1中列出，該表顯示了這些化合物在反映GWT1基因產(chǎn)物活性的報告系統(tǒng)中的抑制活性。這些化合物的結構如下所示實施例B2中所述化合物1-(4-丁基芐基)異喹啉 實施例B60中所述化合物N-(3-(4-(1-異喹啉基甲基)苯基)-2-丙炔基)乙酰胺 實施例B73中所述化合物N-(3-(4-(1-異喹啉基甲基)苯基)丙基)-N-甲基乙酰胺
實施例B85中所述化合物5-丁基-2-(1-異喹啉基甲基)苯酚 實驗結果示于圖3和4。在WO 02/04626表1所列的化合物中，實施例B2和實施例B85中的所述化合物在?；疓PI的斑點強度中顯示出劑量依賴性的減少，所述化合物具有IC50為1μg/ml或更低的抑制活性。實施例B73中所述化合物的斑點強度未觀察到減少，該化合物IC50為50μg/ml。
此結果說明利用GPI?；瘜嶒炏到y(tǒng)可以對抑制GWT1基因產(chǎn)物的酶活性的化合物進行篩選。
工業(yè)實用性利用簡單的轉?；钚詫嶒灒景l(fā)明可以對抑制GPI-錨定蛋白到真菌細胞壁的轉運過程的化合物進行篩選。
序列表<110>衛(wèi)材株式會社(Eisai Co.，Ltd.)獨立行政法人產(chǎn)業(yè)技術總合研究所(National Institute of Advanced Industrial Scienceand Technology)<120>篩選抑制GWT1基因產(chǎn)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>釀酒酵母(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>釀酒酵母(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>煙曲霉(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>煙曲霉(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>煙曲霉(Aspergillus fumigatus)<220>
<221>內含子<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>新生隱球菌(Cryptococcus neoformans)<220>
<221>內含子<222>(137)..(198)<220>
<221>內含子<222>(892)..(942)
<220>
<221>內含子<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>新生隱球菌(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>新生隱球菌(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>釀酒酵母(Saccharomyces cerevisiae)<400>17atggcaacag tacatcagga gaatatgtcg actttaaaac cggatccccg tcgtttaaac 60<210>18<211>60<212>DNA<213>釀酒酵母(Saccharomyces cerevisiae)<400>18ttatagctta atgaatattc tttttctata caagaaaacc gaattcgagc tcgtttaaac 60
權利要求
1.一種篩選具有抗真菌活性的化合物的方法，其中該方法包括下述步驟(1)將試樣與由GWT1基因編碼且過表達的蛋白接觸；(2)檢測GlcN-(?；?PI；和(3)選出減少GlcN-(?；?PI的試樣。
2.權利要求1所述的方法，其中所述GWT1基因是下述任一種(a)編碼包含氨基酸序列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)在嚴謹條件下，與包含核苷酸序列SEQ ID NO1，3，5，7，9，11，12或13的DNA雜交的DNA；和(d)編碼包含氨基酸序列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/或插入一個或多個氨基酸。
3.權利要求1或2所述的方法，其中檢測酰基化GPI的步驟是薄層層析。
4.權利要求1-3中任一項所述的方法，其中該方法進一步包含步驟(4)，測定選出的試樣是否抑制GPI-錨定蛋白轉運到真菌細胞壁的過程，試樣是否抑制GPI-錨定蛋白在真菌細胞表面上的表達，或試樣是否抑制真菌的增殖。
全文摘要
本發(fā)明利用簡單的轉?；罣lcN－PI的實驗，利用表達GWT1蛋白的膜級分，可以對抑制GPI－錨定蛋白轉運到真菌細胞壁的過程的化合物進行篩選。通過抑制GPI－錨定蛋白到真菌細胞壁的轉運，可以獲得抑制真菌細胞壁合成，并且還抑制與宿主細胞粘附的新抗真菌劑。
文檔編號C12N15/55GK1742095SQ20038010911
公開日2006年3月1日 申請日期2003年11月21日 優(yōu)先權日2002年11月22日
發(fā)明者塚原克平, 土谷滿美子, 地神芳文, 仲山賢一, 梅村真理子, 岡本美智代 申請人:衛(wèi)材株式會社, 獨立行政法人產(chǎn)業(yè)技術總合研究所