CN1387566A - 环状缩肽合成酶及其基因、以及环状缩肽的大规模生产系统 - Google Patents
环状缩肽合成酶及其基因、以及环状缩肽的大规模生产系统 Download PDFInfo
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Abstract
本发明提供合成环状缩肽、特别是合成PF1022物质的酶及其基因。这种环状缩肽合成酶含有(a)序列号2的氨基酸序列;或(b)由上述氨基酸序列衍生的氨基酸序列,该序列经过了1个以上选自替换、缺失、添加和插入的修饰,并且具有环状缩肽合成酶活性。本发明的环状缩肽合成酶基因包括编码上述环状缩肽合成酶的核苷酸序列。进而,本发明还提供环状缩肽的大规模生产系统,以及制造环状缩肽合成酶的方法等。
Description
技术领域
本发明涉及环状缩肽合成酶及其基因、以及环状缩肽的大规模生产系统,更具体地说,本发明涉及具有驱虫活性的PF1022物质的合成酶及其基因、以及PF1022物质的大规模生产系统。
背景技术
PF1022物质[环(D-乳酰基-L-N-甲基亮氨酰基-D-3-苯基乳酰基-L-N-甲基亮氨酰基-D-乳酰基-L-N-甲基亮氨酰基-D-3-苯基乳酰基-L-N-甲基亮氨酰基)]是由无孢目(Agonomycetales)的丝状真菌PF1022菌株(无孢菌类(Mycelia sterilia),FERM BP-2671)产生的环状缩肽,该物质对动物寄生性线虫类有极强的驱虫活性(特开平3-35796号,Sasaki,T.,et al.J.Antibiotics.,45,692,(1992))。因此,该物质不仅可用作动物用的驱虫剂,还可作为原材料用于合成活性更高的该物质的衍生物。
通常,由天然分离的微生物所产生的次级代谢产物的量是微量的。因此,要将其用于生产,必须要提高次级代谢产物的产量。为了提高产量,进行了培养方法的研究、培养基成分的研究、添加前体物质的发酵条件的改良、以及利用紫外线照射或诱变剂进行诱变的菌株的改良。最近,除了以上方法以外,还有采用基因重组的手段来提高生产性能的方法。
作为采用基因重组的手段来提高生产性能的方法,例如增强生物合成途径中酶基因的表达、增强生物合成中调控基因的表达、阻断不必要的生物合成途径等(Khetan,A.and Hu,W.-S.Manual ofIndustrial Mocrobiology and Biotechnology 2nd edition,p.717,(1999))。此外,作为特殊的例子,以提高氧的利用能力为目的,已知有将血红蛋白基因在细菌中表达并提高生产性能的方法(Minas,W.et al,Biotechnol.Prog.,14,561,(1998))。
用基因重组的方法来提高生产性能时,最常用的方法是提高生物合成途径中酶基因的表达水平。为了适应这种方法,必须建立对象微生物的转化方法、存在可提高表达水平的启动子和终止子,还必须搞清楚其生物合成的途径、以及要分离出这些基因。对于PF1022物质生产菌,己成功地通过转化导入了外源基因(WO 97/00944号),但其生物合成途径的基因尚未分离出来。
PF1022物质包括L-N-甲基亮氨酸、D-乳酸、以及D-苯基乳酸通过酯键或酰胺键结合的结构,在生产菌中由4个分子的L-亮氨酸、2个分子的D-乳酸、2个分子的D-苯基乳酸通过某种肽合成酶的作用合成。肽合成酶是以氨基酸或羟基酸为底物,合成肽、缩肽、脂肽、肽内酯等微生物次级代谢产物的生物合成酶,现已明确几种肽合成酶的序列(Marahiel,M.A.,et al.Chem.Rev.,97,2651,(1997))。由这种酶所催化的反应,与以mRNA为模板、由核糖体合成蛋白质的系统完全不同。其催化机理可能是,肽合成酶对于每种底物有1个结构域,每种底物在该结构域中由ATP活化,并通过结构域中的磷酸泛酸的介导而结合,最后,这些底物通过各个结构域间的区域的催化作用形成酰胺键或脂键。
发明的概述
本发明的目的在于,提供合成环状缩肽、特别是PF1022物质的酶(以下,称为“环状缩肽合成酶”)。
本发明的第二个目的在于,提供编码环状缩肽合成酶的基因(以下,称为“环状缩肽合成酶基因”)。
本发明的第三个目的在于,提供表达环状缩肽合成酶的重组载体和转化体、以及环状缩肽的大规模生产系统及其制造方法。
本发明的第四个目的在于,提供环状缩肽合成酶的制造方法。
本发明的环状缩肽合成酶是含有选自下列序列构成的组的氨基酸序列的蛋白质。(a)序列号2的氨基酸序列,以及(b)序列号2的氨基酸序列的修饰氨基酸序列,该修饰序列具有1个以上选自置换、缺失、添加和插入的修饰,并具有环状缩肽合成酶活性。
本发明的环状缩肽合成酶基因包括编码环状缩肽合成酶的核苷酸序列。
并且,本发明的环状缩肽合成酶基因由选自下列序列构成的组的核苷酸序列构成。(c)序列号1的DNA序列,(d)与序列号1的DNA序列至少有70%的同源性,并编码具有环状缩肽合成酶活性的蛋白质的核苷酸序列,(e)序列号1的DNA序列的修饰DNA序列,该修饰序列具有1个以上选自置换、缺失、添加和插入的修饰,并编码具有环状缩肽合成酶活性的蛋白质,以及(f)在严格条件下与序列号1的DNA序列杂交,并编码具有环状缩肽合成酶活性的蛋白质的核苷酸序列。
本发明的重组载体是含有本发明的环状缩肽合成酶基因的载体。
本发明的转化体和环状缩肽的大规模生产系统是含有本发明的重组载体的宿主。
本发明的环状缩肽的制造方法包括培养本发明的转化体,并从培养物中提取环状缩肽的步骤。
本发明的环状缩肽合成酶的制造方法包括培养本发明的转化体,并从培养物中提取环状缩肽合成酶的步骤。
按照本发明,可在PF1022物质生产菌株中过表达环状缩肽合成酶,同时可大规模生产PF1022物质。
附图的简单说明
图1表示质粒pABP/PFsyn的构建方法。
图2表示含Abp1基因的6kb Hind III片段的限制性酶图谱。
图3表示pABPd的结构和限制性酶图谱。
图4表示由亲本株和导入pABP/PFsyn的基因导入株提取的蛋白质的电泳结果。
图5表示由亲本株和导入pABP/PFsynN的基因导入株提取的蛋白质的电泳结果。
发明详述 微生物的保藏
实施例1的1中所述的PF1022菌株委托通商产业省工业技术院生命工程工业技术研究所(日本国茨城县筑波市东1丁目1-3号)保藏,保藏日期为1989年1月24日,保藏编号为FERM BP-2671。
实施例2的1.(1)中所述的用质粒pPFsyn转化的大肠杆菌(DH5α)委托通商产业省工业技术院生命工程工业技术研究所(日本国茨城县筑波市东1丁目1-3号)保藏,保藏日期为1999年9月1日,保藏编号为FERM BP-7253。
实施例2的1.(1)中所述的用质粒pPFsynN转化的大肠杆菌(DH5α)委托通商产业省工业技术院生命工程工业技术研究所(日本国茨城县筑波市东1丁目1-3号)保藏,保藏日期为1999年9月1日,保藏编号为FERM BP-7254。基因和蛋白质
本发明提供环状缩肽合成酶,优选提供PF1022物质合成酶及其基因。
本发明的酶可作用于4个分子的L-亮氨酸、2个分子的D-乳酸、以及2个分子的D-苯基乳酸,并合成PF1022物质。在本发明中,通过预先对D-乳酸、L-亮氨酸、以及D-苯基乳酸进行修饰,可制造PF1022物质的衍生物。
作为PF1022物质的衍生物,例如PF1022物中两个苯基的对位被氨基取代的物质。此时,作为PF1022物质的衍生物的合成底物,例如可用D-对氨基苯基乳酸替代D-苯基乳酸。
序列(b)中修饰的数目,例如可以是1~数个,更具体是1~6个。
序列(e)中修饰的数目,例如可以是1~数十个。
序列(b)和(e)中存在多个突变时,导入的突变种类可以相同,也可以不同。
序列(d)与序列号1的DNA序列的同源性可以优选至少为80%,更优选至少为90%,最优选至少为95%。
序列(f)中“严格条件”是指杂交后膜的洗涤操作在高温、低盐浓度中进行,例如洗涤条件为在0.2×SSC浓度(1×SSC:15mM柠檬酸三钠、150mM氯化钠)、0.1%SDS溶液中,60℃洗涤15分钟。
就序列(b)是否“具有环状缩肽合成酶活性”,例如可用以下方法确认,即,将被测蛋白作用于环状缩肽的底物,然后用色谱法确认环状缩肽的生成。
就序列(d)、(e)和(f)是否“编码具有环状缩肽合成酶活性的蛋白质”,例如可用下述方法作出评价,如实施例2所述,在宿主中表达被测核苷酸序列,将所得的蛋白质作用于环状缩肽的底物,然后用色谱法确认环状缩肽的生成。
如果给出本发明合成酶的氨基酸序列,则很容易确定编码该氨基酸序列的核苷酸序列,序列号2中所示的氨基酸序列的各种编码核苷酸序列都可以选择。因此,编码本发明合成酶的核苷酸序列,除了序列号1所示的DNA序列的一部分或全部以外,也指有简并密码子的、编码同一氨基酸的DNA序列,还包括与这些DNA对应的RNA序列。
本发明的基因,例如可由以下方法获得。
从PF1022物质生产菌提取基因组DNA,用适当的限制性酶切断后,用噬菌体载体构建PF1022物质生产菌的基因组文库。以肽合成酶的氨基酸序列保守区,或以由PF1022物质生产菌提取并纯化的环状缩肽合成酶的部分氨基酸序列为基础,合成适当的引物,用此引物,以PF1022物质生产菌的基因组DNA为模板,通过PCR法扩增环状缩肽合成酶基因的DNA片段。用此片段作为探针,对基因组文库进行筛选。由此,可分离出环状缩肽合成酶的全长基因。测定该DNA片段的碱基序列后,在翻译起始密码子的上游和翻译终止密码子的下游通过PCR等方法导入适合的限制性酶切位点,即可得到只含环状缩肽合成酶基因的基因片段。重组载体
本发明提供含有环状缩肽合成酶的编码核苷酸序列的重组载体。
本发明的重组载体可以采用基因工程领域中常用的程序和方法构建。
作为本发明中可以使用的载体,例如整合到宿主染色体DNA中的载体,或具有可自主复制的自主复制序列并以质粒的形式存在于宿主细胞内的载体,例如pUC系列(pUC18或pUC118等)、pBluescript系列(pBluescript II KS+等)、以及pBR322等质粒。宿主细胞内存在的基因拷贝数可以是1个拷贝,也可以是多个拷贝。
本发明的重组载体可通过下述方法构建,例如在编码环状缩肽合成酶的核苷酸序列上游或下分别按可操纵的方式连接启动子或终止子,并根据需要将基因筛选标记和/或其他的调控序列按可操纵的方式连接。
启动子和终止子与本发明的基因的连接,以及在作为表达单位的载体中的插入,均可按公知的方法进行。
对本发明所用的启动子和终止子无特殊的限制,例如3-磷酸甘油酸激酶、甘油醛-3-磷酸脱氢酶、烯醇化酶等糖酵解系统酶基因的调控序列,鸟氨酸氨甲酰基转移酶、色氨酸合成酶等氨基酸合成系统酶基因的调控序列,淀粉酶、蛋白酶、脂肪酶、纤维素酶、乙酰胺酶等水解酶基因的调控序列,硝酸盐还原酶、乳清酸核苷-5`-磷酸脱氢酶、乙醇脱氢酶等氧化还原酶基因的调控序列,以及在PF1022物质生产菌株中高表达的来源于PF1022物质生产菌的Abp1等基因的调控序列。
本发明的基因也可与编码其他蛋白质翻译区的外源基因连接,以融合蛋白形式表达。
基因筛选标记的导入方法如下,例如通过PCR法将适当的限制性酶切位点导入调控序列,将该调控序列插入质粒载体后,再连接抗药性基因或营养缺陷型基因等筛选标记基因。
基因筛选标记可根据转化体的筛选方法适当地进行选择。例如可选用抗药性基因和营养缺陷型基因。作为抗药性基因,例如越霉素、苯菌灵、寡霉素、潮霉素、G418、博莱霉素、bialaphos、杀稻瘟菌素S、腐草霉素、phosphinothricin、氨苄青霉素、卡那霉素等药物的抗药性基因。作为营养缺陷型基因,例如amdS、pyrG、argB、trpC、niaD、TRP1、LEU2、URA3等基因。转化体和环状缩肽的制造
本发明提供由上述载体转化的宿主。
本发明中所使用的宿主只要是能用于基因重组宿主的微生物,则无其他特殊的限制。作为可使用的宿主,例如任一细菌类或真菌类微生物,优选大肠杆菌、杆菌属细菌、放线菌、酵母、丝状真菌,更优选生产PF1022物质的丝状真菌,最优选PF1022菌株(无孢菌类,FERMBP-2671)。
将基因表达用重组载体导入宿主时,可以采用常规的方法。作为导入方法,例如电穿孔法、聚乙二醇法、土壤杆菌法、锂盐法或氯化钙法等,可根据宿主细胞选择有效的方法。使用PF1022物质生产菌作为宿主时,优选使用聚乙二醇法
转化体的培养可选择合适的培养基和培养条件,并按常规方法进行。作为培养基使用惯用的成分,例如作为碳源可以使用葡萄糖、蔗糖、纤维素、麦芽糖、糊精、淀粉、甘油、糖蜜、动植物油等。另外,作为氮源可以使用大豆粉、小麦胚芽、药物介质(pharma media)、玉米浸渍液、棉子渣、牛肉汤、蛋白胨、聚胨、麦芽提取物、酵母提取物、硫酸铵、硝酸钠、尿素等。根据需要还可添加能够生成钠、钾、钙、镁、钴、氯、磷酸根、磷酸根以及其它离子的无机盐类,例如可添加氯化钾、碳酸钙、磷酸氢二钾、硫酸镁、磷酸二氢钾、硫酸锌、硫酸锰、硫酸铜。另外,根据需要也可以添加硫胺素(硫胺素盐酸盐等)等各种维生素,谷氨酸(谷氨酸钠等)、天冬酰胺(DL-天冬酰胺等)等氨基酸,核苷酸等微量营养素,抗生素等筛选药物。另外,可以适当添加有助于菌体发育、促进环状缩肽生产的有机物和无机物。
培养方法可以采用需氧条件下的振荡培养法、通气搅拌培养法或深部需氧培养法,特别是深部需氧培养法最为适合。培养基的pH例如为pH6~pH8左右。培养的合适温度为15℃~40℃,但大多数场合是在26℃~37℃附近进行培养。环状缩肽合成酶和环状缩肽的生产根据培养基、培养条件或所用的宿主而异,但所有培养方法通常都是在2天~25天期间其蓄积量达到最高峰。
培养过程中,在环状缩肽合成酶或环状缩肽的蓄积量达到最高峰时停止培养,并从培养物中分离并纯化环状缩肽合成酶或环状缩肽。
从培养物中提取环状缩肽合成酶或环状缩肽时,可以根据其性质用常规的分离手段,例如溶剂萃取法、离子交换树脂法、吸附或分配柱色谱法、凝胶过滤法、透析法、沉淀法、结晶化法等,这些方法可以单独使用,或以适当的组合方式进行提取和纯化。
环状缩肽合成酶例如可以通过丁基琼脂糖等疏水性色谱有效地提取和纯化。
环状缩肽例如可以用丙酮、甲醇、丁醇、醋酸乙酯、醋酸丁酯等从培养物中萃取。环状缩肽也可通过硅胶、氧化铝等吸附剂,SephadexLH-20(Pharmacia公司)或者Toyopearl HW-40(Toso公司)等色谱进一步纯化。由上述方法,或将上述方法适当组合可得到纯净的环状缩肽。
本发明提供环状缩肽的大规模生产系统。作为环状缩肽的生产系统、特别是PF1022物质生产系统的宿主,优选生产PF1022物质的丝状真菌、最优选PF1022菌株(无孢菌类,FERM BP-2671)。作为转化所用的重组载体,优选的是将PF1022物质生产菌的功能调控序列(启动子、终止子等)按可操纵的方式与环状缩肽合成酶基因连接的表达载体,最优选的是将PF1022菌株(无孢菌类,FERM BP-2671)的功能调控序列按可操纵的方式与环状缩肽合成酶基因连接的表达载体。环状缩肽、特别是PF1022物质,优选通过以下方法制造。即,将PF1022物质生产菌的功能调控序列按可操纵的方式与环状缩肽合成酶基因连接,构建表达载体,培养由该表达载体转化的PF1022物质生产菌,并从培养物中分离出环状缩肽。
对于不能合成PF1022物质的底物L-亮氨酸、D-乳酸或D-苯基乳酸的宿主,通过添加缺乏的底物或底物的衍生物进行培养,则有可能生产出PF1022物质或PF1022物质的衍生物。
实施例
以下结合实施例对本发明进行详细说明,但本发明不受这些实施例的限制。实施例1:从PF1022物质生产菌克隆环状缩肽合成酶基因1.基因组DNA的分离及基因组文库的构建
用UV照射或NTG处理PF1022菌株(无孢菌类,FERM BP-2671),诱发其突变,从提高了PF1022生产性能的PF1022物质生产菌432-26株中提取基因组DNA。将PE1022物质生产菌432-26株在50ml种子培养基[1%酵母提取物、1%麦芽提取物、2%聚胨、2.5%葡萄糖、0.1%磷酸氢二钾、0.05%硫酸镁(pH7.0)]中,于26℃培养2天,然后通过离心分离回收菌体。将得到的菌体用液氮冷冻后,用研钵和研杵磨碎。用ISOPLANT(日本基因公司),按生产商所提供的方案,由此磨碎的菌体分离基因组DNA。用Sau3AI将分离的基因组DNA部分酶切后,通过琼脂糖凝胶电泳回收15kb-20kb的DNA片段,然后用碱性磷酸酶处理,使DNA片段末端脱磷酸化。将此DNA片段插入噬菌体载体的LambdaDASH II(Stratagene公司)。用Gigapack III Gold PackagingExtract(Stratagene公司),按照生产商所提供的方案,对由此得到的重组噬菌体载体进行体外包装。然后,用此重组噬菌体载体感染大肠杆菌XL1-Blue MRA(P2)株,并在平板上培养,使其形成噬菌斑。2.环状缩肽合成酶基因部分DNA片段的分离
对已知的肽合成酶进行多重序列比较,找出保守性较好的区域WTSMYDE(序列号3)和VVQYFPT(序列号4)。根据此序列,合成5′-TGGACIWSNATGTAYGAYGG-3′(序列号5)和5′-GTIGGRAARTAYTGIACNAC-3′(序列号6)的引物。用该引物对,以从PF1022物质生产菌分离的基因组DNA为模板进行PCR扩增。PCR的50μl反应液中有50ng的基因组DNA作为模板,有1.25U的ExTaq DNA聚合酶(宝酒造社),随酶提供的缓冲液和dNTP混合物,以及10μM的引物。PCR反应条件反应为94℃ 3分钟,[94℃ 1分钟、65℃(每个循环下降0.5℃) 1分钟、72℃ 1分钟]×30次,72℃ 3分钟。由此反应扩增出约350bp的DNA片段,用Original TA克隆试剂盒(Invitrogen公司),按照生产商所提供的方案,将扩增的DNA片段插入到pCR2.1质粒载体中。
用DNA Sequencing Kit dRhodamine Terminator CycleSequencing Ready Reaction(Applied Biosystems公司)和ABI PRISM310 Genetic Analyzer(Applied Biosystems公司),按生产商所提供的方案,测定按上述方法克隆的DNA片段的碱基序列。结果表明,分离的DNA的碱基序列与肽合成酶基因有同源性,明确了该片段是目的环状缩肽合成酶基因的一部分。3.环状缩肽合成酶全长基因的克隆
通过PCR将荧光素标记的dUTP加入到DNA片段中,制备筛选基因组文库所用的探针。在50μl的PCR反应液中,以100ng插入环状缩肽合成酶基因DNA片段的pCR2.1质粒载体为模板,加入1.25U的ExTaqDNA聚合酶(宝酒造社)和随酶提供的缓冲液、0.2mM dATP、0.2mMdCTP、0.2mM dGTP、0.02mM dTTP、0.18mM荧光素标记的dUTP(FluoroGreen,Amersham Pharmacia Biotech公司),以及10μM引物(序列号5和序列号6)。PCR反应条件为94℃ 2分钟,(94℃ 30秒、55℃ 1分钟、72℃ 1分钟)×25次,72℃ 3分钟。由此反应制备出约350bp荧光素标记的探针。
将Hybond-N+膜(Amersham Pharmacia Biotech公司)置于实施例1的1中制成的生成噬菌斑的平板上,使噬菌斑印迹在膜上。用碱处理该膜,使膜上的重组噬菌体DNA变性成为单链,并吸附在膜上。将吸附噬菌体DNA的膜放入用Hybridization Buffer Tablets(Amersham Pharmacia Biotech公司)调制的缓冲液中,60℃保温1小时。然后,将上述荧光素标记的探针热变性后加入到其中,60℃下杂交过夜。随后,将膜放入1×SSC(SSC:15mM柠檬酸三钠、150mM氯化钠)-0.1%SDS溶液中,60℃下洗涤15分钟,再在0.2×SSC-0.1%SDS溶液中,于60℃洗涤15分钟。用DIG洗涤及封闭缓冲液(Boehringermannheim公司)、碱性磷酸酶标记的抗荧光素抗体(Anti-fluorescein-AP,Fab fragment,Boehringer mannheim公司)、生色底物氮蓝四唑氯(Boehringer mannheim公司)以及X-磷酸盐(Boehringer mannheim公司),按照生产商提供的方案显象荧光素结合的噬菌斑。由此,筛选出含有与探针相同区域的5′上游区和3′下游区的阳性克隆。4.碱基序列的测定
用噬菌体载体的序列5′-GCGGAATTAACCCTCACTAAAGGGAACGAA-3′(序列号7)和5′-GCGTAATACGACTCACTATAGGGCGAAGAA-3′(序列号8)为引物,用PCR扩增上述分离的阳性克隆中的DNA片段。PCR的50μl的反应液中,以100ng阳性克隆DNA为模板,加入2.5U的LA Taq DNA聚合酶(宝酒造社)、随酶提供的缓冲液、dNTP混合物、2.5mM氯化镁以及0.2μM引物。PCR反应条件为94℃ 1分钟, (98℃ 10秒钟,68℃ 15分钟)×25次,72℃ 15分钟。纯化所得的PCR产物后,经喷雾处理,使其随机分解为0.5kb~2.0kb的片段。用T4 DNA聚合酶补平这些片段的末端,用T4多核苷酸激酶磷酸化后,插入到pT7Blue(Novagen公司)的EcoRV位点,然后导入大肠杆菌JM109株中。将所得的168个菌落用M13引物M4(宝酒造社)和M13引物RV(宝酒造社)直接进行PCR,产物纯化后,用M13引物M4(宝酒造社)为引物进行测序。PCR的50μl反应液中,加入1.25U的ExTaq DNA聚合酶(宝酒造社)、随酶提供的缓冲液、dNTP混合物以及0.5μM的引物。PCR反应条件为94℃ 4分钟,(94℃ 30秒、55℃ 30秒、72℃ 2分钟)×30次,72℃ 3分钟。此外,用DNA Sequencing Kit dRhodamineTerminator Cycle Sequencing Ready Reaction(AppliedBiosystems公司)和ABI PRISM 310 Genetic Analyzer(AppliedBiosystems公司),按生产商所提供的方案进行测序。
对于所得的结果中解析不够充分的区域,以已解析的碱基序列为基础,重新设计引物进行PCR扩增,产物纯化后,用PCR所用的引物进行测序。由此,确定阳性克隆中DNA片段15606bp的碱基序列。
该序列的解析结果表明,序列中存在包括9633bp的开放阅读框架(ORF),由此序列预测的蛋白质具有3210个氨基酸残基,分子量为354kDa,并与肽合成酶有同源性。此外,表现出最高同源性的蛋白质是恩镰孢素合成酶(S39842),其同源性为56%。序列表的序列号1中示出由此分离的本发明环状缩肽合成酶基因ORF的碱基序列。另外,序列号2中示出其氨基酸序列。实施例2:通过环状缩肽合成酶基因的过表达提高PF1022的生产性能1.基因表达用重组载体的构建(图1)(1)环状缩肽合成酶基因区的克隆
用NotI从实施例1的3中所得的阳性克隆中切出插入的DNA片段,插入到pBluescript II KS+(Stratagene公司)中的NotI位点,构建成质粒pPF7。用BanIII和SmaI消化pPF7,然后进行琼脂糖凝胶电泳,并从琼脂糖凝胶中回收约8250bp的DNA片段。将此片段插入pBluescript II KS+中,构建质粒pPF7-1。
以pPF7为模板,用5`-AGCATCGGATCCTAACAATGGGCGTTGAGCAGCAAGCCCTA-3′(序列号9,按从ORF的N末端起第10位的Met开始翻译而设计)或5′-AGCATCGGATCCTAACAATGTCAAACATGGCACCACTCCCTA-3′(序列号11,按从ORF的N末端第1位的Met开始翻译而设计)和5′-TTTGCTTCGTACTCGGGTCCT-3′(序列号10)作为引物,通过PCR扩增从N末端附近至BanIII位点的约440bp(用序列号9和序列号10)或约470bp(用序列号11和序列号10)的片段。另外,用引物5′-GCATCGCGATACTAGAGAAG-3′(序列号12)和5′-AGCATCGAATTCGGATCCCTAAACCAACGCCAAAGCCCGAAT-3′(序列号13)扩增从SmaI位点至C末端的约920bp的片段。此时,上述PCR引物是按照在本发明的环状缩肽合成酶基因的5′端和3′端导入BamHI位点而设计的。PCR的50μl的反应液中,以150ng的质粒DNA为模板,加入2.5U的KOD DNA聚合酶(东洋纺织社)、随酶提供的缓冲液、dNTP混合物、1mM氯化镁以及0.5μM的引物。PCR反应条件为98℃ 30秒,(98℃ 15秒、65℃ 2秒、74℃ 30秒)×10次,74℃ 1分钟。用乙醇沉淀由各种引物所得的PCR反应液,回收PCR产物。用BmaHI和BanIII消化N末端区,用SmaI和BmaHI消化C末端区,然后进行琼脂糖凝胶电泳,并从琼脂糖凝胶中回收DNA片段。
在pPF7-1的SmaI和BmaHI位点间插入上述C末端区的PCR片段,构建质粒pPF7-2。用BanIII和BmaHI消化该质粒后,进行琼脂糖凝胶电泳,并从琼脂糖凝胶中回收约9170bp的DNA片段。将此DNA片段、以及用序列号9和序列号10扩增的N末端区同时插入到pBluescriptII KS+的BmaHI位点,由此重构建本发明的环状缩肽合成酶基因,进而构建质粒pPFsyn(从ORF的N末端起第10位的Met开始翻译)。
另-方面,将从pPF7-2切出的约9170bp的DNA片段、以及用序列号9和序列号11扩增的N末端区同时插入到PHSG299(宝酒造社)的BamHI位点,由此重构建本发明的环状缩肽合成酶基因,进而构建质粒pPFsynN(从ORF的N末端起第1位的Met开始翻译)。由此,制备两端都有BamHI位点的环状缩肽合成酶基因。
用BamHI消化pPFsyn和pPFsynN后,分别从凝胶中回收环状缩肽合成酶基因区。(2)用Abp1基因的表达调控序列构建表达载体PF1022物质生产菌的基因组DNA分离
PF1022物质生产菌(FERM BP-2671)的基因组DNA分离,按照文献(H.Horiuchi et al.,J.Bacteriol.,170,272-278,(1988))所述方法进行。具体方法是,首先将PF1022菌株(FERM BP-2671)在种子培养基(可溶性淀粉2.0%、葡萄糖1.0%、聚胨0.5%、小麦胚芽0.6%、酵母提取物0.3%、大豆饼0.2%以及碳酸钙0.2%,灭菌前pH为7.0;参照WO 97/00944号的实施例1)中培养2天,通过离心分离(3500rpm、10分钟)回收菌体。随后,将所得的菌体冷冻干燥后,悬浮于TE中,在3%SDS溶液中60℃处理30分钟后,用TE饱和酚萃取,除去菌体残渣。萃取液用乙醇沉淀后,用核糖核酸酶A(Sigma公司制)和蛋白酶K(和光纯药社制)处理,再用12%的聚乙二醇6000使核酸沉淀。将此沉淀用TE饱和酚萃取,用乙醇沉淀,将该沉淀溶于TE中,即得到基因组DNA。PF1022物质生产菌的基因组文库的构建
将上述由PF1022物质生产菌制备的基因组DNA用Sau3AI部分消化。用T4连接酶(宝酒造社制连接试剂盒Ver.2)将此消化产物与噬菌体载体、λEMBL3克隆试剂盒(Stratagene公司)的BamHI臂连接。将此连接物用乙醇沉淀后,溶于TE中。用Gigapack III PlusPackaging试剂盒(Stratagene公司)将全部连接混合物感染大肠杆菌LE392株,形成噬菌体噬菌斑。由此方法得到的1.3×104个(2.6×104PFU/ml)噬菌体文库进行Abp1基因的克隆。由PF1022物质生产菌的基因组DNA克隆Abp1基因
用PCR法扩增Abp1基因的翻译区,用作探针。以由上述PF1022物质生产菌制备的基因组DNA为模板,用由8-73U和8-73R组成的合成引物,用LETS GO PCR试剂盒(SAWADY Technology公司)进行PCR反应。PCR反应的条件为,以94℃30秒、50℃30秒、72℃90秒为一个循环,共反复操作25次进行扩增。8-73U和8-73R的DNA序列如下所示。8-73U:CTCAAACCAGGAACTCTTTC(序列号14)8-73R:GACATGTGGAAACCACATTTTG(序列号15)
用ECL Direct System(Amersham Pharmacia Biotech公司制)标记上述所得的PCR产物。将上述制备的噬菌体噬菌斑印迹到HybondN+尼龙转移膜(Amersham PharmaciaBiotech公司制)上,碱变性后,用5倍浓度的SSC(SSC:15mM柠檬酸三钠、150mM氯化钠)洗涤、干燥并固定DNA。按试剂盒附带方案所述的方法,预杂交1小时(42℃)后,加入预先标记的探针,杂交16小时(42℃)。按上述试剂盒附带方案所述的方法洗涤探针。将已洗涤探针的膜在检测溶液中浸渍1分钟后,置于医用X光片上感光,得到1个阳性克隆。该克隆的Southern分析结果表明,至少6kb的HindIII片段与基因组DNA的限制性酶切片段长度一致。该HindIII片段的限制性酶图谱如图2所示。将HindIII片段亚克隆到pUC119中(pRQHin/119),供以后实验之用。表达载体的构建
以pRQHin/119为模板,通过PCR法扩增Abp1基因的启动子区和终止子区。启动子的扩增用ABP-Neco和ABP-Nbam组成的引物,终止子的扩增用ABP-Cbam和ABP-Cxba组成的引物,用PCR Super Mix HighFidelity(Lifetech Oriental公司制)进行PCR反应。PCR反应的条件以94℃30秒、50℃30秒、72℃90秒为一个循环,反复操作25次进行扩增。ABP-Neco、ABP-Nbam、ABP-Cbam以及ABP-Cxba的DNA序列如下所示。ABP-Neco:GGGGAATTCGTGGGTGGTGATATCATGGC(序列号16)ABP-Nbam:GGGGGATCCTTGATGGGTTTTGGG(序列号17)ABP-Cbam:GGGGGATCCTAAACTCCCATCTATAGC(序列号18)ABP-Cxba:GGGTCTAGACGACTCATTGCAGTGAGTGG(序列号19)
用Microspin S-400柱(Amersham Pharmacia Biotech公司制)将各种PCR产物纯化,用乙醇沉淀后,启动子用EcoRI和BamHI消化,终止子用BamHI和XbaI消化,并与用同样的酶消化的pBluescript IIKS+依次连接。将其用XbaI消化后,插入来源于pMKD01(WO 98/03667号)的越霉素抗性组件,构建pABPd(图3)。pABPd中具有Abp1基因的启动子和终止子。
将上述从凝胶中回收的环状缩肽合成酶基因区插入到pABPd中的BamHI位点,构建可使环状缩肽合成酶基因表达的表达载体pABP/PFsyn(从ORF的N末端起第10位的Met开始翻译)以及pABP/PFsynN(从ORF的N末端起第1位的Met开始翻译)。2.PF1022物质生产菌中环状缩肽合成酶基因的导入和表达
按照WO 97/00944号的实施例1中所述方法,将表达载体导入PF1022菌株(无孢菌类,FERM BP-2671)中,之后筛选潮霉素B抗性高的菌株。确认这些菌株中是否导入了目的基因,可根据Abp1启动子设计引物5′-TGATATGCTGGAGCTTCCCT-3′(序列号20)和根据环状缩肽合成酶基因的序列设计引物5′-GCACAACCTCTTTCCAGGCT-3′(序列号21),进行PCR反应来确认。由此,筛选潮霉素B抗性高的、导入了本发明环状缩肽合成酶基因的基因导入株。
将基因导入株和亲本株(无孢菌类,FERM BP-2671)分别在50ml的种子培养基中26℃培养2天后,每种培养液取1ml分别接种于50ml的生产培养基[6%麦芽糖、2.6%淀粉、2%小麦胚芽、1%药物介质(Pharmamedia)、0.2%硫酸镁7水合物、0.2%碳酸钙、0.3%氯化钠(pH7.5)]中,26℃下培养4天。培养液在4500rpm下离心5分钟,收集菌体,所得的每种菌体用0.3M氯化钾洗涤。用液氮将每种菌体分别冷冻后,进行冷冻干燥。
以下所述的抽提操作是在冰上或在4℃低温室内进行。将10mg冷冻干燥的菌体和1.0ml的玻璃珠(直径0.5mm)加入2ml的微型离心管内,然后添加1.0ml抽提缓冲液[50mM Tris-HCl(pH8.0)、0.3M氯化钾、60%甘油、10mM乙二胺四乙酸钠、5mM二硫苏糖醇、10μM亮肽素、1mM甲苯磺酸、60μg/ml糜蛋白酶]。将此微型离心管置于Mini-Bead-Beater-8(Biospeck公司)中,以最高速度转3分钟进行抽提。将此管在15000rpm离心5分钟后,将100μl的上清液加到100μl的三氯乙酸溶液中,并混合。放置15分钟以后,在15000rpm离心10分钟,将所得的沉淀溶于15μl的碱溶液(2%碳酸钠、0.4%氢氧化钠)中,并加入60μl的样品缓冲液(125mM Tris-HCl(pH6.8)、20%甘油、4%十二烷基硫酸钠、10%2-巯基乙醇、50mg/l溴酚蓝)。将其在沸水中加热5分钟后,通过电泳系统(Difco公司),用4%~20%的聚丙烯酰胺凝胶进行电泳[十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)]。电泳后的聚丙烯酰胺凝胶用Quick-CBB(和光纯药社),按生产商所提供的方案染色。由亲本株和导入pABP/PFsyn的基因导入株提取的蛋白质的电泳结果如图4所示。此外,由亲本株和导入pABP/PFsynN的基因导入株提取的蛋白质的电泳结果如图5所示。
由此可见,基因导入株的环状缩肽合成酶的表达量明显高于亲本株。3.PF1022物质的提取和定量
将基因导入株和亲本株分别在50ml的种子培养基中,26℃培养2天后,每种培养液取1ml分别接种于50ml生产培养基中,于26℃培养6天。每种培养液取10ml,分别在3000rpm离心10分钟,分别收集菌体。在每种菌体中加入10ml甲醇,激烈振荡后静置30分钟。然后,再进行振荡,3000rpm离心10分钟后,将每种菌体提取液上清中的PF1022物质通过液相色谱分别进行定量。所用的柱为LiChrospher100 RP-18(e)(关东化学社),柱的温度为40℃,流动相为80%的乙腈,流速为1.0ml/分钟,根据其在210nm的吸收检测PF1022物质。在此条件下,PF1022物质的保留时间为5分钟~6分钟。实验反复进行两次,由亲本株和导入pABP/PFsyn的基因导入株提取的PF1022物质定量结果的平均值如表1所示。
表1
PF1022物质(μg/ml)
亲本株 88
基因导入株 222
基因导入株的PF1022生产性能为亲本株的约2.5倍。这表明通过过表达本发明的环状缩肽合成酶,提高了PF1022物质的生产性能。
由亲本株和导入pABP/PFsynN的基因导入株提取的PF1022物质定量结果的平均值如表2所示。
表2
PF1022物质(μg/ml)
亲本株 29
基因导入株1 123
基因导入株2 136
基因导入株3 172
表2的结果表明,基因导入株的PF1022生产性能为亲本株的4.3~6.0倍。这表明通过过表达本发明的环状缩肽合成酶,提高了PF1022物质的生产性能。
序列表<110>明治制果株式会社(Meiji Seika Kaisha,Ltd)<120>环状缩肽合成酶及其基因、以及环状缩肽的大规模生产系统<130>127184<150>JP 253040/1999<151>1999-09-07<150>JP 104291/2000<151>2000-04-06<160>21<170>PatentIn Ver.2.1<210>1<211>9633<212>DNA<213>无孢菌类(Mycelia sterilia)<220><221>CDS<222>(1)..(9633)<223>PF1022肽合成酶<220><221>成熟肽<222>(13)..(9630)<400>1atg tca aac atg gca cca ctc cct acg atg ggc gtt gag cag caa gcc 48Met Ser Asn Met Ala Pro Leu Pro Thr Met Gly Val Glu Gln Gln Ala
-1 1 5 10cta tca ctt tca tgc ccc tta ctc cct cat gac gat gag aaa cac tca 96Leu Ser Leu Ser Cys Pro Leu Leu Pro His Asp Asp Glu Lys His Ser
15 20 25gac aac ctt tac gag caa gca act cgg cac ttc ggc ttg agc cga gac 144Asp Asn Leu Tyr Glu Gln Ala Thr Arg His Phe Gly Leu Ser Arg Asp
30 35 40aag atc gaa aat gtc tta cca tgt act tcc ttt caa tgt gat gtc ata 192Lys Ile Glu Asn Val Leu Pro Cys Thr Ser Phe Gln Cys Asp Val Ile45 50 55 60gat tgc gcc gtc gac gat cgg cgg cat gct atc ggt cac gtc gtc tat 240Asp Cys Ala Val Asp Asp Arg Arg His Ala Ile Gly His Val Val Tyr
65 70 75gat atc ccc aat aca gtg gac atc cag cgt tta gcc gca gcc tgg aaa 288Asp Ile Pro Asn Thr Val Asp Ile Gln Arg Leu Ala Ala Ala Trp Lys
80 85 90gag gtt gtg cgg cag aca cca atc ttg agg acc ggc atc ttt aca tca 336Glu Val Val Arg Gln Thr Pro Ile Leu Arg Thr Gly Ile Phe Thr Ser
95 100 105gaa acc ggc gac tct ttt cag atc gtc ttg aaa gaa ggc tgc cta ccg 384Glu Thr Gly Asp Ser Phe Gln Ile Val Leu Lys Glu Gly Cys Leu Pro
110 115 120tgg atg tac gcg aca tgt ctc ggc atg aag ggg gca gtg ata caa gat 432Trp Met Tyr Ala Thr Cys Leu Gly Met Lys Gly Ala Val Ile Gln Asp125 130 135 140gaa gca gtc gcc gct atg act gga ccg cgt tgc aat cga tat gtc gtc 480Glu Ala Val Ala Ala Met Thr Gly Pro Arg Cys Asn Arg Tyr Val Val
145 150 155ctg gag gac ccg agt acg aag caa agg ctg ctc atc tgg aca ttc agc 528Leu Glu Asp Pro Ser Thr Lys Gln Arg Leu Leu Ile Trp Thr Phe Ser
160 165 170cat gct tta gtg gat tat aca gtc cag gaa cgc atc ctt cag cgg gtt 576His Ala Leu Val Asp Tyr Thr Val Gln Glu Arg Ile Leu Gln Arg Val
175 180 185ctc aca gta tac gac ggc cgg gac gtc gag tgc cct cgc atc aag gat 624Leu Thr Val Tyr Asp Gly Arg Asp Val Glu Cys Pro Arg Ile Lys Asp
190 195 200aca gaa cat gtc tct cgg ttt tgg caa caa cac ttt gaa ggc tta gat 672Thr Glu His Val Ser Arg Phe Trp Gln Gln His Phe Glu Gly Leu Asp205 210 215 220gcc tcc gta ttt ccc ctt cta cca tct cac cta act gtg tgc aat ccc 720Ala Ser Val Phe Pro Leu Leu Pro Ser His Leu Thr Val Cys Asn Pro
225 230 235aat gcg cgc gca gaa cat cat atc tca tac acg gga cca gtc cag agg 768Asn Ala Arg Ala Glu His His Ile Ser Tyr Thr Gly Pro Val Gln Arg
240 245 250aag tgg tcc cat aca agt atc tgt cgg gct gca ctc gca gtt ctt cta 816Lys Trp Ser His Thr Ser Ile Cys Arg Ala Ala Leu Ala Val Leu Leu
255 260 265tct cgc ttt aca cac tct tcg gag gcc ctc ttc ggt gtt gtg aca gaa 864Ser Arg Phe Thr His Ser Ser Glu Ala Leu Phe Gly Val Val Thr Glu
270 275 280caa tct cac aac tcc gag gac caa aga cgg tca att gat ggc ccc gca 912Gln Ser His Asn Ser Glu Asp Gln Arg Arg Ser Ile Asp Gly Pro Ala285 290 295 300agg aca gta gtg cct atc cgc gtc ctt tgt gcc cca gat caa tat gtg 960Arg Thr Val Val Pro Ile Arg Val Leu Cys Ala Pro Asp Gln Tyr Val
305 310 315tcg gat gtc att ggg gca atc acc gca cac gaa cac gcc atg cgc ggg 1008Ser Asp Val Ile Gly Ala Ile Thr Ala His Glu His Ala Met Arg Gly
320 325 330ttt gag cac gct gga ctt cgc aat atc cgc cgt acc gga gac gac ggg 1056Phe Glu His Ala Gly Leu Arg Asn Ile Arg Arg Thr Gly Asp Asp Gly
335 340 345tct gct gct tgt gga ttc cag acc gtg cta ctg gtg act gac ggt gat 1104Ser Ala Ala Cys Gly Phe Gln Thr Val Leu Leu Val Thr Asp Gly Asp
350 355 360gct ccc aag acc cct ggg agt gta ctt cat cga agt gta gaa gaa tcg 1152Ala Pro Lys Thr Pro Gly Ser Val Leu His Arg Ser Val Glu Glu Ser365 370 375 380gat aga ttc atg ccc tgc gct aat cgt gcc ctt ctg ctc gac tgc cag 1200Asp Arg Phe Met Pro Cys Ala Asn Arg Ala Leu Leu Leu Asp Cys Gln
385 390 395atg gct ggc aac tcg gca tcc cta gtc gca cga tat gat cat aat gtg 1248Met Ala Gly Asn Ser Ala Ser Leu Val Ala Arg Tyr Asp His Asn Val
400 405 410atc gac cca cgc cag atg tct cgc ttc ctg cga cag cta gga tac ctg 1296Ile Asp Pro Arg Gln Met Ser Arg Phe Leu Arg Gln Leu Gly Tyr Leu
415 420 425atc caa caa ttt cat cat cac gtc gat ctg cct ctg gtc aaa gaa ctg 1344Ile Gln Gln Phe His His His Val Asp Leu Pro Leu Val Lys Glu Leu
430 435 440gac gtc gtg acg gcg gag gat tgt gcg gaa atc gag aaa tgg aat tca 1392Asp Val Val Thr Ala Glu Asp Cys Ala Glu Ile Glu Lys Trp Asn Ser445 450 455 460gaa cgc cta aca atg caa gac gcc tta atc cac gac acc ata tcc aag 1440Glu Arg Leu Thr Met Gln Asp Ala Leu Ile His Asp Thr Ile Ser Lys
465 470 475tgg gct gct ggc gat ccc aac aaa gct gca gtt ttc gct tgg gat ggg 1488Trp Ala Ala Gly Asp Pro Asn Lys Ala Ala Val Phe Ala Trp Asp Gly
480 485 490gaa tgg aca tac gcc gag cta gac aac ata tcc tcc cgt ctc gcc gtg 1536Glu Trp Thr Tyr Ala Glu Leu Asp Asn Ile Ser Ser Arg Leu Ala Val
495 500 505tat atc caa tcc ctg gac ttg aga cca gga caa gca ata ctc cca ctc 1584Tyr Ile Gln Ser Leu Asp Leu Arg Pro Gly Gln Ala Ile Leu Pro Leu
510 515 520tgc ttc gag aag tca aaa tgg gtc gtc gcc aca att ctc gcc gtc ctc 1632Cys Phe Glu Lys Ser Lys Trp Val Val Ala Thr Ile Leu Ala Val Leu525 530 535 540aaa gtc ggt cgg gca ttc aca ctc atc gac ccg tgc gac ccc tcg gca 1680Lys Val Gly Arg Ala Phe Thr Leu Ile Asp Pro Cys Asp Pro Ser Ala
545 550 555agg atg gcc cag gtc tgt cag cag acc tcc gcc aca gtc gct ctc acc 1728Arg Met Ala Gln Val Cys Gln Gln Thr Ser Ala Thr Val Ala Leu Thr
560 565 570tcc aaa ctc cac aac acc acc tta cgt tcc gtc gtt tcc cgc tgc att 1776Ser Lys Leu His Asn Thr Thr Leu Arg Ser Val Val Ser Arg Cys Ile
575 580 585gtg gtc gac gac gac ctc ctt cgg tcc tta ccc cac gcc gat ggc cgc 1824Val Val Asp Asp Asp Leu Leu Arg Ser Leu Pro His Ala Asp Gly Arg
590 595 600tta aag gcc acc gtg aag cca cag gac ttg gcc tat gtt att ttc aca 1872Leu Lys Ala Thr Val Lys Pro Gln Asp Leu Ala Tyr Val Ile Phe Thr605 610 615 620tct ggc agc aca gga gag ccg aaa ggc atc atg atc gaa cat cgg ggg 1920Ser Gly Ser Thr Gly Glu Pro Lys Gly Ile Met Ile Glu His Arg Gly
625 630 635ttc gtg tcg tgt gct atg aaa ttt ggc ccc gcg ctc gga atg gat gag 1968Phe Val Ser Cys Ala Met Lys Phe Gly Pro Ala Leu Gly Met Asp Glu
640 645 650cac acg cgc gct ctt caa ttc gcc tca tat gcg ttt ggc gct tgt ctg 2016His Thr Arg Ala Lcu Gln Phe Ala Ser Tyr Ala Phe Gly Ala Cys Leu
655 660 665gta gaa gtt gtg aca gct ctg atg cac ggc ggc tgc gtc tgc atc cct 2064Val Glu Val Val Thr Ala Leu Met His Gly Gly Cys Val Cys Ile Pro
670 675 680tcc gat gac gat cgc ttg aac aat gta ccg gag ttc atc aaa agg gcc 2112Ser Asp Asp Asp Arg Leu Asn Asn Val Pro Glu Phe Ile Lys Arg Ala685 690 695 700cag gtg aac tgg gtg ata ctc act ccg tcg tat atc ggg aca ttc cag 2160Gln Val Asn Trp Val Ile Leu Thr Pro Ser Tyr Ile Gly Thr Phe Gln
705 710 715ccg gaa gat gtc cct gga cta caa aca ctg gta ttg gtt gga gaa cct 2208Pro Glu Asp Val Pro Gly Leu Gln Thr Leu Val Leu Val Gly Glu Pro
720 725 730atc tca gcg tct att cgg gat acc tgg gcc tcc cag gtt cga ctc ctg 2256Ile Ser Ala Ser Ile Arg Asp Thr Trp Ala Ser Gln Val Arg Leu Leu
735 740 745aat gcc tac ggt cag agt gaa agc tca act atg tgc agc gtc acg gaa 2304Asn Ala Tyr Gly Gln Ser Glu Ser Ser Thr Met Cys Ser Val Thr Glu
750 755 760gtc agc ccg ctc tcc ctc gaa ccg aac aat atc ggt cgg gct gta ggc 2352Val Ser Pro Leu Ser Leu Glu Pro Asn Asn Ile Gly Arg Ala Val Gly765 770 775 780gca cga tcc tgg atc att gat ccc gac gag cct gat cgt ctt gct cca 2400Ala Arg Ser Trp Ile Ile Asp Pro Asp Glu Pro Asp Arg Leu Ala Pro
785 790 795att ggc tgc att gga gag cta gtg atc gaa agt ccg ggc att gcg cgc 2448Ile Gly Cys Ile Gly Glu Leu Val Ile Glu Ser Pro Gly Ile Ala Arg
800 805 810gac tat atc atc gcg ccg ccg ccg gac aag tcc ccc ttt ctc cta gca 2496Asp Tyr Ile Ile Ala Pro Pro Pro Asp Lys Ser Pro Phe Leu Leu Ala
815 820 825ccc ccg gcc tgg tat cca gcc ggg aaa tta tcc aac gcc ttt aaa ttt 2544Pro Pro Ala Trp Tyr Pro Ala Gly Lys Leu Ser Asn Ala Phe Lys Phe
830 835 840tac aag act gga gat ctc gtg cgt tat gga cct gac ggc acc atc gtc 2592Tyr Lys Thr Gly Asp Leu Val Arg Tyr Gly Pro Asp Gly Thr Ile Val845 850 855 860tgc ctg gga cgc aaa gat tcg caa gtg aag atc cga ggg cag cgc gta 2640Cys Leu Gly Arg Lys Asp Ser Gln Val Lys Ile Arg Gly Gln Arg Val
865 870 875gag atc agc gca gtg gaa gcc agt cta cga cga caa cta cct agt gac 2688Glu Ile Ser Ala Val Glu Ala Ser Leu Arg Arg Gln Leu Pro Ser Asp
880 885 890atc atg ccc gtg gcc gaa gct atc aaa cgc tcg gat tcg tca ggc agc 2736Ile Met Pro Val Ala Glu Ala Ile Lys Arg Ser Asp Ser Ser Gly Ser
895 900 905aca gtc ttg act gcc ttc ttg ata ggg tca tct aag agc gga gat ggt 2784Thr Val Leu Thr Ala Phe Leu Ile Gly Ser Ser Lys Ser Gly Asp Gly
910 915 920aat ggc cac gct tta tct gcg gca gac gcc gtt atc ttg gat cac ggt 2832Asn Gly His Ala Leu Ser Ala Ala Asp Ala Val Ile Leu Asp His Gly925 930 935 940gct acc aac gag ata aac gcg aag ttg cag caa atc ctt ccc cag cat 2880Ala Thr Asn Glu Ile Asn Ala Lys Leu Gln Gln Ile Leu Pro Gln His
945 950 955tcc gtt cca tcc tat tat atc cac atg gaa aat ctt cct cga act gcc 2928Ser Val Pro Ser Tyr Tyr Ile His Met Glu Asn Leu Pro Arg Thr Ala
960 965 970acc ggc aaa gcg gac agg aaa atg ctt cga tct att gct agc aag cta 2976Thr Gly Lys Ala Asp Arg Lys Met Leu Arg Ser Ile Ala Ser Lys Leu
975 980 985ttg ggt gaa ttg tct cag aac gtg aca tct caa ccg att gag aag cac 3024Leu Gly Glu Leu Ser Gln Asn Val Thr Ser Gln Pro Ile Glu Lys His
990 995 1000gat gcc cca gca act ggt ata gag gtc aag ctg aag gag ctt tgg ttt 3072Asp Ala Pro Ala Thr Gly Ile Glu Val Lys Leu Lys Glu Leu Trp Phe1005 1010 1015 1020ctg agc ttg aat ctt aat ccc aac tct caa gat gtc gga gcg agt ttc 3120Leu Ser Leu Asn Leu Asn Pro Asn Ser Gln Asp Val Gly Ala Ser Phe
1025 1030 1035ttt gac tta ggc gga aat tcc att atc gcc atc aaa atg gta aac atg 3168Phe Asp Leu Gly Gly Asn Ser Ile Ile Ala Ile Lys Met Val Asn Met
1040 1045 1050gcg agg tca gct ggg ata gca ctg aag gta tcc gac ata ttc cag aat 3216Ala Arg Ser Ala Gly Ile Ala Leu Lys Val Ser Asp Ile Phe Gln Asn
1055 1060 1065ccc acg ctc gcc ggc ctt gtg gat gtc atc ggg cga gac ccg gct ccg 3264Pro Thr Leu Ala Gly Leu Val Asp Val Ile Gly Arg Asp Pro Ala Pro1070 1075 1080tac aac ctc atc cca aca aca gca tac agc gga cct gtt gag cag tcg 3312Tyr Asn Leu Ile Pro Thr Thr Ala Tyr Ser Gly Pro Val Glu Gln Ser1085 1090 1095 1100ttc gcc cag ggc cgt cta tgg ttc ttg gac cag atc gaa ctc gat gcg 3360Phe Ala Gln Gly Arg Leu Trp Phe Leu Asp Gln Ile Glu Leu Asp Ala
1105 1110 1115ttg tgg tac ctt cta cca tac gcc gtt cgc atg cgc ggg cca ttg cat 3408Leu Trp Tyr Leu Leu Pro Tyr Ala Val Arg Met Arg Gly Pro Leu His
1120 1125 1130att gat gcg ctc act att gcg ttg cta gct ata cag caa cga cat gaa 3456Ile Asp Ala Leu Thr Ile Ala Leu Leu Ala Ile Gln Gln Arg His Glu
1135 1140 1145acc ttg cgg aca acc ttt gag gag cag gac ggc gta ggc gtt cag gtt 3504Thr Leu Arg Thr Thr Phe Glu Glu Gln Asp Gly Val Gly Val Gln Val1150 1155 1160gtc cat gcg agc ccc atc tcc gac ttg agg ata atc gac gta tca ggc 3552Val His Ala Ser Pro Ile Ser Asp Leu Arg Ile Ile Asp Val Ser Gly1165 1170 1175 1180gac cga aac agt gac tac ctc cag ttg cta cac caa gag cag acg act 3600Asp Arg Asn Ser Asp Tyr Leu Gln Leu Leu His Gln Glu Gln Thr Thr
1185 1190 1195cca ttc att cta gca tgt cag gca gga tgg agg gta tca ctg att aga 3648Pro Phe Ile Leu Ala Cys Gln Ala Gly Trp Arg Val Ser Leu Ile Arg
1200 1205 1210cta gga gaa gat gat cac atc ctc tct atc gta atg cat cac atc atc 3696Leu Gly Glu Asp Asp His Ile Leu Ser Ile Val Met His His Ile Ile
1215 1220 1225tcc gac ggc tgg tct atc gac att cta cgc cgg gaa cta agc aat ttc 3744Ser Asp Gly Trp Ser Ile Asp Ile Leu Arg Arg Glu Leu Ser Asn Phe1230 1235 1240tat tca gcc gct ctc cgg ggc tct gat cct cta tcg gtg gtg agc cca 3792Tyr Ser Ala Ala Leu Arg Gly Ser Asp Pro Leu Ser Val Val Ser Pro1245 1250 1255 1260ctc cca ctc cac tac cgc gac ttt tcc gtt tgg caa aag cag gtc gaa 3840Leu Pro Leu His Tyr Arg Asp Phe Ser Val Trp Gln Lys Gln Val Glu
1265 1270 1275cag gag acc gaa cat gag cgg caa ctc gaa tac tgg gtc aag cag ctc 3888Gln Glu Thr Glu His Glu Arg Gln Leu Glu Tyr Trp Val Lys Gln Leu
1280 1285 1290gca gac agc tcg gcc gcc gaa ttc cta acc gac ttc ccc cga ccc aac 3936Ala Asp Ser Ser Ala Ala Glu Phe Leu Thr Asp Phe Pro Arg Pro Asn
1295 1300 1305ata ctg tcc ggt gaa gca ggt tcc gtg cca gtg acg atc gaa ggc gaa 3984Ile Leu Ser Gly Glu Ala Gly Ser Val Pro Val Thr Ile Glu Gly Glu1310 1315 1320ctg tat gaa agg ctc caa gaa ttc tgt aaa gta gag caa atg acg cct 4032Leu Tyr Glu Arg Leu Gln Glu Phe Cys Lys Val Glu Gln Met Thr Pro1325 1330 1335 1340ttc gcc gtg ttg tta ggg gcc ttc cgc gcg acc cat tat cgt ctc acc 4080Phe Ala Val Leu Leu Gly Ala Phe Arg Ala Thr His Tyr Arg Leu Thr
1345 1350 1355ggc gcc gaa gac tcg atc atc ggc acg ccc atc gcg aac cgc aac cgc 4128Gly Ala Glu Asp Ser Ile Ile Gly Thr Pro Ile Ala Asn Arg Asn Arg
1360 1365 1370cag gag ctt gaa aac atg atc ggc ttc ttc gtc aac acc caa tgc atg 4176Gln Glu Leu Glu Asn Met Ile Gly Phe Phe Val Asn Thr Gln Cys Met
1375 1380 1385cga atc acg gtc gac ggc gac gac act ttt gaa agc ctg gtg cga caa 4224Arg Ile Thr Val Asp Gly Asp Asp Thr Phe Glu Ser Leu Val Arg Gln1390 1395 1400gtt cgg acc acg gcg acg gcg gca ttc gag cac caa gac gtc ccc ttt 4272Val Arg Thr Thr Ala Thr Ala Ala Phe Glu His Gln Asp Val Pro Phe1405 1410 1415 1420gag cgc gtc gtg acg gca ctc ctt cca cgc tcg aga gac cta tcc cga 4320Glu Arg Val Val Thr Ala Leu Leu Pro Arg Ser Arg Asp Leu Ser Arg
1425 1430 1435aac cca cta gca cag ctc acc ttc gct ctt cat tct caa cag gac ctc 4368Asn Pro Leu Ala Gln Leu Thr Phe Ala Leu His Ser Gln Gln Asp Leu
1440 1445 1450ggc aag ttc gag ctg gag ggt ctc gta gcg gaa ccc gtc tcg aac aag 4416Gly Lys Phe Glu Leu Glu Gly Leu Val Ala Glu Pro Val Ser Asn Lys
1455 1460 1465gta tac acc agg ttc gac gtg gag ttt cac ctg ttc caa gaa gcc gga 4464Val Tyr Thr Arg Phe Asp Val Glu Phe His Leu Phe Gln Glu Ala Gly1470 1475 1480aga cta agc ggt aac gtg gca ttt gcg gca gat cta ttc aag cct gag 4512Arg Leu Ser Gly Asn Val Ala Phe Ala Ala Asp Leu Phe Lys Pro Glu1485 1490 1495 1500acc att agc aat gta gtc gcc ata ttt ttc caa atc ctg cga caa ggc 4560Thr Ile Ser Asn Val Val Ala Ile Phe Phe Gln Ile Leu Arg Gln Gly
1505 1510 1515att cgc cag cct cgg act cca atc gct gtt ctc ccg ctt acc gat ggg 4608Ile Arg Gln Pro Arg Thr Pro Ile Ala Val Leu Pro Leu Thr Asp Gly
1520 1525 1530tta gcg gac ctt cgt gcc atg ggc ttg ctt gag atc gag aag gca gaa 4656Leu Ala Asp Leu Arg Ala Met Gly Leu Leu Glu Ile Glu Lys Ala Glu
1535 1540 1545tac ccg cgg gag tcg agc gtc gtc gac gtc ttc cgc aag cag gtg gcc 4704Tyr Pro Arg Glu Ser Ser Val Val Asp Val Phe Arg Lys Gln Val Ala1550 1555 1560gct cac ccg cac gct ttt gcc gtt gtc gat tcg gca tcg cgc ctc aca 4752Ala His Pro His Ala Phe Ala Val Val Asp Ser Ala Ser Arg Leu Thr1565 1570 1575 1580tat gct gat ctc gat cgt caa tcc gat caa ctc gcg acc tgg ctc ggt 4800Tyr Ala Asp Leu Asp Arg Gln Ser Asp Gln Leu Ala Thr Trp Leu Gly
1585 1590 1595cgg cgc aat atg acg gct gag acg ctg gtc ggg gtg tta gca ccg cgg 4848Arg Arg Asn Met Thr Ala Glu Thr Leu Val Gly Val Leu Ala Pro Arg
1600 1605 1610tca tgt caa aca gtt gtt gcc att tta ggt atc ctg aaa gcg aat ctc 4896Ser Cys Gln Thr Val Val Ala Ile Leu Gly Ile Leu Lys Ala Asn Leu
1615 1620 1625gca tat ctc ccg ctt gat gtg aat tgt cct acc gcc cgc ctg caa aca 4944Ala Tyr Leu Pro Leu Asp Val Asn Cys Pro Thr Ala Arg Leu Gln Thr1630 1635 1640atc cta tct aca ttg aat cgg cac aag ttg gtc cta ctc ggc tct aac 4992Ile Leu Ser Thr Leu Asn Arg His Lys Leu Val Leu Leu Gly Ser Asn1645 1650 1655 1660gca act act ccg gat gtc cag ata cct gat gta gag ctg gta cga atc 5040Ala Thr Thr Pro Asp Val Gln Ile Pro Asp Val Glu Leu Val Arg Ile
1665 1670 1675agc gat atc tta gat cgc ccc atc aat ggc cag gca aag cta aat ggt 5088Ser Asp Ile Leu Asp Arg Pro Ile Asn Gly Gln Ala Lys Leu Asn Gly
1680 1685 1690cat aca aaa tcg aat ggc tac tca aag cca aac ggc tat acg cat ctg 5136His Thr Lys Ser Asn Gly Tyr Ser Lys Pro Asn Gly Tyr Thr His Leu
1695 1700 1705aaa ggc tat tca aac cta aac ggt tat tca aaa caa aat ggt tat gca 5184Lys Gly Tyr Ser Asn Leu Asn Gly Tyr Ser Lys Gln Asn Gly Tyr Ala1710 1715 1720caa ctc aac ggc cat aga gag cgt aac aat tat tta gat cta aat ggg 5232Gln Leu Asn Gly His Arg Glu Arg Asn Asn Tyr Leu Asp Leu Asn Gly1725 1730 1735 1740cac tca ctg cta aat ggg aat tca gac atc acc aca tca ggg ccc tca 5280His Ser Leu Leu Asn Gly Asn Ser Asp Ile Thr Thr Ser Gly Pro Ser
1745 1750 1755gca aca agc ctt gcc tac gtg atc ttc aca tcc ggc tca acc gga aag 5328Ala Thr Ser Leu Ala Tyr Val Ile Phe Thr Ser Gly Ser Thr Gly Lys
1760 1765 1770ccc aaa gga gtc atg gtc gaa cac cgc agc atc atc cga ctt gca aag 5376Pro Lys Gly Val Met Val Glu His Arg Ser Ile Ile Arg Leu Ala Lys
1775 1780 1785aag aac aga atc ata tcc agg ttc cca tct gta gcc aag gta gct cac 5424Lys Asn Arg Ile Ile Ser Arg Phe Pro Ser Val Ala Lys Val Ala His1790 1795 1800ctc tca aac atc gcc ttt gac gcc gcc act tgg gaa atg ttc gca gcc 5472Leu Ser Asn Ile Ala Phe Asp Ala Ala Thr Trp Glu Met Phe Ala Ala1805 1810 1815 1820ctt cta aac ggc gga acg ctg gtc tgt atc gac tat atg acc acc ctg 5520Leu Leu Asn Gly Gly Thr Leu Val Cys Ile Asp Tyr Met Thr Thr Leu
1825 1830 1835gac agc aaa acg ctc gag gcc gcg ttt gca cga gaa caa atc aac gcc 5568Asp Ser Lys Thr Leu Glu Ala Ala Phe Ala Arg Glu Gln Ile Asn Ala
1840 1845 1850gcg tta ctc acg ccc gct ttg ttg aag cag tgc cta gcc aac att ccc 5616Ala Leu Leu Thr Pro Ala Leu Leu Lys Gln Cys Leu Ala Asn Ile Pro
1855 1860 1865act acc cta ggc agg ctg agt gca ctc gtt att gga ggt gat agg ctt 5664Thr Thr Leu Gly Arg Leu Ser Ala Leu Val Ile Gly Gly Asp Arg Leu1870 1875 1880gac ggc caa gac gcg atc gca gca cat gcg ctt gtc ggt gct ggc gtg 5712Asp Gly Gln Asp Ala Ile Ala Ala His Ala Leu Val Gly Ala Gly Val1885 1890 1895 1900tat aat gcg tat ggc ccg acc gaa aac gga gtg atc agt acg att tat 5760Tyr Asn Ala Tyr Gly Pro Thr Glu Asn Gly Val Ile Ser Thr Ile Tyr
1905 1910 1915aat atc act aaa aac gac tcg ttc atc aac gga gtc ccc atc ggc tgt 5808Asn Ile Thr Lys Asn Asp Ser Phe Ile Asn Gly Val Pro Ile Gly Cys
1920 1925 1930gca atc agc aat tcc ggc gcc tac atc aca gac cca gac cag cag ctc 5856Ala Ile Ser Asn Ser Gly Ala Tyr Ile Thr Asp Pro Asp Gln Gln Leu
1935 1940 1945gta cct cct ggc gtc atg ggt gaa ctc gtc gtt acc ggt gac ggg ctc 5904Val Pro Pro Gly Val Met Gly Glu Leu Val Val Thr Gly Asp Gly Leu1950 1955 1960gcg cgg ggg tat aca gac cca gca cta gac gcg ggc cgc ttc gtc cag 5952Ala Arg Gly Tyr Thr Asp Pro Ala Leu Asp Ala Gly Arg Phe Val Gln1965 1970 1975 1980atc atg atc aat gac aag gcc gtg agg gcg tac cga acg ggt gac cgg 6000Ile Met Ile Asn Asp Lys Ala Val Arg Ala Tyr Arg Thr Gly Asp Arg
1985 1990 1995gca cga tat cgc gta gga gac ggt cag atc gag ttc ttc gga cgc atg 6048Ala Arg Tyr Arg Val Gly Asp Gly Gln Ile Glu Phe Phe Gly Arg Met
2000 2005 2010gat cag caa gtc aag atc cga ggt cac cgc att gaa cca gcc gaa gtg 6096Asp Gln Gln Val Lys Ile Arg Gly His Arg Ile Glu Pro Ala Glu Val
2015 2020 2025gag cgt gct att ctc gac caa gac tcg gcc cgc gac gcc gtc gtt gtc 6144Glu Arg Ala Ile Leu Asp Gln Asp Ser Ala Arg Asp Ala Val Val Val2030 2035 2040atc cgg cac caa gaa ggt gaa gaa ccg gag atg gtt ggt ttc gtc gcg 6192Ile Arg His Gln Glu Gly Glu Glu Pro Glu Met Val Gly Phe Val Ala2045 2050 2055 2060acc cac ggc gat cac tct gcc gaa caa gag gaa gca gac gac cag gtt 6240Thr His Gly Asp His Ser Ala Glu Gln Glu Glu Ala Asp Asp Gln Val
2065 2070 2075gaa ggt tgg aaa gac ttc ttc gag agc aat aca tat gcc gac atg gat 6288Glu Gly Trp Lys Asp Phe Phe Glu Ser Asn Thr Tyr Ala Asp Met Asp
2080 2085 2090acc atc ggc cag tct gct ata ggc aac gac ttt acg ggc tgg acg tcc 6336Thr Ile Gly Gln Ser Ala Ile Gly Asn Asp Phe Thr Gly Trp Thr Ser
2095 2100 2105atg tac gac ggg agc gag atc aac aag gcc gag atg cag gag tgg ctc 6384Met Tyr Asp Gly Ser Glu Ile Asn Lys Ala Glu Met Gln Glu Trp Leu2110 2115 2120gac gac acc atg cgc aca ctc ctc gat ggc caa gcg ccg ggt cac gta 6432Asp Asp Thr Met Arg Thr Leu Leu Asp Gly Gln Ala Pro Gly His Val2125 2130 2135 2140ctc gaa ata ggc aca ggc agt ggc atg gta ttg ttt aac tta ggg gcc 6480Leu Glu Ile Gly Thr Gly Ser Gly Met Val Leu Phe Asn Leu Gly Ala
2145 2150 2155ggg cta caa agc tac gta ggt ctt gaa cca tct aga tct gca gcc acg 6528Gly Leu Gln Ser Tyr Val Gly Leu Glu Pro Ser Arg Ser Ala Ala Thr
2160 2165 2170ttt gtt acc aaa gcg atc aat tcc acc cca gct ctt gca gga aag gcc 6576Phe Val Thr Lys Ala Ile Asn Ser Thr Pro Ala Leu Ala Gly Lys Ala
2175 2180 2185gaa gtg cac gtc ggc aca gcg aca gac ata aac cga ctt cgt gga ctt 6624Glu Val His Val Gly Thr Ala Thr Asp Ile Asn Arg Leu Arg Gly Leu2190 2195 2200cgt ccc gat cta gtt gtg ctc aac tcg gta gtc cag tat ttc ccc acg 6672Arg Pro Asp Leu Val Val Leu Asn Ser Val Val Gln Tyr Phe Pro Thr2205 2210 2215 2220ccc gag tac cta cta gag gtc gtg gag agt ctc gtc cgg att ccg ggc 6720Pro Glu Tyr Leu Leu Glu Val Val Glu Ser Leu Val Arg Ile Pro Gly
2225 2230 2235gtc aag cgc gtg gtc ttc ggc gac ata cga tct cac gcc acg aac aga 6768Val Lys Arg Val Val Phe Gly Asp Ile Arg Ser His Ala Thr Asn Arg
2240 2245 2250cat ttt ctt gct gcc agg gcg ctg cat tcg ctg ggc tcc aag gcg acc 6816His Phe Leu Ala Ala Arg Ala Leu His Ser Leu Gly Ser Lys Ala Thr
2255 2260 2265aaa gat gct ata cgt caa aag atg acg gag atg gaa gag cgc gag gaa 6864Lys Asp Ala Ile Arg Gln Lys Met Thr Glu Met Glu Glu Arg Glu Glu2270 2275 2280gag ctg ctc gtc gac ccg gcc ttc ttc acg gcg ctg ctg cag ggc cag 6912Glu Leu Leu Val Asp Pro Ala Phe Phe Thr Ala Leu Leu Gln Gly Gln2285 2290 2295 2300ctt gcc gat cga atc aag cac gtc gag atc ctc ccg aag aac atg cgc 6960Leu Ala Asp Arg Ile Lys His Val Glu Ile Leu Pro Lys Asn Met Arg
2305 2310 2315gcc acg aac gag ctg agc gcg tac cgg tat aca gcc gtc att cac gta 7008Ala Thr Asn Glu Leu Ser Ala Tyr Arg Tyr Thr Ala Val Ile His Val
2320 2325 2330cgc ggc cca gag gaa cag tcg cgg ccc gtg tat ccg atc caa gtg aac 7056Arg Gly Pro Glu Glu Gln Ser Arg Pro Val Tyr Pro Ile Gln Val Asn
2335 2340 2345gac tgg atc gac ttt cag gcc tca cgc att gac cgc cgc gcc ctt ctc 7104Asp Trp Ile Asp Phe Gln Ala Ser Arg Ile Asp Arg Arg Ala Leu Leu2350 2355 2360cga ctc cta cag cgc tcg gca gac gcc gcg acc gtc gcc gtc agc aac 7152Arg Leu Leu Gln Arg Ser Ala Asp Ala Ala Thr Val Ala Val Ser Asn2365 2370 2375 2380atc ccc tac agc aag acg att gta gaa cgc cat gtc gtc gag tcc ctt 7200Ile Pro Tyr Ser Lys Thr Ile Val Glu Arg His Val Val Glu Ser Leu
2385 2390 2395gac aat aac aac agg gag aat acg cat aga gca cca gac ggc gcg gct 7248Asp Asn Asn Asn Arg Glu Asn Thr His Arg Ala Pro Asp Gly Ala Ala
2400 2405 2410tgg atc tcg gcc gtc cgc tcc aag gcc gag cgc tgc acg tcc ctc tcc 7296Trp Ile Ser Ala Val Arg Ser Lys Ala Glu Arg Cys Thr Ser Leu Ser
2415 2420 2425gtg acc gat ctt gtg cag ctc ggg gaa gaa gcc ggc ttt cgc gta gaa 7344Val Thr Asp Leu Val Gln Leu Gly Glu Glu Ala Gly Phe Arg Val Glu2430 2435 2440gtc agc gca gcg cgg cag tgg tct caa agc ggc gcg ctc gat gcc gtc 7392Val Ser Ala Ala Arg Gln Trp Ser Gln Ser Gly Ala Leu Asp Ala Val2445 2450 2455 2460ttt cac cgc tat aat ttg ccc act caa agc aat agt cgc gtt ctg att 7440Phe His Arg Tyr Asn Leu Pro Thr Gln Ser Asn Ser Arg Val Leu Ile
2465 2470 2475cag ttc cct acc gaa gat ggc cag acg cga aga tcc gcc act ctg aca 7488Gln Phe Pro Thr Glu Asp Gly Gln Thr Arg Arg Ser Ala Thr Leu Thr
2480 2485 2490aac cga cca cta cag cgt ctg cag agc cgt cgg ttc gca tca cag atc 7536Asn Arg Pro Leu Gln Arg Leu Gln Ser Arg Arg Phe Ala Ser Gln Ile
2495 2500 2505cgc gaa cag ctg aag gcc gtg ctc ccg tca tac atg atc ccc tcc cgc 7584Arg Glu Gln Leu Lys Ala Val Leu Pro Ser Tyr Met Ile Pro Ser Arg2510 2515 2520atc gtg gtc ata gac cag atg cct ctc aat gcc aat ggc aag gtc gac 7632Ile Val Val Ile Asp Gln Met Pro Leu Asn Ala Asn Gly Lys Val Asp2525 2530 2535 2540cgg aaa gaa ctt acc aga agg gcc caa atc gcg ccg aaa tct cag gcg 7680Arg Lys Glu Leu Thr Arg Arg Ala Gln Ile Ala Pro Lys Ser Gln Ala
2545 2550 2555gct ccc gcc aaa ccc gtc aaa cag gtc gat ccg ttc gtc aac ctg gaa 7728Ala Pro Ala Lys Pro Val Lys Gln Val Asp Pro Phe Val Asn Leu Glu
2560 2565 2570gcc att tta tgt gag gag ttc gcg gag gtg ctg ggc atg gaa gtc ggc 7776Ala Ile Leu Cys Glu Glu Phe Ala Glu Val Leu Gly Met Glu Val Gly
2575 2580 2585gtg aac gac cac ttc ttc caa cta ggc gga cac tct ctc ttg gcc acg 7824Val Asn Asp His Phe Phe Gln Leu Gly Gly His Ser Leu Leu Ala Thr2590 2595 2600aag ctc gtc gcg cgt ctc agt cgt cgg cta aac ggt cgt gtg tct gtg 7872Lys Leu Val Ala Arg Leu Ser Arg Arg Leu Asn Gly Arg Val Ser Val2605 2610 2615 2620agg gat gtg ttc gac cag cct gtg att tcc gac ctc gca gtc acc ctc 7920Arg Asp Val Phe Asp Gln Pro Val Ile Ser Asp Leu Ala Val Thr Leu
2625 2630 2635cgc cag gga ctg acc ttg gaa aac gcc att ccc gca acg ccg gac agc 7968Arg Gln Gly Leu Thr Leu Glu Asn Ala Ile Pro Ala Thr Pro Asp Ser
2640 2645 2650ggg tat tgg gag cag aca atg tcc gca ccg aca acc ccg agc gac gac 8016Gly Tyr Trp Glu Gln Thr Met Ser Ala Pro Thr Thr Pro Ser Asp Asp
2655 2660 2665atg gag gcc gtg cta tgc aag gag ttt gcg gat gtg ctt ggc gtc gaa 8064Met Glu Ala Val Leu Cys Lys Glu Phe Ala Asp Val Leu Gly Val Glu2670 2675 2680gtc agc gcc acc gac agc ttc ttc gat ctc ggt ggg cat tcc ctc atg 8112Val Ser Ala Thr Asp Ser Phe Phe Asp Leu Gly Gly His Ser Leu Met2685 2690 2695 2700gct acg aag ctc gct gcg cgt att agc cgt cgg cta gat gta ccg gtg 8160Ala Thr Lys Leu Ala Ala Arg Ile Ser Arg Arg Leu Asp Val Pro Val
2705 2710 2715tca atc aaa gac ata ttc gat cac tca gtt cct cta aac ctt gcg agg 8208Ser Ile Lys Asp Ile Phe Asp His Ser Val Pro Leu Asn Leu Ala Arg
2720 2725 2730aag att cgg ctc act caa gca aaa ggc cac gaa gcg acc aat gga gta 8256Lys Ile Arg Leu Thr Gln Ala Lys Gly His Glu Ala Thr Asn Gly Val
2735 2740 2745caa atc gcc aac gac gcc cca ttc caa ctc att tcc gta gaa gat cca 8304Gln Ile Ala Asn Asp Ala Pro Phe Gln Leu Ile Ser Val Glu Asp Pro2750 2755 2760gag ata ttc gtc caa cgt gaa atc gcc cct caa cta caa tgc tca ccc 8352Glu Ile Phe Val Gln Arg Glu Ile Ala Pro Gln Leu Gln Cys Ser Pro2765 2770 2775 2780gag aca atc cta gac gtc tac ccc gcc acg caa atg caa agg gtc ttc 8400Glu Thr Ile Leu Asp Val Tyr Pro Ala Thr Gln Met Gln Arg Val Phe
2785 2790 2795ctc ctc aac cca gta aca gga aag ccg cgc tca cca acg cca ttt cac 8448Leu Leu Asn Pro Val Thr Gly Lys Pro Arg Ser Pro Thr Pro Phe His
2800 2805 2810ata gac ttc ccg ccg gac gca gac tgc gcc agc ctg atg cgg gca tgt 8496Ile Asp Phe Pro Pro Asp Ala Asp Cys Ala Ser Leu Met Arg Ala Cys
2815 2820 2825gca tct cta gcg aag cat ttc gat atc ttc agg acg gtg ttc ctc gaa 8544Ala Ser Leu Ala Lys His Phe Asp Ile Phe Arg Thr Val Phe Leu Glu2830 2835 2840gcc aga ggc gaa ctc tac caa gta gtt cta aaa cac gtc gac gtg ccc 8592Ala Arg Gly Glu Leu Tyr Gln Val Val Leu Lys His Val Asp Val Pro2845 2850 2855 2860atc gag atg ctc cag acc gaa gaa aac atc aac agc gcg acc cgg tcg 8640Ile Glu Met Leu Gln Thr Glu Glu Asn Ile Asn Ser Ala Thr Arg Ser
2865 2870 2875ttc ctg gac gta gac gca gaa aaa ccc atc cgg cta ggc cag cca ctg 8688Phe Leu Asp Val Asp Ala Glu Lys Pro Ile Arg Leu Gly Gln Pro Leu
2880 2885 2890atc cgc atc gcg ata cta gag aag ccc ggg tcc acg ctg cgc gtc atc 8736Ile Arg Ile Ala Ile Leu Glu Lys Pro Gly Ser Thr Leu Arg Val Ile
2895 2900 2905cta cga tta tcc cac gcc tta tac gac ggc ctg agc cta gag cac atc 8784Leu Arg Leu Ser His Ala Leu Tyr Asp Gly Leu Ser Leu Glu His Ile2910 2915 2920ctg cac tct ctg cac atc ctc ttt ttc ggc ggc agt ctt ccc ccg ccg 8832Leu His Ser Leu His Ile Leu Phe Phe Gly Gly Ser Leu Pro Pro Pro2925 2930 2935 2940ccc aag ttc gcc ggg tac atg caa cac gtc gcg agc agt cgc aga gaa 8880Pro Lys Phe Ala Gly Tyr Met Gln His Val Ala Ser Ser Arg Arg Glu
2945 2950 2955ggc tac gat ttc tgg cgt tcc gtt ctc cga gat tcg tct atg aca gtc 8928Gly Tyr Asp Phe Trp Arg Ser Val Leu Arg Asp Ser Ser Met Thr Val
2960 2965 2970atc aaa ggc aac aat aat aca act cca cca cct cct cct caa caa caa 8976Ile Lys Gly Asn Asn Asn Thr Thr Pro Pro Pro Pro Pro Gln Gln Gln
2975 2980 2985tcc acc ccc tcc gga gcc cac cac gcc tcc aaa gta gtc act atc cca 9024Ser Thr Pro Ser Gly Ala His His Ala Ser Lys Val Val Thr Ile Pro2990 2995 3000acc caa gcc aac aca gac agc cgg atc acg cgc gcc acg atc ttc act 9072Thr Gln Ala Asn Thr Asp Ser Arg Ile Thr Arg Ala Thr Ile Phe Thr3005 3010 3015 3020acc gct tgc gca cta atg ctc gcg aaa gaa gac aac tcc agc gac gtc 9120Thr Ala Cys Ala Leu Met Leu Ala Lys Glu Asp Asn Ser Ser Asp Val
3025 3030 3035gtc ttc ggg cgt acg gta tcg ggg cgt caa ggc ctg ccc cta gcc cac 9168Val Phe Gly Arg Thr Val Ser Gly Arg Gln Gly Leu Pro Leu Ala His
3040 3045 3050caa aac gtg atc gga cca tgt ctc aac caa gtg ccc gtg cgc gcg cgc 9216Gln Asn Val Ile Gly Pro Cys Leu Asn Gln Val Pro Val Arg Ala Arg
3055 3060 3065ggt tta aac cga gga acc act cac cac cga gaa ctt ctc cgc gag atg 9264Gly Leu Asn Arg Gly Thr Thr His His Arg Glu Leu Leu Arg Glu Met3070 3075 3080caa gag caa tat ctc aac agt ctc gct ttc gaa act ctc ggg tac gac 9312Gln Glu Gln Tyr Leu Asn Ser Leu Ala Phe Glu Thr Leu Gly Tyr Asp3085 3090 3095 3100gag atc aag gcg cac tgc aca gac tgg ccg gac gtg cca gcg acc gcg 9360Glu Ile Lys Ala His Cys Thr Asp Trp Pro Asp Val Pro Ala Thr Ala
3105 3110 3115agc ttc ggg tgc tgc atc gtg tac cag aac ttc gat tcg cac ccg gac 9408Ser Phe Gly Cys Cys Ile Val Tyr Gln Asn Phe Asp Ser His Pro Asp
3120 3125 3130agc cga gtc gaa gag cag cgg ctg cag atc ggg gtc ttg tcg cgg aac 9456Ser Arg Val Glu Glu Gln Arg Leu Gln Ile Gly Val Leu Ser Arg Asn
3135 3140 3145tac gag gct att aat gag ggg ctc gtg cat gat ctt gtt att gct ggg 9504Tyr Glu Ala Ile Asn Glu Gly Leu Val His Asp Leu Val Ile Ala Gly3150 3155 3160gag tcg gag cct gat ggg gat gat ttg agg gtt act gtt gtg gcg aat 9552Glu Ser Glu Pro Asp Gly Asp Asp Leu Arg Val Thr Val Val Ala Asn3165 3170 3175 3180cgg agg ttg tgc gat gag gaa agg ttg aag agg atg ctg gag gag ctg 9600Arg Arg Leu Cys Asp Glu Glu Arg Leu Lys Arg Met Leu Glu Glu Leu
3185 3190 3195tgt ggg aat att cgg gct ttg gcg ttg gtt tag 9633Cys Gly Asn Ile Arg Ala Leu Ala Leu Val
3200 3205<210>2<211>3210<212>PRT<213>无孢菌类(Mycelia sterilia)<400>2Met Ser Asn Met Ala Pro Leu Pro Thr Met Gly Val Glu Gln Gln Ala1 5 10 15Leu Ser Leu Ser Cys Pro Leu Leu Pro His Asp Asp Glu Lys His Ser
20 25 30Asp Asn Leu Tyr Glu Gln Ala Thr Arg His Phe Gly Leu Ser Arg Asp
35 40 45Lys Ile Glu Asn Val Leu Pro Cys Thr Ser Phe Gln Cys Asp Val Ile
50 55 60Asp Cys Ala Val Asp Asp Arg Arg His Ala Ile Gly His Val Val Tyr65 70 75 80Asp Ile Pro Asn Thr Val Asp Ile Gln Arg Leu Ala Ala Ala Trp Lys
85 90 95Glu Val Val Arg Gln Thr Pro Ile Leu Arg Thr Gly Ile Phe Thr Ser
100 105 110Glu Thr Gly Asp Ser Phe Gln Ile Val Leu Lys Glu Gly Cys Leu Pro
115 120 125Trp Met Tyr Ala Thr Cys Leu Gly Met Lys Gly Ala Val Ile Gln Asp
130 135 140Glu Ala Val Ala Ala Met Thr Gly Pro Arg Cys Asn Arg Tyr Val Val145 150 155 160Leu Glu Asp Pro Ser Thr Lys Gln Arg Leu Leu Ile Trp Thr Phe Ser
165 170 175His Ala Leu Val Asp Tyr Thr Val Gln Glu Arg Ile Leu Gln Arg Val
180 185 190Leu Thr Val Tyr Asp Gly Arg Asp Val Glu Cys Pro Arg Ile Lys Asp
195 200 205Thr Glu His Val Ser Arg Phe Trp Gln Gln His Phe Glu Gly Leu Asp
210 215 220Ala Ser Val Phe Pro Leu Leu Pro Ser His Leu Thr Val Cys Asn Pro225 230 235 240Asn Ala Arg Ala Glu His His Ile Ser Tyr Thr Gly Pro Val Gln Arg
245 250 255Lys Trp Ser His Thr Ser Ile Cys Arg Ala Ala Leu Ala Val Leu Leu
260 265 270Ser Arg Phe Thr His Ser Ser Glu Ala Leu Phe Gly Val Val Thr Glu
275 280 285Gln Ser His Asn Ser Glu Asp Gln Arg Arg Ser Ile Asp Gly Pro Ala
290 295 300Arg Thr Val Val Pro Ile Arg Val Leu Cys Ala Pro Asp Gln Tyr Val305 310 315 320Ser Asp Val Ile Gly Ala Ile Thr Ala His Glu His Ala Met Arg Gly
325 330 335Phe Glu His Ala Gly Leu Arg Asn Ile Arg Arg Thr Gly Asp Asp Gly
340 345 350Ser Ala Ala Cys Gly Phe Gln Thr Val Leu Leu Val Thr Asp Gly Asp
355 360 365Ala Pro Lys Thr Pro Gly Ser Val Leu His Arg Ser Val Glu Glu Ser
370 375 380Asp Arg Phe Met Pro Cys Ala Asn Arg Ala Leu Leu Leu Asp Cys Gln385 390 395 400Met Ala Gly Asn Ser Ala Ser Leu Val Ala Arg Tyr Asp His Asn Val
405 410 415Ile Asp Pro Arg Gln Met Ser Arg Phe Leu Arg Gln Leu Gly Tyr Leu
420 425 430Ile Gln Gln Phe His His His Val Asp Leu Pro Leu Val Lys Glu Leu
435 440 445Asp Val Val Thr Ala Glu Asp Cys Ala Glu Ile Glu Lys Trp Asn Ser
450 455 460Glu Arg Leu Thr Met Gln Asp Ala Leu Ile His Asp Thr Ile Ser Lys465 470 475 480Trp Ala Ala Gly Asp Pro Asn Lys Ala Ala Val Phe Ala Trp Asp Gly
485 490 495Glu Trp Thr Tyr Ala Glu Leu Asp Asn Ile Ser Ser Arg Leu Ala Val
500 505 510Tyr Ile Gln Ser Leu Asp Leu Arg Pro Gly Gln Ala Ile Leu Pro Leu
515 520 525Cys Phe Glu Lys Ser Lys Trp Val Val Ala Thr Ile Leu Ala Val Leu
530 535 540Lys Val Gly Arg Ala Phe Thr Leu Ile Asp Pro Cys Asp Pro Ser Ala545 550 555 560Arg Met Ala Gln Val Cys Gln Gln Thr Ser Ala Thr Val Ala Leu Thr
565 570 575Ser Lys Leu His Asn Thr Thr Leu Arg Ser Val Val Ser Arg Cys Ile
580 585 590Val Val Asp Asp Asp Leu Leu Arg Ser Leu Pro His Ala Asp Gly Arg
595 600 605Leu Lys Ala Thr Val Lys Pro Gln Asp Leu Ala Tyr Val Ile Phe Thr
610 615 620Ser Gly Ser Thr Gly Glu Pro Lys Gly Ile Met Ile Glu His Arg Gly625 630 635 640Phe Val Ser Cys Ala Met Lys Phe Gly Pro Ala Leu Gly Met Asp Glu
645 650 655His Thr Arg Ala Leu Gln Phe Ala Ser Tyr Ala Phe Gly Ala Cys Leu
660 665 670Val Glu Val Val Thr Ala Leu Met His Gly Gly Cys Val Cys Ile Pro
675 680 685Ser Asp Asp Asp Arg Leu Asn Asn Val Pro Glu Phe Ile Lys Arg Ala
690 695 700Gln Val Asn Trp Val Ile Leu Thr Pro Ser Tyr Ile Gly Thr Phe Gln705 710 715 720Pro Glu Asp Val Pro Gly Leu Gln Thr Leu Val Leu Val Gly Glu Pro
725 730 735Ile Ser Ala Ser Ile Arg Asp Thr Trp Ala Ser Gln Val Arg Leu Leu
740 745 750Asn Ala Tyr Gly Gln Ser Glu Ser Ser Thr Met Cys Ser Val Thr Glu
755 760 765Val Ser Pro Leu Ser Leu Glu Pro Asn Asn Ile Gly Arg Ala Val Gly
770 775 780Ala Arg Ser Trp Ile Ile Asp Pro Asp Glu Pro Asp Arg Leu Ala Pro785 790 795 800Ile Gly Cys Ile Gly Glu Leu Val Ile Glu Ser Pro Gly Ile Ala Arg
805 810 815Asp Tyr Ile Ile Ala Pro Pro Pro Asp Lys Ser Pro Phe Leu Leu Ala
820 825 830Pro Pro Ala Trp Tyr Pro Ala Gly Lys Leu Ser Asn Ala Phe Lys Phe
835 840 845Tyr Lys Thr Gly Asp Leu Val Arg Tyr Gly Pro Asp Gly Thr Ile Val
850 855 860Cys Leu Gly Arg Lys Asp Ser Gln Val Lys Ile Arg Gly Gln Arg Val865 870 875 880Glu Ile Ser Ala Val Glu Ala Ser Leu Arg Arg Gln Leu Pro Ser Asp
885 890 895Ile Met Pro Val Ala Glu Ala Ile Lys Arg Ser Asp Ser Ser Gly Ser
900 905 910Thr Val Leu Thr Ala Phe Leu Ile Gly Ser Ser Lys Ser Gly Asp Gly
915 920 925Asn Gly His Ala Leu Ser Ala Ala Asp Ala Val Ile Leu Asp His Gly
930 935 940Ala Thr Asn Glu Ile Asn Ala Lys Leu Gln Gln Ile Leu Pro Gln His945 950 955 960Ser Val Pro Ser Tyr Tyr Ile His Met Glu Asn Leu Pro Arg Thr Ala
965 970 975Thr Gly Lys Ala Asp Arg Lys Met Leu Arg Ser Ile Ala Ser Lys Leu
980 985 990Leu Gly Glu Leu Ser Gln Asn Val Thr Ser Gln Pro Ile Glu Lys His
995 1000 1005Asp Ala Pro Ala Thr Gly Ile Glu Val Lys Leu Lys Glu Leu Trp Phe1010 1015 1020Leu Ser Leu Asn Leu Asn Pro Asn Ser Gln Asp Val Gly Ala Ser Phe025 1030 1035 1040Phe Asp Leu Gly Gly Asn Ser Ile Ile Ala Ile Lys Met Val Asn Met
1045 1050 1055Ala Arg Ser Ala Gly Ile Ala Leu Lys Val Ser Asp Ile Phe Gln Asn
1060 1065 1070Pro Thr Leu Ala Gly Leu Val Asp Val Ile Gly Arg Asp Pro Ala Pro
1075 1080 1085Tyr Asn Leu Ile Pro Thr Thr Ala Tyr Ser Gly Pro Val Glu Gln Ser1090 1095 1100Phe Ala Gln Gly Arg Leu Trp Phe Leu Asp Gln Ile Glu Leu Asp Ala105 1110 1115 1120Leu Trp Tyr Leu Leu Pro Tyr Ala Val Arg Met Arg Gly Pro Leu His
1125 1130 1135Ile Asp Ala Leu Thr Ile Ala Leu Leu Ala Ile Gln Gln Arg His Glu
1140 1145 1150Thr Leu Arg Thr Thr Phe Glu Glu Gln Asp Gly Val Gly Val Gln Val
1155 1160 1165Val His Ala Ser Pro Ile Ser Asp Leu Arg Ile Ile Asp Val Ser Gly1170 1175 1180Asp Arg Asn Ser Asp Tyr Leu Gln Leu Leu His Gln Glu Gln Thr Thr185 1190 1195 1200Pro Phe Ile Leu Ala Cys Gln Ala Gly Trp Arg Val Ser Leu Ile Arg
1205 1210 1215Leu Gly Glu Asp Asp His Ile Leu Ser Ile Val Met His His Ile Ile
1220 1225 1230Ser Asp Gly Trp Ser Ile Asp Ile Leu Arg Arg Glu Leu Ser Asn Phe
1235 1240 1245Tyr Ser Ala Ala Leu Arg Gly Ser Asp Pro Leu Ser Val Val Ser Pro1250 1255 1260Leu Pro Leu His Tyr Arg Asp Phe Ser Val Trp Gln Lys Gln Val Glu265 1270 1275 1280Gln Glu Thr Glu His Glu Arg Gln Leu Glu Tyr Trp Val Lys Gln Leu
1285 1290 1295Ala Asp Ser Ser Ala Ala Glu Phe Leu Thr Asp Phe Pro Arg Pro Asn
1300 1305 1310Ile Leu Ser Gly Glu Ala Gly Ser Val Pro Val Thr Ile Glu Gly Glu
1315 1320 1325Leu Tyr Glu Arg Leu Gln Glu Phe Cys Lys Val Glu Gln Met Thr Pro1330 1335 1340Phe Ala Val Leu Leu Gly Ala Phe Arg Ala Thr His Tyr Arg Leu Thr345 1350 1355 1360Gly Ala Glu Asp Ser Ile Ile Gly Thr Pro Ile Ala Asn Arg Asn Arg
1365 1370 1375Gln Glu Leu Glu Asn Met Ile Gly Phe Phe Val Asn Thr Gln Cys Met
1380 1385 1390Arg Ile Thr Val Asp Gly Asp Asp Thr Phe Glu Ser Leu Val Arg Gln
1395 1400 1405Val Arg Thr Thr Ala Thr Ala Ala Phe Glu His Gln Asp Val Pro Phe1410 1415 1420Glu Arg Val Val Thr Ala Leu Leu Pro Arg Ser Arg Asp Leu Ser Arg425 1430 1435 1440Asn Pro Leu Ala Gln Leu Thr Phe Ala Leu His Ser Gln Gln Asp Leu
1445 1450 1455Gly Lys Phe Glu Leu Glu Gly Leu Val Ala Glu Pro Val Ser Asn Lys
1460 1465 1470Val Tyr Thr Arg Phe Asp Val Glu Phe His Leu Phe Gln Glu Ala Gly
1475 1480 1485Arg Leu Ser Gly Asn Val Ala Phe Ala Ala Asp Leu Phe Lys Pro Glu1490 1495 1500Thr Ile Ser Asn Val Val Ala Ile Phe Phe Gln Ile Leu Arg Gln Gly505 1510 1515 1520Ile Arg Gln Pro Arg Thr Pro Ile Ala Val Leu Pro Leu Thr Asp Gly
1525 1530 1535Leu Ala Asp Leu Arg Ala Met Gly Leu Leu Glu Ile Glu Lys Ala Glu
1540 1545 1550Tyr Pro Arg Glu Ser Ser Val Val Asp Val Phe Arg Lys Gln Val Ala
1555 1560 1565Ala His Pro His Ala Phe Ala Val Val Asp Ser Ala Ser Arg Leu Thr1570 1575 1580Tyr Ala Asp Leu Asp Arg Gln Ser Asp Gln Leu Ala Thr Trp Leu Gly585 1590 1595 1600Arg Arg Asn Met Thr Ala Glu Thr Leu Val Gly Val Leu Ala Pro Arg
1605 1610 1615Ser Cys Gln Thr Val Val Ala Ile Leu Gly Ile Leu Lys Ala Asn Leu
1620 1625 1630Ala Tyr Leu Pro Leu Asp Val Asn Cys Pro Thr Ala Arg Leu Gln Thr
1635 1640 1645Ile Leu Ser Thr Leu Asn Arg His Lys Leu Val Leu Leu Gly Ser Asn1650 1655 1660Ala Thr Thr Pro Asp Val Gln Ile Pro Asp Val Glu Leu Val Arg Ile665 1670 1675 1680Ser Asp Ile Leu Asp Arg Pro Ile Asn Gly Gln Ala Lys Leu Asn Gly
1685 1690 1695His Thr Lys Ser Asn Gly Tyr Ser Lys Pro Asn Gly Tyr Thr His Leu
1700 1705 1710Lys Gly Tyr Ser Asn Leu Asn Gly Tyr Ser Lys Gln Asn Gly Tyr Ala
1715 1720 1725Gln Leu Asn Gly His Arg Glu Arg Asn Asn Tyr Leu Asp Leu Asn Gly1730 1735 1740His Ser Leu Leu Asn Gly Asn Ser Asp Ile Thr Thr Ser Gly Pro Ser745 1750 1755 1760Ala Thr Ser Leu Ala Tyr Val Ile Phe Thr Ser Gly Ser Thr Gly Lys
1765 1770 1775Pro Lys Gly Val Met Val Glu His Arg Ser Ile Ile Arg Leu Ala Lys
1780 1785 1790Lys Asn Arg Ile Ile Ser Arg Phe Pro Ser Val Ala Lys Val Ala His
1795 1800 1805Leu Ser Asn Ile Ala Phe Asp Ala Ala Thr Trp Glu Met Phe Ala Ala1810 1815 1820Leu Leu Asn Gly Gly Thr Leu Val Cys Ile Asp Tyr Met Thr Thr Leu825 1830 1835 1840Asp Ser Lys Thr Leu Glu Ala Ala Phe Ala Arg Glu Gln Ile Asn Ala
1845 1850 1855Ala Leu Leu Thr Pro Ala Leu Leu Lys Gln Cys Leu Ala Asn Ile Pro
1860 1865 1870Thr Thr Leu Gly Arg Leu Ser Ala Leu Val Ile Gly Gly Asp Arg Leu
1875 1880 1885Asp Gly Gln Asp Ala Ile Ala Ala His Ala Leu Val Gly Ala Gly Val1890 1895 1900Tyr Asn Ala Tyr Gly Pro Thr Glu Asn Gly Val Ile Ser Thr Ile Tyr905 1910 1915 1920Asn Ile Thr Lys Asn Asp Ser Phe Ile Asn Gly Val Pro Ile Gly Cys
1925 1930 1935Ala Ile Ser Asn Ser Gly Ala Tyr Ile Thr Asp Pro Asp Gln Gln Leu
1940 1945 1950Val Pro Pro Gly Val Met Gly Glu Leu Val Val Thr Gly Asp Gly Leu
1955 1960 1965Ala Arg Gly Tyr Thr Asp Pro Ala Leu Asp Ala Gly Arg Phe Val Gln1970 1975 1980Ile Met Ile Asn Asp Lys Ala Val Arg Ala Tyr Arg Thr Gly Asp Arg985 1990 1995 2000Ala Arg Tyr Arg Val Gly Asp Gly Gln Ile Glu Phe Phe Gly Arg Met
2005 2010 2015Asp Gln Gln Val Lys Ile Arg Gly His Arg Ile Glu Pro Ala Glu Val
2020 2025 2030Glu Arg Ala Ile Leu Asp Gln Asp Ser Ala Arg Asp Ala Val Val Val
2035 2040 2045Ile Arg His Gln Glu Gly Glu Glu Pro Glu Met Val Gly Phe Val Ala2050 2055 2060Thr His Gly Asp His Ser Ala Glu Gln Glu Glu Ala Asp Asp Gln Val065 2070 2075 2080Glu Gly Trp Lys Asp Phe Phe Glu Ser Asn Thr Tyr Ala Asp Met Asp
2085 2090 2095Thr Ile Gly Gln Ser Ala Ile Gly Asn Asp Phe Thr Gly Trp Thr Ser
2100 2105 2110Met Tyr Asp Gly Ser Glu Ile Asn Lys Ala Glu Met Gln Glu Trp Leu
2115 2120 2125Asp Asp Thr Met Arg Thr Leu Leu Asp Gly Gln Ala Pro Gly His Val2130 2135 2140Leu Glu Ile Gly Thr Gly Ser Gly Met Val Leu Phe Asn Leu Gly Ala145 2150 2155 2160Gly Leu Gln Ser Tyr Val Gly Leu Glu Pro Ser Arg Ser Ala Ala Thr
2165 2170 2175Phe Val Thr Lys Ala Ile Asn Ser Thr Pro Ala Leu Ala Gly Lys Ala
2180 2185 2190Glu Val His Val Gly Thr Ala Thr Asp Ile Asn Arg Leu Arg Gly Leu
2195 2200 2205Arg Pro Asp Leu Val Val Leu Asn Ser Val Val Gln Tyr Phe Pro Thr2210 2215 2220Pro Glu Tyr Leu Leu Glu Val Val Glu Ser Leu Val Arg Ile Pro Gly225 2230 2235 2240Val Lys Arg Val Val Phe Gly Asp Ile Arg Ser His Ala Thr Asn Arg
2245 2250 2255His Phe Leu Ala Ala Arg Ala Leu His Ser Leu Gly Ser Lys Ala Thr
2260 2265 2270Lys Asp Ala Ile Arg Gln Lys Met Thr Glu Met Glu Glu Arg Glu Glu
2275 2280 2285Glu Leu Leu Val Asp Pro Ala Phe Phe Thr Ala Leu Leu Gln Gly Gln2290 2295 2300Leu Ala Asp Arg Ile Lys His Val Glu Ile Leu Pro Lys Asn Met Arg305 2310 2315 2320Ala Thr Asn Glu Leu Ser Ala Tyr Arg Tyr Thr Ala Val Ile His Val
2325 2330 2335Arg Gly Pro Glu Glu Gln Ser Arg Pro Val Tyr Pro Ile Gln Val Asn
2340 2345 2350Asp Trp Ile Asp Phe Gln Ala Ser Arg Ile Asp Arg Arg Ala Leu Leu
2355 2360 2365Arg Leu Leu Gln Arg Ser Ala Asp Ala Ala Thr Val Ala Val Ser Asn2370 2375 2380Ile Pro Tyr Ser Lys Thr Ile Val Glu Arg His Val Val Glu Ser Leu385 2390 2395 2400Asp Asn Asn Asn Arg Glu Asn Thr His Arg Ala Pro Asp Gly Ala Ala
2405 2410 2415Trp Ile Ser Ala Val Arg Ser Lys Ala Glu Arg Cys Thr Ser Leu Ser
2420 2425 2430Val Thr Asp Leu Val Gln Leu Gly Glu Glu Ala Gly Phe Arg Val Glu
2435 2440 2445Val Ser Ala Ala Arg Gln Trp Ser Gln Ser Gly Ala Leu Asp Ala Val2450 2455 2460Phe His Arg Tyr Asn Leu Pro Thr Gln Ser Asn Ser Arg Val Leu Ile465 2470 2475 2480Gln Phe Pro Thr Glu Asp Gly Gln Thr Arg Arg Ser Ala Thr Leu Thr
2485 2490 2495Asn Arg Pro Leu Gln Arg Leu Gln Ser Arg Arg Phe Ala Ser Gln Ile
2500 2505 2510Arg Glu Gln Leu Lys Ala Val Leu Pro Ser Tyr Met Ile Pro Ser Arg
2515 2520 2525Ile Val Val Ile Asp Gln Met Pro Leu Asn Ala Asn Gly Lys Val Asp2530 2535 2540Arg Lys Glu Leu Thr Arg Arg Ala Gln Ile Ala Pro Lys Ser Gln Ala545 2550 2555 2560Ala Pro Ala Lys Pro Val Lys Gln Val Asp Pro Phe Val Asn Leu Glu
2565 2570 2575Ala Ile Leu Cys Glu Glu Phe Ala Glu Val Leu Gly Met Glu Val Gly
2580 2585 2590Val Asn Asp His Phe Phe Gln Leu Gly Gly His Ser Leu Leu Ala Thr
2595 2600 2605Lys Leu Val Ala Arg Leu Ser Arg Arg Leu Asn Gly Arg Val Ser Val2610 2615 2620Arg Asp Val Phe Asp Gln Pro Val Ile Ser Asp Leu Ala Val Thr Leu625 2630 2635 2640Arg Gln Gly Leu Thr Leu Glu Asn Ala Ile Pro Ala Thr Pro Asp Ser
2645 2650 2655Gly Tyr Trp Glu Gln Thr Met Ser Ala Pro Thr Thr Pro Ser Asp Asp
2660 2665 2670Met Glu Ala Val Leu Cys Lys Glu Phe Ala Asp Val Leu Gly Val Glu
2675 2680 2685Val Ser Ala Thr Asp Ser Phe Phe Asp Leu Gly Gly His Ser Leu Met2690 2695 2700Ala Thr Lys Leu Ala Ala Arg Ile Ser Arg Arg Leu Asp Val Pro Val705 2710 2715 2720Ser Ile Lys Asp Ile Phe Asp His Ser Val Pro Leu Asn Leu Ala Arg
2725 2730 2735Lys Ile Arg Leu Thr Gln Ala Lys Gly His Glu Ala Thr Asn Gly Val
2740 2745 2750Gln Ile Ala Asn Asp Ala Pro Phe Gln Leu Ile Ser Val Glu Asp Pro
2755 2760 2765Glu Ile Phe Val Gln Arg Glu Ile Ala Pro Gln Leu Gln Cys Ser Pro2770 2775 2780Glu Thr Ile Leu Asp Val Tyr Pro Ala Thr Gln Met Gln Arg Val Phe785 2790 2795 2800Leu Leu Asn Pro Val Thr Gly Lys Pro Arg Ser Pro Thr Pro Phe His
2805 2810 2815Ile Asp Phe Pro Pro Asp Ala Asp Cys Ala Ser Leu Met Arg Ala Cys
2820 2825 2830Ala Ser Leu Ala Lys His Phe Asp Ile Phe Arg Thr Val Phe Leu Glu
2835 2840 2845Ala Arg Gly Glu Leu Tyr Gln Val Val Leu Lys His Val Asp Val Pro2850 2855 2860Ile Glu Met Leu Gln Thr Glu Glu Asn Ile Asn Ser Ala Thr Arg Ser865 2870 2875 2880Phe Leu Asp Val Asp Ala Glu Lys Pro Ile Arg Leu Gly Gln Pro Leu
2885 2890 2895Ile Arg Ile Ala Ile Leu Glu Lys Pro Gly Ser Thr Leu Arg Val Ile
2900 2905 2910Leu Arg Leu Ser His Ala Leu Tyr Asp Gly Leu Ser Leu Glu His Ile
2915 2920 2925Leu His Ser Leu His Ile Leu Phe Phe Gly Gly Ser Leu Pro Pro Pro2930 2935 2940Pro Lys Phe Ala Gly Tyr Met Gln His Val Ala Ser Ser Arg Arg Glu945 2950 2955 2960Gly Tyr Asp Phe Trp Arg Ser Val Leu Arg Asp Ser Ser Met Thr Val
2965 2970 2975Ile Lys Gly Asn Asn Asn Thr Thr Pro Pro Pro Pro Pro Gln Gln Gln
2980 2985 2990Ser Thr Pro Ser Gly Ala His His Ala Ser Lys Val Val Thr Ile Pro
2995 3000 3005Thr Gln Ala Asn Thr Asp Ser Arg Ile Thr Arg Ala Thr Ile Phe Thr3010 3015 3020Thr Ala Cys Ala Leu Met Leu Ala Lys Glu Asp Asn Ser Ser Asp Val025 3030 3035 3040Val Phe Gly Arg Thr Val Ser Gly Arg Gln Gly Leu Pro Leu Ala His
3045 3050 3055Gln Asn Val Ile Gly Pro Cys Leu Asn Gln Val Pro Val Arg Ala Arg
3060 3065 3070Gly Leu Asn Arg Gly Thr Thr His His Arg Glu Leu Leu Arg Glu Met
3075 3080 3085Gln Glu Gln Tyr Leu Asn Ser Leu Ala Phe Glu Thr Leu Gly Tyr Asp3090 3095 3100Glu Ile Lys Ala His Cys Thr Asp Trp Pro Asp Val Pro Ala Thr Ala105 3110 3115 3120Ser Phe Gly Cys Cys Ile Val Tyr Gln Asn Phe Asp Ser His Pro Asp
3125 3130 3135Ser Arg Val Glu Glu Gln Arg Leu Gln Ile Gly Val Leu Ser Arg Asn
3140 3145 3150Tyr Glu Ala Ile Asn Glu Gly Leu Val His Asp Leu Val Ile Ala Gly
3155 3160 3165Glu Ser Glu Pro Asp Gly Asp Asp Leu Arg Val Thr Val Val Ala Asn3170 3175 3180Arg Arg Leu Cys Asp Glu Glu Arg Leu Lys Arg Met Leu Glu Glu Leu185 3190 3195 3200Cys Gly Asn Ile Arg Ala Leu Ala Leu Val
3205 3210<210>3<211>7<212>PRT<213>人工序列(Artificial Sequence)<220><223>人工序列的描述:共有序列<400>3Trp Thr Ser Met Tyr Asp Gly1 5<210>4<211>7<212>PRT<213>人工序列(Artificial Sequence)<220><223>人工序列的描述:共有序列<400>4Val Val Gln Tyr Phe Pro Thr1 5<210>5<211>20<212>DNA<213>人工序列(Artificial Sequence)<220><223>人工序列的描述:用于扩增环状缩肽合成酶基因的引物<400>5tggacnwsna tgtaygaygg 20<210>6<211>20<212>DNA<213>人工序列(Artificial Sequence)<220><223>人工序列的描述:用于扩增环状缩肽合成酶基因的引物<400>6gtnggraart aytgnacnac 20<210>7<211>30<212>DNA<213>人工序列(Artificial Sequence)<220><223>人工序列的描述:用于扩增环状缩肽合成酶基因的引物<400>7gcggaattaa ccctcactaa agggaacgaa 30<210>8<211>30<212>DNA<213>人工序列(Artificial Sequence)<220><223>人工序列的描述:用于扩增环状缩肽合成酶基因的引物<400>8gcgtaatacg actcactata gggcgaagaa 30<210>9<211>41<212>DNA<213>人工序列(Artificial Sequence)<220><223>人工序列的描述:用于扩增环状缩肽合成酶基因的引物<400>9agcatcggat cctaacaatg ggcgttgagc agcaagccct a 41<210>10<211>21<212>DNA<213>人工序列(Artificial Sequence)<220><223>人工序列的描述:用于扩增环状缩肽合成酶基因的引物<400>10tttgcttcgt actcgggtcc t 21<210>11<211>42<212>DNA<213>人工序列(Artificial Sequence)<220><223>人工序列的描述:用于扩增环状缩肽合成酶基因的引物<400>11agcatcggat cctaacaatg tcaaacatgg caccactccc ta 42<210>12<211>20<212>DNA<213>人工序列(Artificial Sequence)<220><223>人工序列的描述:用于扩增环状缩肽合成酶基因的引物<400>12gcatcgcgat actagagaag 20<210>13<211>42<212>DNA<213>人工序列(Artificial Sequence)<220><223>人工序列的描述:用于扩增环状缩肽合成酶基因的引物<400>13agcatcgaat tcggatccct aaaccaacgc caaagcccga at 42<210>14<211>20<212>DNA<213>人工序列(Artificial Sequence)<220><223>人工序列的描述:用于扩增Abp1基因的引物<400>14ctcaaaccag gaactctttc 20<210>15<211>22<212>DNA<213>人工序列(Artificial Sequence)<220><223>人工序列的描述:用于扩增Abp1基因的引物<400>15gacatgtgga aaccacattt tg 22<210>16<211>29<212>DNA<213>人工序列(Artificial Sequence)<220><223>人工序列的描述:用于扩增Abp1基因的引物<400>16ggggaattcg tgggtggtga tatcatggc 29<210>17<211>24<212>DNA<213>人工序列(Artificial Sequence)<220><223>人工序列的描述:用于扩增Abp1基因的引物<400>17gggggatcct tgatgggttt tggg 24<210>18<211>27<212>DNA<213>人工序列(Artificial Sequence)<220><223>人工序列的描述:用于扩增Abp1基因的引物<400>18gggggatcct aaactcccat ctatagc 27<210>19<211>29<212>DNA<213>人工序列(Artificial Sequence)<220><223>人工序列的描述:用于扩增Abp1基因的引物<400>19gggtctagac gactcattgc agtgagtgg 29<210>20<211>20<212>DNA<213>人工序列(Artificial Sequence)<220><223>人工序列的描述:用于扩增Abp1基因启动子的引物<400>20tgatatgctg gagcttccct 20<210>21<211>20<212>DNA<213>人工序列(Artificial Sequence)<220><223>人工序列的描述:用于扩增环状缩肽合成酶基因的引物<400>21gcacaacctc tttccaggct 20
Claims (14)
1.一种蛋白质,它含有选自下列序列构成的组的氨基酸序列,
(a)序列号2的氨基酸序列,以及
(b)序列号2的氨基酸序列的修饰氨基酸序列,该修饰序列具有1个以上选自置换、缺失、添加和插入的修饰,而且具有环状缩肽合成酶活性。
2.一种多核苷酸,它编码权利要求1所述的蛋白质。
3.权利要求2所述的多核苷酸,它包括序列号1的DNA序列。
4.一种多核苷酸,它选自下列序列构成的组,
(c)序列号1的DNA序列,
(d)与序列号1的DNA序列至少具有70%的同源性,而且编码具有环状缩肽合成酶活性的蛋白质的核苷酸序列,
(e)序列号1的DNA序列的修饰DNA序列,该修饰序列具有1个以上选自置换、缺失、添加和插入的修饰,而且编码具有环状缩肽合成酶活性的蛋白质,
(f)与序列号1的DNA序列在严格条件下杂交,而且编码具有环状缩肽合成酶活性的蛋白质的核苷酸序列。
5.权利要求4所述的多核苷酸,其中,(d)是与序列号1的DNA序列至少具有80%同源性的核苷酸序列。
6.权利要求4所述的多核苷酸,其中,(d)是与序列号1的DNA序列至少具有90%同源性的核苷酸序列。
7.一种重组载体,它含有权利要求2~6任一项所述的多核苷酸。
8.一种宿主,它含有权利要求7所述的重组载体。
9.权利要求8所述的宿主,它可表达环状缩肽合成酶。
10.权利要求8或9所述的宿主,该宿主为PF1022物质生产菌。
11.一种制造环状缩肽的方法,该方法包括培养权利要求8~10任一项所述的宿主,然后从培养物中提取环状缩肽的步骤。
12.权利要求11所述的制造方法,其中,环状缩肽是PF1022物质及其衍生物。
13.一种制造环状缩肽合成酶的方法,该方法包括培养 8~10任一项所述的宿主,然后从培养物中提取环状缩肽合成酶的步骤。
14.权利要求13所述的制造方法,其中,环状缩肽是PF1022物质及其衍生物。
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JP25304099 | 1999-09-07 | ||
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US (1) | US7285404B1 (zh) |
EP (1) | EP1215281B1 (zh) |
JP (1) | JP3961289B2 (zh) |
KR (1) | KR100702728B1 (zh) |
CN (1) | CN1183248C (zh) |
AT (1) | ATE327321T1 (zh) |
AU (1) | AU784466B2 (zh) |
CA (1) | CA2384122C (zh) |
DE (1) | DE60028217T2 (zh) |
NO (1) | NO330902B1 (zh) |
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Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
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CN106414481A (zh) * | 2014-03-20 | 2017-02-15 | 柏林工业大学 | 用于在丝状真菌中生产嵌合环寡缩酚酸肽的方法 |
CN106749569A (zh) * | 2017-03-03 | 2017-05-31 | 重庆乾泰生物医药有限公司 | 一种pf1022a的分离纯化方法 |
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AU2001252608B2 (en) * | 2000-04-26 | 2005-09-29 | Meiji Seika Kaisha, Ltd. | Novel (r)-2-hydroxy-3-phenylpropionate (d-phenyllactate) dehydrogenase and gene encoding the same |
US7326546B2 (en) | 2005-03-10 | 2008-02-05 | Ajinomoto Co., Inc. | Purine-derived substance-producing bacterium and a method for producing purine-derived substance |
EP1924699A4 (en) * | 2005-08-26 | 2009-11-11 | Aureogen Biosciences | Aureobasidin-A-SYNTHETASE |
PL2530149T3 (pl) | 2010-01-26 | 2018-04-30 | Meiji Seika Pharma Co., Ltd. | Struktura kwasu nukleinowego zawierająca klaster genów biosyntezy pirypiropenu i gen markerowy |
WO2014010714A1 (en) | 2012-07-09 | 2014-01-16 | Meiji Seika Pharma Co., Ltd. | Uk-2 biosynthetic genes and method for improving uk-2 productivity using the same |
WO2018166899A1 (en) * | 2017-03-14 | 2018-09-20 | Acidophil Ltd | Methods for production of pf1022a derivatives |
CN113046332B (zh) * | 2021-03-29 | 2023-08-01 | 华东理工大学 | 一类二倍半萜骨架化合物及其合成基因及制备方法 |
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NO176766C (no) * | 1989-02-07 | 1995-05-24 | Meiji Seika Kaisha | Fremgangsmåte for fremstilling av en forbindelse med anthelmintaktivitet |
EP0578616A3 (en) * | 1992-07-09 | 1994-06-01 | Sandoz Ltd | Cylosporin synthetase |
EP0780468A4 (en) * | 1995-06-22 | 2000-07-19 | Meiji Seika Kaisha | TRANSFORMANT WHICH PRODUCES THE SUBSTANCE PF1022 AND METHOD FOR TRANSFORMING MICROORGANISMS IN THE CLASS OF THE HYPOMYCETES |
US6057491A (en) * | 1997-05-29 | 2000-05-02 | Borad Of Regents For University Of Oklahoma | Protein having insecticidal activities and method of use |
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2000
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- 2000-09-07 US US10/070,387 patent/US7285404B1/en not_active Expired - Fee Related
- 2000-09-07 CA CA2384122A patent/CA2384122C/en not_active Expired - Fee Related
- 2000-09-07 KR KR1020027002704A patent/KR100702728B1/ko not_active IP Right Cessation
- 2000-09-07 JP JP2001522391A patent/JP3961289B2/ja not_active Expired - Fee Related
- 2000-09-07 NZ NZ517588A patent/NZ517588A/en not_active IP Right Cessation
- 2000-09-07 CN CNB008153019A patent/CN1183248C/zh not_active Expired - Fee Related
- 2000-09-07 AU AU68741/00A patent/AU784466B2/en not_active Ceased
- 2000-09-07 DE DE60028217T patent/DE60028217T2/de not_active Expired - Lifetime
- 2000-09-07 WO PCT/JP2000/006103 patent/WO2001018179A1/ja active IP Right Grant
- 2000-09-07 AT AT00957009T patent/ATE327321T1/de not_active IP Right Cessation
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Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN106414481A (zh) * | 2014-03-20 | 2017-02-15 | 柏林工业大学 | 用于在丝状真菌中生产嵌合环寡缩酚酸肽的方法 |
CN106749569A (zh) * | 2017-03-03 | 2017-05-31 | 重庆乾泰生物医药有限公司 | 一种pf1022a的分离纯化方法 |
CN106749569B (zh) * | 2017-03-03 | 2021-10-15 | 重庆乾泰生物医药有限公司 | 一种pf1022a的分离纯化方法 |
Also Published As
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WO2001018179A1 (fr) | 2001-03-15 |
JP3961289B2 (ja) | 2007-08-22 |
AU6874100A (en) | 2001-04-10 |
NO20021100L (no) | 2002-05-07 |
ATE327321T1 (de) | 2006-06-15 |
KR100702728B1 (ko) | 2007-04-03 |
US7285404B1 (en) | 2007-10-23 |
CA2384122C (en) | 2010-05-04 |
EP1215281B1 (en) | 2006-05-24 |
DE60028217D1 (de) | 2006-06-29 |
NO20021100D0 (no) | 2002-03-06 |
NZ517588A (en) | 2005-01-28 |
DE60028217T2 (de) | 2007-04-26 |
KR20020029767A (ko) | 2002-04-19 |
EP1215281A1 (en) | 2002-06-19 |
CA2384122A1 (en) | 2001-03-15 |
NO330902B1 (no) | 2011-08-15 |
CN1183248C (zh) | 2005-01-05 |
AU784466B2 (en) | 2006-04-06 |
EP1215281A4 (en) | 2003-07-02 |
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