CN107098977A - 免疫原性组合物 - Google Patents

免疫原性组合物 Download PDF

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CN107098977A
CN107098977A CN201710239709.0A CN201710239709A CN107098977A CN 107098977 A CN107098977 A CN 107098977A CN 201710239709 A CN201710239709 A CN 201710239709A CN 107098977 A CN107098977 A CN 107098977A
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C.卡斯塔多
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Abstract

本发明涉及免疫原性组合物，以及包含来自艰难梭菌的毒素A和/或毒素B的片段的融合蛋白。具体而言，本发明涉及包含第一片段和第二片段的蛋白，其中(v)所述第一片段是毒素A重复结构域片段；(vi)所述第二片段是毒素B重复结构域片段；(vii)所述第一片段具有第一近端；(viii)所述第二片段具有第二近端；并且其中所述第一片段和所述第二片段彼此邻近，并且其中所述多肽诱导中和毒素A或毒素B或两者的抗体。

Description

免疫原性组合物
本申请是申请日为2012年05月25日，申请号为201280037472.3，发明名称为“免疫原性组合物”的发明专利申请的分案申请。
技术领域
本发明涉及来自艰难梭菌(Clostridium difficile)的抗原。具体而言，本发明涉及包含毒素A和/或毒素B的片段的重组蛋白抗原。本发明还涉及包含这些抗原的免疫原性组合物或疫苗，以及本发明的疫苗和免疫原性组合物在预防或治疗中的用途。本发明还涉及使用本发明的组合物的免疫方法，以及本发明的组合物在药物制造中的用途。
背景技术
艰难梭菌是医院肠道感染的最重要原因并且是人中假膜性结肠炎的主要原因(Bartlett等人，Am. J. Clin.Nutr.11 suppl:2521-6 (1980))。感染艰难梭菌的个体的总体相关死亡率在诊断的3个月内计算为5.99%，并且更高的死亡率与高龄相关，在超过80岁的患者中为13.5% (karas 等人，Journal of Infection 561:1-9 (2010))。目前对艰难梭菌感染的治疗是施用抗生素(甲硝唑和万古霉素)，然而，已经存在对这些抗生素具有抗性的菌株的证据 (Shah等人，Expert Rev. Anti Infect.Ther.8(5)555-564 (2010))。因此，存在对能够诱导针对艰难梭菌的抗体和/或保护性免疫应答的免疫原性组合物的需求。
发明内容
艰难梭菌的肠毒性主要由于毒素A和毒素B两种毒素的作用。这些均是有效的细胞毒素(Lyerly等人，Current Microbiology 21:29-32 (1990)。毒素A和毒素B的C末端结构域包含重复单位，例如毒素A的C末端由连续的重复单位构成(Dove等人，Infect. Immun. 58:480-499 (1990))，由于这一原因，C末端结构域也可以称为“重复结构域”。如Ho等人在(PNAS 102:18373-18378 (2005))中描述，这些重复部分可以进一步分为短重复(SRs)和长重复(LRs)。
已经确定了毒素A重复结构域的C末端的127-aa片段的结构(Ho等人，PNAS 102:18373-18378 (2005))。该片段形成β-螺线管类折叠，主要由β链和低比例的α螺旋构成。
已经表明毒素A具体为C末端结构域的片段能够导致在仓鼠中的保护性免疫应答(Lyerly等人，Current Microbiology 21:29-32 (1990))、WO96/12802和WO00/61762。
已知设计在表达过程中能够正确折叠的融合蛋白是困难的。本发明的多肽是融合蛋白，其中，保持天然β-螺线管类结构，并且发现其能够在小鼠中提供针对毒素A和毒素B二者的免疫应答。
在本发明的第一个方面，提供包含第一片段和第二片段的多肽，其中
(i) 所述第一片段是毒素A重复结构域片段；
(ii) 所述第二片段是毒素B重复结构域片段；
(iii) 所述第一片段具有第一近端；
(iv) 所述第二片段具有第二近端；并且
多肽诱导中和毒素A或毒素B或两者的抗体。
在本发明的第二个方面，提供多肽，其包括：
具有至少90%、95%、98%、99%或100%相似性的变体；或
的至少或个氨基酸的片段。
在本发明的第三个方面，提供编码本发明的多肽的多核苷酸。
在本发明的第四个方面，提供包含与诱导型启动子连接的本发明的多核苷酸的载体。
在本发明的第五个方面，提供包含本发明的载体或本发明的多核苷酸的宿主细胞。
在本发明的第六个方面，提供包含本发明的多肽和药学上可接受的赋形剂的免疫原性组合物。
在本发明的第七个方面，提供包含本发明的免疫原性组合物的疫苗。
在本发明的第八个方面，提供本发明的免疫原性组合物或本发明的疫苗在治疗或预防艰难梭菌疾病中的用途。
在本发明的第九个方面，提供本发明的免疫原性组合物或本发明的疫苗在制备用于预防或治疗艰难梭菌疾病的药物中的用途。
在本发明的第十个方面，提供预防或治疗艰难梭菌疾病的方法，其包括向患者施用本发明的免疫原性组合物或本发明的疫苗。
附图说明
图1－本发明的多肽的序列表。
图2－ToxA和ToxB的C末端结构域的图示，SR重复表示为白色方框并且LR盒表示为黑色方框。
图3－融合体1中使用ToxA的第三个SR VIII和Tox B的第四个SR II之间的连接的图示。
图4－融合体2中使用ToxA的第二个SR VIII和Tox B的第三个SR II之间的连接的图示。
图5－融合体3(仅包含部分的ToxA的LRVII和部分的ToxB的LR II)中使用的ToxA的LRVII和ToxB的LRII之间的连接的图示。
图6－融合体4中使用ToxA的第二个SR VIII和ToxB的第三个SR I之间的连接的图示。
图7－融合体5中使用的包含ToxA蛋白序列的最后一个残基和ToxB的第四个SRII的起点之间的甘氨酸接头的连接的图示。
图8－描绘通过沉降速度分析超速离心测定的艰难梭菌ToxA-ToxB融合体1-5的分布的图。图a)描绘了融合体1的分布，图b)描绘了融合体2的分布，图c)描绘了融合体3的分布，图d)描绘了融合体4的分布和图e)描绘了融合体5的分布。
图9－描绘使用圆二色性测定的融合体2、3、4和5的远UV谱的图。融合体2的谱用具有表示为小矩形的点的线表示，融合体3的谱用具有表示为小菱形的点的线表示，融合体4用具有表示为圆形的点的线表示，并且融合体5用具有表示为十字形的点的线表示。
图10－描绘使用圆二色性测定的融合体2、3、4和5的近UV谱的图。融合体2的谱用具有表示为十字形的点的线表示，融合体3的谱用具有表示为圆形的点的线表示，融合体4的谱用具有表示为三角形的点的线表示，并且融合体5的谱用具有表示为小菱形的点的线表示。
图11－显示在用毒素A的C末端片段(aa 2387-2706)、毒素B的C末端片段(aa1750-2360)或融合体1、2、3、4或5免疫的小鼠中的抗ToxA免疫原性的图。
图12－显示在用毒素A的C末端片段(aa 2387-2706)、毒素B的C末端片段(aa1750-2360)或融合体1、2、3、4或5免疫的小鼠中的血细胞凝集抑制的图。
图13－显示在用毒素A的C末端片段(aa 2387-2706)、毒素B的C末端片段(aa1750-2360)或融合体1、2、3、4或5免疫的小鼠中的抗ToxB免疫原性的图。
图14－来自用毒素A的C末端片段(aa 2387-2706)、毒素B的C末端片段(aa 1750-2360)或融合体1、2、3、4或5免疫的小鼠的细胞毒性(Cyotoxicity)抑制效价。
图15－描绘通过沉降速度分析超速离心测定的艰难梭菌ToxA-ToxB融合体F52New、F54Gly、F54New和F5ToxB的分布的图。图a)描绘了F52New的分布，图b)描绘了F54Gly的分布，图c)描绘了F54New的分布并且图d)描绘了F5ToxB的分布。
图16－描绘使用圆二色性测定的融合体F52New、F54Gly、F54New和F5ToxB的远UV谱的图。F52New的谱用具有表示为双十字的点的线表示，F54Gly的谱用具有表示为三角形的点的线表示，F54New用具有表示为矩形的点的线表示，并且F5ToxB用具有表示为十字形的点的线表示。
图17－描绘使用圆二色性测定的融合体F52New、F54Gly、F54New和F5ToxB的近UV谱的图。F52New的谱用具有表示为双十字的点的线表示，F54Gly的谱用具有表示为三角形的点的线表示，F54New用具有表示为矩形的点的线表示，并且F5ToxB用具有表示为十字形的点的线表示。
图18－显示用F2、F52New、F54Gly、G54New或F5ToxB融合体免疫的小鼠的抗ToxAELISA结果的图。
图19－显示用F2、F52New、F54Gly、F54New或F5ToxB融合体免疫的小鼠的抗ToxBELISA结果的图。
图20－显示用F2、F52New、F54Gly、F54New或F5ToxB融合体免疫的小鼠中的血细胞凝集抑制的图。
图21－显示来自用F2、F52New、F54Gly、F54New或F5ToxB融合体免疫的小鼠的HT29细胞中的细胞毒性效价的图。
图22－显示来自用F2、F52New、F54Gly、F54New或F5ToxB融合体免疫的小鼠的IMR90细胞中的细胞毒性效价的图。
发明详述
多肽
本发明涉及包含第一片段和第二片段的多肽，其中
(i) 所述第一片段是毒素A重复结构域片段；
(ii) 所述第二片段是毒素B重复结构域片段；
(iii) 所述第一片段具有第一近端；
(iv) 所述第二片段具有第二近端；并且
其中第一片段和第二片段彼此邻近，并且其中该多肽诱导中和毒素A或毒素B或两者的抗体。
术语多肽指氨基酸的连续序列。
术语“毒素A重复结构域”指来自艰难梭菌的毒素A蛋白的C末端结构域，其包含重复序列。该结构域指来自菌株VPI10463 (ATCC43255)的毒素A的氨基酸1832-2710以及它们在不同菌株中的等同物，来自菌株VPI10463 (ATCC43255)的氨基酸1832-2710的序列对应于SEQ ID NO:1的氨基酸1832-2710。
术语“毒素B重复结构域”指来自艰难梭菌的毒素B蛋白的C末端结构域。该结构域指来自菌株VPI10463 (ATCC43255)的氨基酸1834-2366以及它们在不同菌株中的等同物，来自菌株VPI10463 (ATCC43255)的氨基酸1834-2366的序列对应于SEQ ID NO:2的氨基酸1834-2366。
艰难梭菌毒素A和B是保守蛋白，然而，该序列在菌株间有少量差别，此外，不同菌株中的毒素A和B的氨基酸序列可能在许多氨基酸上有所不同。
因此，本发明的术语毒素A重复结构域和/或毒素B重复结构域指这样的序列，其是与SEQ ID NO:1的氨基酸1832-2710具有90%、95%、98%、99%或100%序列同一性的变体或与SEQ ID NO:2的氨基酸1834-2366具有90%、95%、98%、99%或100%序列同一性的变体。在一个实施方案中，“变体”是通过保守性氨基酸取代(由此，残基由具有相同物理化学性质的另一个残基所取代)而与参照多肽有所不同的多肽。通常这样的取代在Ala、Val、Leu和Ile之间；在Ser和Thr之间；在酸性残基Asp和Glu之间；在Asn和Gln之间，并在碱性残基Lys和Arg之间；或在芳香族残基Phe和Tyr之间。在一个实施方案中，“片段”是包含多肽的至少250个氨基酸的连续部分的多肽。
此外，在来自一种菌株的毒素A(或毒素B)与来自另一种菌株的毒素A(或毒素B)的C末端结构域之间，氨基酸编号可以有所不同。由于这一原因，术语“不同菌株中的等同物”是指，对应于参考菌株(例如艰难梭菌VPI10463)的那些，但发现于来自不同菌株的毒素中并且可能因此而编号不同的氨基酸。可以通过比对来自不同菌株的毒素的序列来确定“等同物”氨基酸的区域。全文中提供的氨基酸编号指菌株VPI10463的编号。
术语多肽或蛋白的“片段”指来自该多肽或蛋白的至少100、200、230、250、300、350、380、400、450、480、500、530、550、580或600个氨基酸的连续部分。术语“第一片段”指毒素A重复结构域的至少100、250、300、350、380、400、450、480、500、530、550、580或600个氨基酸的连续部分。术语“第二片段”指毒素B重复结构域的至少100、200、230、250、280、300、350、400、450或500个氨基酸的连续部分。
术语“第一近端”指第一片段(Tox A片段)的末端，其与第二片段(ToxB片段)共价连接或与第一和第二片段之间的接头序列共价连接，并且与第二片段在一级结构上最接近。术语“第二近端”指第二片段的末端，其与第一片段(ToxA片段)共价连接或与第一和第二片段之间的接头序列共价连接，并且与第一片段在一级结构上最接近。
多肽可以是更大的蛋白诸如前体或融合蛋白的一部分。包括含有有助于纯化的额外氨基酸序列，诸如多组氨酸残基，或用于重组生产过程中稳定性的额外序列通常是有利的。此外，还考虑加入外源多肽或脂质尾或多核苷酸序列以提高最终分子的免疫原性潜力。
片段可以这样安置，从而使第一片段的N末端邻近第二片段的C末端，或者第一片段的C末端可以邻近第二片段的N末端，或者第一片段的C末端可以邻近第二片段的C末端，或者第一片段的N末端可以邻近第二片段的N末端。
词语“邻近”表示在一级结构中由少于或正好20、15、10、8、5、2、1或0个氨基酸而分隔。
本发明的多肽诱导中和毒素A或毒素B或两者的抗体。在一个实施方案中，多肽诱导中和毒素A的抗体。在进一步的实施方案中，多肽诱导中和毒素B的抗体。在进一步的实施方案中，多肽诱导中和毒素A和毒素B的抗体。
可以通过用包含该多肽的免疫原性组合物免疫小鼠、收集血清并使用ELISA分析血清中的抗毒素效价来测定多肽是否诱导针对毒素的抗体。应该将血清与获取自未进行免疫的小鼠的参考样品进行比较。该技术的实例可见于实施例6。如果针对多肽的血清的ELISA读数产生了高于参考样品超过10%、20%、30%、50%、70%、80%、90%或100%，则本发明中的多肽诱导了中和毒素A的抗体。
在进一步的实施方案中，本发明的多肽在哺乳动物宿主中诱导了针对艰难梭菌菌株的保护性免疫应答。在一个实施方案中，哺乳动物宿主选自小鼠、兔、豚鼠、非人灵长类动物、猴和人。在一个实施方案中，哺乳动物宿主是小鼠。在进一步的实施方案中，哺乳动物宿主是人。
可以使用激发测定(challenge assay)来确定多肽是否在哺乳动物宿主中诱导了针对艰难梭菌菌株的保护性免疫应答。在此类试验中，用该多肽对哺乳动物宿主进行接种，并通过接触艰难梭菌进行激发，将激发后哺乳动物存活的时间与未经该多肽免疫的参考哺乳动物存活的时间进行比较。如果在用艰难梭菌激发后，经该多肽免疫的哺乳动物存活时间比未经免疫的参考哺乳动物长至少10%、20%、30%、50%、80%、80%、90%或100%，则该多肽诱导了保护性免疫应答。
毒素A和B的C末端结构域的天然结构由扩展的β螺线管类结构组成。如Ho等人(PNAS 102:18373-18378 (2005))所见，该结构主要由β折叠结构组成，同时具有少量的α螺旋结构。所存在的二级结构可以由圆二色性测定。例如，在远UV区(190-250nm)测定CD谱的形状和大小，并且将结果与已知结构的数值进行比较。例如，如下文实施例5中所示，这可以在Jasco J-720旋光分光计上在178-250nm使用0.01cm的光程以1nm的分辨率和带宽来测定。
在一个实施方案中，第一片段包含少于25%、23%、20%、18%、15%、10%或7%的α螺旋二级结构。在一个实施方案中，第二片段包含少于28%、25%、23%、20%、18%、15%、10%或7%的α螺旋二级结构。在进一步的实施方案中，第一片段和第二片段均包含少于28%、25%、23%、20%、18%、15%、10%或7%的α螺旋二级结构。
在一个实施方案中，第一片段包含多于20%、25%、28%、30%、33%、35%、38%、40%或42%的β折叠结构。在一个实施方案中，第二片段包含多于20%、25%、28%、30%、33%、35%、38%、40%或42%的β折叠结构。在进一步的实施方案中，第一片段和第二片段均包含多于20%、25%、28%、30%、33%、35%、38%、40%或42%的β折叠结构。
图2显示了ToxA和ToxB的C末端结构域的构成。毒素A的C末端结构域由8个重复部分(命名为重复部分I、重复部分II、重复部分III、重复部分IV、重复部分V、重复部分VI、重复部分VII和重复部分VIII)构成，这些重复部分的每一个可以进一步分为在图2中表示为白色方框的短重复(SRs)和在图2中表示为黑色方框的长重复(LRs)(除了Tox A重复部分VIII，其不具有长重复)。长重复的每一个与其他长重复具有某些结构和序列相似性。类似地，短重复彼此之间具有某些序列和结构相似性。毒素B的C末端由5个细分为SRs和LRs的重复部分构成。每一个重复部分包含一个LR和2-5个SRs(除了Tox B重复部分V，其不具有长重复)。对于本公开的目的，短语“重复部分”指ToxA的八个重复部分(命名为I、II、III、IV、V、VI、VII和VIII)中的一个或ToxB的五个重复部分(命名为I、II、III、IV或VI)中的一个。如本文所用，术语“第一重复部分”指来自毒素A重复结构域的重复部分(或部分的重复部分)。术语“第二重复部分”指来自毒素B重复结构域的重复部分(或部分的重复部分)。对于本公开的目的，术语“长重复”指如图2中黑色方框所示的LR结构域之一。对于本公开的目的，术语“短重复”指如图2中白色方框所示的SR结构域之一。
因此，例如，ToxA的重复部分I包含三个SRs和一个LR，其可以分别称为ToxA的第一个SRI、ToxA的第二个SRI、ToxA的第三个SRI和ToxA的LRI。
如果第一片段在处于该重复部分内的氨基酸处终止(即第一近端仅包含部分的重复部分序列)，则认为第一个近端在“重复部分”内。类似地，如果第二片段在处于该重复部分内的氨基酸处终止，则认为第二近端在“重复部分”内。例如，如果第一片段以VPI10463的氨基酸1832-1924 (包括在内的)或它们在另一菌株中的等同物的任何一个氨基酸终止，则第一近端在“ToxA的重复部分I”内。如果第一片段以并不在短重复-长重复-短重复部分内的氨基酸结束，则第一近端不在该短重复-长重复-短重复部分内。
已经确定了来自菌株VPI10463 (ATCC43255)的毒素A和毒素B的每一结构域的氨基酸位置。这些如下：
。
为此原因，术语“重复部分”可以指毒素A (SEQ ID NO:1)的氨基酸
或、或毒素B (SEQID NO:2)的氨基酸或、或它们在艰难梭菌的不同菌株中的等同物。
为此原因，术语“短重复”可以指毒素A (SEQ ID NO:1)的氨基酸
或或毒素B (SEQ ID NO:2)的氨基酸
或或它们在艰难梭菌的不同菌株中的等同物。
类似地，术语“长重复”可以指毒素A (SEQ ID NO:1)的氨基酸1894-1924、2028-2058、2162-2192、2276-2306、2410-2440、2523-2553或2614-2644或毒素B (SEQ ID NO:2)的氨基酸1897-1926、2028-2057、2160-2189或2294-2323或它们在艰难梭菌的不同菌株中的等同物。
类似地，术语“短重复-长重复-短重复部分”可以指毒素A (SEQ ID NO:1)的氨基酸1874-1944、2008-2078、2142-2212、2254-2326、2390-2460、2503-2573或2595-2664，或毒素B (SEQ ID NO:2)的氨基酸1877-1946、2008-2078、2140-2212或2274-2343，或它们在艰难梭菌的不同菌株中的等同物。术语“不破坏短重复-长重复-短重复部分”表示近端处于不破坏短重复-长重复-短重复部分的结构的区域中，通常这表示近端不在长重复内并且不在构成短重复-长重复-短重复部分的短重复内，除了近端可以在离序列中该长重复最远的短重复的1、2、3、4、5或6个氨基酸的区域中。在一个实施方案中，术语“不破坏短重复-长重复-短重复部分”表示近端不在短重复-长重复-短重复部分内。
在一个实施方案中，第一近端在短重复内。在一个实施方案中，第二近端在短重复内。在一个实施方案中，第一近端和第二近端在短重复内。在一个实施方案中，第一近端不破坏短重复-长重复-短重复部分。在一个实施方案中，第二近端不破坏短重复-长重复-短重复部分。在一个实施方案中，第一近端和第二近端不破坏短重复-长重复-短重复部分。
在一个实施方案中，第一近端不在毒素A (SEQ ID NO:1)的氨基酸
或或它们在艰难梭菌不同菌株中的等同物内。在第二个实施方案中，第二近端不在毒素B (SEQ ID NO:2)的氨基酸1881-1942、2012-2074、2144-2208或2278-2339或它们在艰难梭菌不同菌株中的等同物内。在进一步的实施方案中，第一近端不在毒素A (SEQ ID NO:1)的氨基酸 或或它们在艰难梭菌不同菌株中的等同物内，并且第二近端不在毒素B (SEQ ID NO:2)的氨基酸1881-1942、2012-2074、2144-2208或2278-2339或它们在艰难梭菌不同菌株中的等同物内。
在一个实施方案中，第一近端在毒素A的重复部分V(SEQ ID NO:1的氨基酸2307-2440或它们在不同菌株中的等同物)、VI(SEQ ID NO:1的氨基酸2441-2553或它们在不同菌株中的等同物)、VII(SEQ ID NO:1的氨基酸2554-2644或它们在不同菌株中的等同物)或VIII(SEQ ID NO:1的氨基酸2645-2710或它们在不同菌株中的等同物)中。在进一步的实施方案中，第一近端在毒素A的重复部分VII(SEQ ID NO:1的氨基酸2554-2644或它们在不同菌株中的等同物)内。在进一步的实施方案中，第一近端在毒素A的重复部分VIII(SEQ IDNO:1的氨基酸2645-2710或它们在不同菌株中的等同物)内。
在一个实施方案中，第二近端在毒素B的重复部分I(SEQ ID NO:2的氨基酸1834-1926或它们在不同菌株中的等同物)、II(SEQ ID NO:2的氨基酸1927-2057或它们在不同菌株中的等同物)、或iii(SEQ ID NO:2的氨基酸2058-2189或它们在不同菌株中的等同物)中。在进一步的实施方案中，第二近端在毒素B的重复部分II(SEQ ID NO:2的氨基酸1927-2057或它们在不同菌株中的等同物)内。在进一步的实施方案中，第二近端在毒素B的重复部分I(SEQ ID NO:2的氨基酸1834-1926或它们在不同菌株中的等同物)内。
在一个实施方案中，第一近端在毒素A的重复部分VIII(SEQ ID NO:1的氨基酸2645-2710或它们在不同菌株中的等同物)内，并且第二近端在毒素B的重复部分I(SEQ IDNO:2的氨基酸1834-1926或它们在不同菌株中的等同物)内。在进一步的实施方案中，第一近端在毒素A的重复部分VIII(SEQ ID NO:1的氨基酸2645-2710或它们在不同菌株中的等同物)内，并且第二近端在毒素B的重复部分II(SEQ ID NO:2的氨基酸1927-2057或它们在不同菌株中的等同物)内。在进一步的实施方案中，第一近端在毒素A的重复部分VII(SEQID NO:1的氨基酸2554-2644或它们在不同菌株中的等同物)内，并且第二近端在毒素B的重复部分I(SEQ ID NO:2的氨基酸1834-1926或它们在不同菌株中的等同物)内。在进一步的实施方案中，第一近端在毒素A的重复部分VII(SEQ ID NO:1的氨基酸2554-2644或它们在不同菌株中的等同物)内，并且第二近端在毒素B的重复部分II(SEQ ID NO:2的氨基酸1927-2057或它们在不同菌株中的等同物)内。在进一步的实施方案中，第一近端在重复部分VI(SEQ ID NO:1的氨基酸2441-2553或它们在不同菌株中的等同物)内，并且第二近端在毒素B的重复部分I(SEQ ID NO:2的氨基酸1834-1926或它们在不同菌株中的等同物)内。在进一步的实施方案中，第一近端在重复部分VI(SEQ ID NO:1的氨基酸2441-2553或它们在不同菌株中的等同物)内，并且第二近端在毒素B的重复部分II(SEQ ID NO:2的氨基酸1927-2057或它们在不同菌株中的等同物)内。在进一步的实施方案中，第一近端在重复部分V(SEQ ID NO:1的氨基酸2307-2440或它们在不同菌株中的等同物)内，并且第二近端在毒素B的重复部分I(SEQ ID NO:2的氨基酸1834-1926或它们在不同菌株中的等同物)内。在进一步的实施方案中，第一近端在重复部分V(SEQ ID NO:1的氨基酸2307-2440或它们在不同菌株中的等同物)内，并且第二近端在毒素B的重复部分II(SEQ ID NO:2的氨基酸1927-2057或它们在不同菌株中的等同物)内。
在一个实施方案中，第一近端在SEQ ID NO:1的氨基酸2690-2710、或2695-2710、或2700-2710或它们在不同菌株中的等同物内。在进一步的实施方案中，第一近端在SEQ IDNO:1的氨基酸2670-2700、或2675-2695、或2680-2690或它们在不同菌株中的等同物内。在一个实施方案中，第二近端在毒素B的氨基酸1860-1878或它们在不同菌株中的等同物内。在一个实施方案中，第二近端在SEQ ID NO:2的氨基酸1950-1980、1955-1975或1960-1970或它们在不同菌株中的等同物内。在进一步的实施方案中，第二近端在SEQ ID NO:2的氨基酸1978-2008、1983-2003或1988-1998或它们在不同菌株中的等同物内。在进一步的实施方案中，第二近端在SEQ ID NO:2的氨基酸1860-1878、1854-1876、1857-1887、1862-1882或1867-1877或它们在不同菌株中的等同物内。
在一个实施方案中，第一片段由整个毒素A重复结构域(氨基酸1832-2710)组成。在一个实施方案中，第二片段由整个毒素B重复结构域(氨基酸1833-2366)组成。
在一个实施方案中，第一近端在毒素A的重复部分VIII的短重复3(SEQ ID NO:1的氨基酸2687-2710或它们在不同菌株中的等同物)内，并且第二近端在毒素B的重复部分II的短重复4(SEQ ID NO:2的氨基酸1988-2007或它们在不同菌株中的等同物)内。在一个实施方案中，第一近端在毒素A的重复部分VIII的短重复3(SEQ ID NO:1的氨基酸2687-2710或它们在不同菌株中的等同物)内，并且第二近端在毒素B的重复部分II的短重复3(SEQ IDNO:2的氨基酸1968-1987或它们在不同菌株中的等同物)内。在一个实施方案中，第一近端在毒素A的重复部分VIII的短重复3(SEQ ID NO:1的氨基酸2687-2710或它们在不同菌株中的等同物)内，并且第二近端在毒素B的重复部分II的短重复2(SEQ ID NO:2的氨基酸1947-1967或它们在不同菌株中的等同物)内。在一个实施方案中，第一近端在毒素A的重复部分VIII的短重复3(SEQ ID NO:1的氨基酸2687-2710或它们在不同菌株中的等同物)内，并且第二近端在毒素B的重复部分I的短重复3(SEQ ID NO:2的氨基酸1877-1896或它们在不同菌株中的等同物)内。在一个实施方案中，第一近端在毒素A的重复部分VIII的短重复3(SEQID NO:1的氨基酸2687-2710或它们在不同菌株中的等同物)内，并且第二近端在毒素B的重复部分I的短重复2(SEQ ID NO:2的氨基酸1855-1876或它们在不同菌株中的等同物)内。在一个实施方案中，第一近端在毒素A的重复部分VIII的短重复3(SEQ ID NO:1的氨基酸2687-2710或它们在不同菌株中的等同物)内，并且第二近端在毒素B的重复部分I的短重复1(SEQ ID NO:2的氨基酸1834-1854或它们在不同菌株中的等同物)内。在一个实施方案中，第一近端在毒素A的重复部分VIII的短重复2(SEQ ID NO:1的氨基酸2665-2686或它们在不同菌株中的等同物)内，并且第二近端在毒素B的重复部分II的短重复4(SEQ ID NO:2的氨基酸1988-2007或它们在不同菌株中的等同物)内。在一个实施方案中，第一近端在毒素A的重复部分VIII的短重复2(SEQ ID NO:1的氨基酸2665-2686或它们在不同菌株中的等同物)内，并且第二近端在毒素B的重复部分II的短重复3(SEQ ID NO:2的氨基酸1968-1987或它们在不同菌株中的等同物)内。在一个实施方案中，第一近端在毒素A的重复部分VIII的短重复2(SEQ ID NO:1的氨基酸2665-2686或它们在不同菌株中的等同物)内，并且第二近端在毒素B的重复部分II的短重复2(SEQ ID NO:2的氨基酸1947-1967或它们在不同菌株中的等同物)内。在一个实施方案中，第一近端在毒素A的重复部分VIII的短重复2(SEQ ID NO:1的氨基酸2665-2686或它们在不同菌株中的等同物)内，并且第二近端在毒素B的重复部分I的短重复3(毒素B的氨基酸1877-1896或它们在不同菌株中的等同物)内。在一个实施方案中，第一近端在毒素A的重复部分VIII的短重复2(SEQ ID NO:1的氨基酸2665-2686或它们在不同菌株中的等同物)内，并且第二近端在毒素B的重复部分I的短重复2(SEQ ID NO:2的氨基酸1855-1876或它们在不同菌株中的等同物)内。在一个实施方案中，第一近端在毒素A的重复部分VIII的短重复2(SEQ ID NO:1的氨基酸2665-2686或它们在不同菌株中的等同物)内，并且第二近端在毒素B的重复部分I的短重复1(SEQ ID NO:2的氨基酸1834-1854或它们在不同菌株中的等同物)内。在一个实施方案中，第一近端在毒素A的重复部分vii的短重复2(SEQ ID NO:1的氨基酸2574-2594或它们在不同菌株中的等同物)内，并且第二近端在毒素B的重复部分II的短重复4(SEQ ID NO:2的氨基酸1988-2007或它们在不同菌株中的等同物)内。在一个实施方案中，第一近端在毒素A的重复部分vii的短重复2(SEQ ID NO:1的氨基酸2574-2594或它们在不同菌株中的等同物)内，并且第二近端在毒素B的重复部分II的短重复3(SEQ ID NO:2的氨基酸1668-1987或它们在不同菌株中的等同物)内。在一个实施方案中，第一近端在毒素A的重复部分vii的短重复2(SEQ ID NO:1的氨基酸2574-2594或它们在不同菌株中的等同物)内，并且第二近端在毒素B的重复部分II的短重复2(SEQ IDNO:2的氨基酸1947-1967或它们在不同菌株中的等同物)内。在一个实施方案中，第一近端在毒素A的重复部分VII的短重复2(SEQ ID NO:1的氨基酸2574-2594或它们在不同菌株中的等同物)内，并且第二近端在毒素B的重复部分I的短重复2(SEQ ID NO:2的氨基酸1855-1876或它们在不同菌株中的等同物)内。在一个实施方案中，第一近端在毒素A的重复部分VII的短重复2(SEQ ID NO:1的氨基酸2574-2594或它们在不同菌株中的等同物)内，并且第二近端在毒素B的重复部分I的短重复1(SEQ ID NO:2的氨基酸1834-1854或它们在不同菌株中的等同物)内。在一个实施方案中，第一近端在毒素A的重复部分VI的短重复3(SEQ IDNO:1的氨基酸2482-2502或它们在不同菌株中的等同物)内，并且第二近端在毒素B的重复部分II的短重复4(SEQ ID NO:2的氨基酸1988-2007或它们在不同菌株中的等同物)内。在一个实施方案中，第一近端在毒素A的重复部分VI的短重复3(SEQ ID NO:1的氨基酸2482-2502或它们在不同菌株中的等同物)内，并且第二近端在毒素B的重复部分II的短重复3(SEQ ID NO:2的氨基酸1968-1987或它们在不同菌株中的等同物)内。在一个实施方案中，第一近端在毒素A的重复部分VI的短重复3(SEQ ID NO:1的氨基酸2482-2502或它们在不同菌株中的等同物)内，并且第二近端在毒素B的重复部分II的短重复2(SEQ ID NO:2的氨基酸1947-1967或它们在不同菌株中的等同物)内。在一个实施方案中，第一近端在毒素A的重复部分VI的短重复3(SEQ ID NO:1的氨基酸2482-2502或它们在不同菌株中的等同物)内，并且第二近端在毒素B的重复部分I的短重复2(SEQ ID NO:2的氨基酸1855-1876或它们在不同菌株中的等同物)内。在一个实施方案中，第一近端在毒素A的重复部分VI的短重复3(SEQ ID NO:1的氨基酸2482-2502或它们在不同菌株中的等同物)内，并且第二近端在毒素B的重复部分I的短重复1(SEQ ID NO:2的氨基酸1834-1854或它们在不同菌株中的等同物)内。在一个实施方案中，第一近端在毒素A的重复部分VI的短重复2(SEQ ID NO:1的氨基酸2461-2481或它们在不同菌株中的等同物)内，并且第二近端在毒素B的重复部分II的短重复4(SEQ ID NO:2的氨基酸1988-2007或它们在不同菌株中的等同物)内。在一个实施方案中，第一近端在毒素A的重复部分VI的短重复2(SEQ ID NO:1的氨基酸2461-2481或它们在不同菌株中的等同物)内，并且第二近端在毒素B的重复部分II的短重复3(SEQ ID NO:2的氨基酸1968-1987或它们在不同菌株中的等同物)内。在一个实施方案中，第一近端在毒素A的重复部分VI的短重复2(SEQ ID NO:1的氨基酸2461-2481或它们在不同菌株中的等同物)内，并且第二近端在毒素B的重复部分II的短重复2(SEQ ID NO:2的氨基酸1947-1967或它们在不同菌株中的等同物)内。在一个实施方案中，第一近端在毒素A的重复部分VI的短重复2(SEQ ID NO:1的氨基酸2461-2481或它们在不同菌株中的等同物)内，并且第二近端在毒素B的重复部分I的短重复2(SEQ ID NO:2的氨基酸1855-1876或它们在不同菌株中的等同物)内。在一个实施方案中，第一近端在毒素A的重复部分VI的短重复2(SEQ ID NO:1的氨基酸2461-2481或它们在不同菌株中的等同物)内，并且第二近端在毒素B的重复部分I的短重复1(SEQ ID NO:2的氨基酸1834-1854或它们在不同菌株中的等同物)内。
在一个实施方案中，第一近端在SEQ ID NO:1的氨基酸2690-2710、或2695-2710、或2700-2710或它们在不同菌株中的等同物内，并且第二近端在SEQ ID NO:2的氨基酸1950-1980、1955-1975或1960-1970或它们在不同菌株中的等同物内。在一个实施方案中，第一近端在SEQ ID NO:1的氨基酸2690-2710、或2695-2710、或2700-2710或它们在不同菌株中的等同物内，并且第二近端在SEQ ID NO:2的氨基酸1978-2008、1983-2003或1988-1998或它们在不同菌株中的等同物内。在一个实施方案中，第一近端在SEQ ID NO:1的氨基酸2690-2710、或2695-2710、或2700-2710或它们在不同菌株中的等同物内，并且第二近端在SEQ ID NO:2的氨基酸1857-1887、1862-1882或1867-1877或它们在不同菌株中的等同物内。在一个实施方案中，第一近端在SEQ ID NO:1的氨基酸2670-2700、或2675-2695、或2680-2690或它们在不同菌株中的等同物内，并且第二近端在SEQ ID NO:2的氨基酸1950-1980、1955-1975或1960-1970或它们在不同菌株中的等同物内。在一个实施方案中，第一近端在SEQ ID NO:1的氨基酸2670-2700、或2675-2695、或2680-2690或它们在不同菌株中的等同物内，并且第二近端在SEQ ID NO:2的氨基酸1978-2008、1983-2003或1988-1998或它们在不同菌株中的等同物内。在一个实施方案中，第一近端在SEQ ID NO:1的氨基酸2670-2700、或2675-2695、或2680-2690或它们在不同菌株中的等同物内，并且第二近端在SEQ IDNO:2的氨基酸1857-1887、1862-1882、1860-1878或1867-1877或它们在不同菌株中的等同物内。
在一个实施方案中，第一片段包含至少100、200、300、400或450个氨基酸。在一个实施方案中，第二片段包含至少100、200、300或400个氨基酸。
在一个实施方案中，多肽还包含接头。该接头可以在第一近端和第二近端之间，或者该接头可以将第一片段和/或第二片段的远端与进一步的氨基酸序列连接。
可以使用肽接头序列以分隔第一片段和第二片段。使用本领域熟知的标准技术将这样的肽接头序列整合入融合蛋白中。可以根据以下因素选择合适的肽接头序列：(1)它们采用柔性延伸构象的能力；(2)它们不能采用能够与第一片段和/或第二片段上的功能性表位相互作用的二级结构；和(3)缺少可以与Tox A和/或ToxB功能性表位相互作用的疏水性或带电残基。肽接头序列可包含Gly、Asn和Ser残基。其他的近中性氨基酸诸如Thr和Ala也可用于接头序列中。可有效地用作接头的氨基酸序列包括Maratea等人，Gene 40:39-46(1985)、Murphy等人，Proc. Natl. Acad. Sci. USA 83:8258-8262 (1986)、美国专利号4,935,233和美国专利号4,751,180中公开的那些。
在一个实施方案中，接头包含1-19、1-15、1-10、1-5、1-2、5-20、5-15、5-15、10-20或10-15个之间的氨基酸。在一个实施方案中，接头是甘氨酸接头，该接头可以包含多个(1、2、3、4、5、6、7、8、9、10、12、15、18或19个)连续甘氨酸残基，或者该接头可以包含一些甘氨酸残基和一些其他氨基酸(诸如丙氨酸)的残基。在进一步的实施方案中，接头包含单个甘氨酸残基。
在一个实施方案中，本发明的多肽是更大的融合蛋白的一部分。融合蛋白还可以包含编码进一步的蛋白抗原的免疫原性部分的氨基酸。例如，融合蛋白还可以包含获自或来源自选自以下的细菌的蛋白抗原的免疫原性部分：肺炎链球菌(S.pneumoniae)、流感嗜血杆菌(H.influenzae)、脑膜炎奈瑟氏菌(N.meningitidis)、大肠杆菌(E.coli)、卡他莫拉菌(M.cattarhalis)、破伤风杆菌(C.tentani)、白喉棒杆菌(C.diphtheriae)、百日咳杆菌(B.pertussis)、表皮葡萄球菌(S.epidermidis)、肠球菌(enterococci)、金黄色葡萄球菌(S.aureus)和铜绿假单胞菌(Pseudomonas aeruginosa)。在这种情况下，接头可以在第一片段或第二片段与蛋白抗原的进一步的免疫原性部分之间。
术语“其免疫原性部分”或“免疫原性片段”指多肽的片段，其中该片段包含被细胞毒性T淋巴细胞、辅助T淋巴细胞或B细胞识别的表位。合适地，免疫原性部分将包含至少30％、合适地至少50％、尤其至少75％和具体至少90％(例如95％或98％)的参考序列中的氨基酸。免疫原性部分将合适地包含参考序列所有的表位区。
在一个实施方案中，多肽包含
或的免疫原性片段。在一个实施方案中，多肽包含
或的至少 或个氨基酸的免疫原性片段。在进一步的实施方案中，多肽包含
或的变体，在进一步的实施方案中，多肽包含与SEQ ID NO:3-SEQ ID NO:7具有至少80%、85%、90%、92%、95%、98%、99%或100%序列同一性的变体。
在一个实施方案中，多肽包含多于450、475、500、525、575、600、625、650、675、700、725、750、775、800、825或850个来自毒素A的氨基酸。在一个实施方案中，多肽包含少于850、825、800、775、750、725、700、675、650、625或600个来自毒素A的氨基酸。在一个实施方案中，多肽包含多于350、375、400、425、450、475、500或525个来自毒素B的氨基酸。在一个实施方案中，多肽包含少于525、500、475或450个来自毒素B的氨基酸。
术语“同一性”是本领域已知的，是两条或多条多肽序列之间或者两条或多条多核苷酸序列之间的关系，作为示例可以是如通过比较序列确定。在本领域，“同一性”也表示多肽或多核苷酸序列之间序列相关性的程度，作为示例可以是如通过此类序列的字符串之间的匹配确定。“同一性”可以容易地通过已知方法计算，包括但不限于描述于以下中的那些
。设计测定同一性的方法以产生检测序列之间的最大匹配。此外，测定同一性的方法编程于公开可得的计算机程序中。用于测定两条序列之间的同一性的计算机程序方法包括但不限于Needle程序BLASTP、BLASTN(Altschul, S.F.等人，J. Molec.Biol.215:403-410 (1990)，和FASTA (Pearson和LipmanProc. Natl. Acad. Sci. USA 85; 2444-2448 (1988)。BLAST家族程序可以从NCBI和其他来源公开获得(BLAST Manual, Altschul, S.等人，NCBI NLM NIH Bethesda, MD 20894;Altschul, S.等人，J. Mol. Biol. 215:403-410 (1990)。众所周知的Smith Waterman算法也可以用于测定同一性。
用于多肽序列比较的参数包括以下内容：
算法：Needleman和Wunsch，J. Mol Biol.48:443-453 (1970)
比较矩阵：来自Henikoff和Henikoff的BLOSSUM62，Proc.Natl.Acad.Sci.USA.89:10915-10919 (1992)
缺口罚分：10
缺口延伸罚分：0.5。
使用这些参数的程序作为来自EMBOSS程序包中的‘needle’程序是公开可得的(Rice P.等人，Trends in Genetics 2000 col.16(6):276-277)。前述参数是用于多肽比较的缺省参数(连同对末端缺口无罚分)。
为了确定参考序列与SEQ ID NO:1的同一性，在一个实施方案中，计算对参考序列全长的序列同一性。在进一步的实施方案中，计算对SEQ ID NO:1中的序列全长的序列同一性。为了确定参考序列与SEQ ID NO:2的同一性，在一个实施方案中，计算对参考序列全长的序列同一性。在进一步的实施方案中，计算对SEQ ID NO:2中的序列全长的序列同一性。
在本发明的进一步的方面，提供多肽，其包含：
或或，(ii)与SEQ ID NO:10-19具有至少80%、85%、88%、90%、92%、95%、98%、99%或100%同一性的变体；或(iii)
或或的至少
或个氨基酸的片段。在进一步的实施方案中，多肽包含：
或或，ii)与
或
或具有至少80%、85%、88%、90%、92%、95%、98%、99%或100%同一性的变体；或(iii)
或或的至少
或个氨基酸的片段。在进一步的实施方案中，多肽包含：
或或，
ii)与
或或
具有至少80%、85%、88%、90%、92%、95%、98%、99%或100%同一性的变体；或(iii)
或或的至少
或个氨基酸的片段。
在一个实施方案中，多肽包含多于450、475、500、525、575、600、625、650、675、700、725、750、775、800、825或850个来自毒素A的氨基酸。在一个实施方案中，多肽包含少于850、825、800、775、750、725、700、675、650、625或600个来自毒素A的氨基酸。在一个实施方案中，多肽包含多于350、375、400、425、450、475、500或525个来自毒素B的氨基酸。在一个实施方案中，多肽包含少于525、500、475或450个来自毒素B的氨基酸。
在进一步的实施方案中，多肽诱导中和毒素A或毒素B或两者的中和抗体。在进一步的实施方案中，多肽诱导中和毒素A的抗体。在进一步的实施方案中，多肽诱导中和毒素B的抗体。在进一步的实施方案中，多肽诱导中和毒素A和毒素B的抗体。如果针对本发明的多肽的血清的ELISA读数高于参考样品超过10%、20%、30%、50%、70%、80%、90%或100%，则该多肽诱导了中和毒素A的抗体。
在进一步的实施方案中，本发明的多肽在哺乳动物宿主中诱导针对艰难梭菌菌株的保护性免疫应答。在一个实施方案中，哺乳动物宿主选自小鼠、兔、豚鼠、猴、非人灵长类动物和人。在一个实施方案中，哺乳动物宿主是小鼠。在进一步的实施方案中，哺乳动物宿主是人。
可以使用激发测定来确定多肽是否在哺乳动物宿主中诱导针对艰难梭菌菌株的保护性免疫应答。在此类测定中，将哺乳动物宿主用该多肽接种并通过暴露于艰难梭菌进行激发，将哺乳动物在激发后存活的时间与未用该多肽免疫的参考哺乳动物存活的时间进行比较。如果在用艰难梭菌激发后，用该多肽免疫的哺乳动物存活长于未免疫的参考哺乳动物至少10%、20%、30%、50%、70%、80%、90%或100%，则多肽诱导了保护性免疫应答。在一个实施方案中，本发明的多肽诱导在选自小鼠、豚鼠、猴和人的哺乳动物中针对艰难梭菌菌株的保护性免疫应答。在一个实施方案中，哺乳动物是小鼠，在进一步的实施方案中，哺乳动物是人。
来自毒素A和B的C末端(重复)结构域的天然结构由延伸的β螺线管类结构组成。如Ho 等人，(PNAS 102:18373-18378 (2005))中所见，该结构主要由β折叠结构组成，具有少量的α螺旋结构。可以使用圆二色性测定存在的二级结构。例如，测定在远UV区(190-250nm)中CD谱的形状和大小并且将结果与已知结构的那些比较。例如，如下文实施例5中所见，这可以在Jasco J-720旋光分光计上从178-250nm使用0.01cm的光程以1nm分辨率和带宽来进行。
在一个实施方案中，多肽包含少于25%、23%、20%、28%、15%、10%或7%的α螺旋二级结构。在进一步的实施方案中，多肽包含多于20%、25%、28%、30%、33%、35%、38%、40%或42%的β折叠结构。
多核苷酸
本发明还提供编码本发明的多肽的多核苷酸。对于本发明的目的，术语“一种或多种多核苷酸”通常指任何多核糖核苷酸或多脱氧核糖核苷酸，其可以是包括单链和双链区/形式的未修饰的RNA或DNA或修饰的RNA或DNA。
如本文所用术语“编码肽的多核苷酸”包括编码本发明的肽或多肽的序列的多核苷酸。该术语还包括这样的多核苷酸，其包括编码肽或多肽的单个连续区或非连续区(例如，由整合的噬菌体、整合的插入序列、整合的载体序列、整合的转座子序列、或由于RNA编辑或基因组DNA再组织而中断的多核苷酸)，连同额的外区域，其也可包含编码和/或非编码序列。
本领域普通技术人员将理解作为遗传密码简并性的结果，存在许多编码如本文描述的多肽的核苷酸序列。这些多核苷酸中的一些与任何天然(即，天然存在的)基因的核苷酸序列具有最小相似性。尽管如此，由于密码子使用中的差异而不同的多核苷酸明确地由本发明所考虑，例如对人和/或灵长类动物和/或大肠杆菌密码子选择优化的多核苷酸。
可以使用本领域中熟知的化学方法全部或部分合成编码期望的多肽的序列(见Caruthers, M. H. 等人，Nucl. Acids Res. Symp. Ser. 第215-223页 (1980)，Horn等人，Nucl. Acids Res .Symp. Ser.第225-232页 (1980))。或者，蛋白本身可以使用合成多肽或其部分的氨基酸序列的化学方法来产生。例如，肽合成可以使用多种固相技术(Roberge 等人，Science 269:202-204 (1995))进行，并且可以实现自动合成，例如使用ASI 431 A 肽合成仪(Perkin Elmer, Palo Alto, CA)。
此外，出于多种原因，本发明的多核苷酸序列可以使用本领域通常已知的方法来工程化以改变多肽编码序列，包括但不限于修饰基因产物的克隆、加工和/或表达的改变。例如，可以使用通过随机断裂的DNA改组和基因片段和合成寡核苷酸的PCR重装配来工程化核苷酸序列。此外，可以使用基因定点诱变来插入新的限制位点、变更糖基化模式、改变密码子偏好、产生剪接变体或引入突变等。
载体
在本发明的进一步的方面，本发明涉及载体，其包含与诱导型启动子连接的本发明的多核苷酸，从而当诱导该启动子时表达由该多核苷酸编码的多肽。
本发明的进一步的方面包含所述载体，其中通过优选向生长培养基中加入足量的IPTG(异丙基-β-D-1-硫代半乳糖苷)激活诱导型启动子。任选地，这处于0.1-10mM、0.1-5mM、0.1-2.5mM、0.2-10mM、0.2-5mM、0.2-2.5mM、0.4-10mM、1-10mM、1-5mM、2.5-10mM、2.5-5mM、5-10mM的浓度。或者，通过改变温度或pH诱导启动子。
宿主细胞
对于本发明的多肽的重组产生，可以将宿主细胞遗传工程化以掺入表达系统或其部分或本发明的多核苷酸。向宿主细胞中引入多核苷酸可以通过许多标准实验室手册中描述的方法实现，诸如Davis等人，Basic Methods in Molecular Biology, (1986)和Sambrook等人，Molecular Cloning: A Laboratory Manual，第二版, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.(1989)，诸如，磷酸钙转染、DEAE-葡聚糖介导的转染、基因转应作用(transvection)、显微注射、阳离子脂质-介导的转染、电穿孔、接合、转导、刮擦上样(scrape loading)、弹道导入(ballistic introduction)和感染。
合适宿主的代表性示例包括革兰氏阴性细菌细胞，诸如大肠杆菌(E. coli)、不动杆菌属(Acinetobacter)、放线杆菌属(Actinobacillus)、博德特氏菌属(Bordetella)、布鲁氏菌属(Brucella)、弯曲杆菌属(Campylobacter)、蓝细菌属(Cyanobacteria)、肠杆菌属(Enterobacter)、欧文氏菌属(Erwinia)、弗朗西丝氏菌属(Franciscella)、螺杆菌属(Helicobacter)、嗜血菌属(Hemophilus)、克雷伯氏菌属(Klebsiella)、军团菌属(Legionella)、莫拉氏菌属(Moraxella)、奈瑟氏球菌属(Neisseria)、巴斯德氏菌属(Pasteurella)、变形菌属(Proteus)、假单胞菌属(Pseudomonas)、沙门氏菌属(Salmonella)、沙雷氏菌属(Serratia)、志贺氏菌属(Shigella)、密螺旋体属(Treponema)、弧菌属(Vibrio)、耶尔森氏菌属(Yersinia)的细胞。在一个实施方案中，宿主细胞是大肠杆菌细胞。或者，还可以使用革兰氏阳性细菌细胞。可以使用许多种表达系统来产生本发明的多肽。在一个实施方案中，载体源自细菌质粒。在这方面，通常适合于在宿主中保持、繁殖或表达多核苷酸和/或表达多肽的任何系统或载体都可以用于表达。可以通过多种众所周知的和常规的技术的任何一种将合适的DNA序列插入表达系统中，诸如，例如Sambrook 等人，Molecular Cloning，A Laboratory Manual(见上文)中描述的那些。
免疫原性组合物和疫苗
本发明还提供包含本发明的多肽和药学上可接受的赋形剂的免疫原性组合物。
在一个实施方案中，免疫原性组合物还包含佐剂。用于与使用本发明的方法制成的细菌毒素或缀合物混合的合适佐剂的选择在本领域技术人员的知识范围内。合适的佐剂包括铝盐诸如氢氧化铝凝胶或磷酸铝或明矾，但还可以是其他金属盐诸如钙盐、镁盐、铁盐或锌盐，或者可以是酰化酪氨酸、或酰化糖类、阳离子或阴离子衍生的糖类、或聚磷腈的不溶性悬浮液。
在一个实施方案中，免疫原性组合物还包含额外的抗原。在一个实施方案中，额外抗原是来源自选自下述细菌的抗原：肺炎链球菌、流感嗜血杆菌、脑膜炎奈瑟氏菌、大肠杆菌、卡他拉莫氏菌、破伤风、白喉、百日咳、表皮葡萄球菌、肠球菌、金黄色葡萄球菌和铜绿假单胞菌。在进一步的实施方案中，本发明的免疫原性组合物可以包含来自艰难梭菌的进一步的抗原，例如S层蛋白(WO01/73030)。
还提供包含免疫原性组合物的疫苗，该疫苗还可以包含药学上可接受的赋形剂。
可以通过经全身或粘膜途径施用包含本发明的免疫原性组合物的疫苗制剂，使用所述疫苗来保护对艰难梭菌感染易感的哺乳动物或治疗患有艰难梭菌感染的哺乳动物。这些施用可以包括经肌内、腹膜内、皮内或皮下途径注射；或通过向口腔/消化道、呼吸道、生殖泌尿道的粘膜施用。尽管本发明的疫苗可以作为单一剂量施用，但其组分也可以在同一时间或以不同次数一同共施用(例如，肺炎球菌糖缀合物可以单独、在疫苗的任何细菌蛋白组分施用的同一时间或其后1-2周施用，以协调对于彼此的免疫应答)。除了单一的施用途径之外，可以使用2种不同的施用途径。例如，糖类或糖缀合物可以肌内(IM)或皮内(ID)施用，并且细菌蛋白可以鼻内(IN)或皮内(ID)施用。此外，本发明的疫苗，对于初免剂量可以肌内施用，并且对于加强剂量，可以鼻内施用。
疫苗中毒素的含量通常将在1-250μg的范围内，任选5-50μg，最常见在5 - 25μg的范围内。初始接种后，受试者可以接受一次或适当分隔开的几次加强免疫。疫苗制剂通常描述于Vaccine Design ("The subunit and adjuvant approach" (Powell M. F.和NewmanM.J.编辑) (1995) Plenum Press New York)中。在脂质体内封装由Fullerton的美国专利4,235,877描述。
在本发明的一个方面，提供疫苗试剂盒，其包括含有本发明的免疫原性组合物的小瓶，任选以冷冻干燥形式，并且还包括含有如本文描述的佐剂的小瓶。预想在本发明的该方面中，将使用佐剂来重构冷冻干燥的免疫原性组合物。
本发明的进一步的方面是预防或治疗艰难梭菌感染的方法，其包括向宿主施用免疫保护性剂量的本发明的免疫原性组合物或疫苗或试剂盒。在一个实施方案中，提供预防或治疗艰难梭菌感染的原发性和/或复发事件的方法，其包括向宿主施用免疫保护性剂量的本发明的免疫原性组合物或疫苗或试剂盒。
本发明的进一步的方面是用于治疗或预防艰难梭菌疾病的本发明的免疫原性组合物。在一个实施方案中，提供用于治疗或预防艰难梭菌疾病的原发性和/或复发事件的本发明的免疫原性组合物。
本发明的进一步的方面是本发明的免疫原性组合物或疫苗或试剂盒在制造用于治疗或预防艰难梭菌疾病的药物中的用途。在一个实施方案中，提供用于制造用于治疗或预防艰难梭菌疾病的原发性和/或复发事件的药物中的本发明的免疫原性组合物。
将“左右”或“大约”定义为在本发明的目的的给定数值的10％或多或少之内。
在每一种情况下，本发明人意在本文的术语“包含(comprising)”、“包含(comprise)”和“包含(comprises)”可以任选地分别由术语“组成(consisting of)”、“组成(consist of)”和“组成(consists of)”所替换。术语“包含(comprises)”表示“包括(includes)”。因此，除非上下文另外需要，将理解词语“包含(comprises)”及变式诸如“包含(comprise)”和“包含(comprising)”意指包括陈述的化合物或组合物(例如核酸、多肽、抗原)或步骤，或化合物或步骤的组，但不排除任何其他的化合物、组合物、步骤、或其组。缩写“例如(e.g.)”来源于拉丁文例如(exempli gratia)，并用于本文以表示非限定性实例。因此，缩写“例如(e.g.)”与术语“例如(for example)”同义。
本文中涉及本发明的“疫苗组合物”的实施方案也适用于涉及本发明的“免疫原性组合物”的实施方案，并且反之亦然。
除非另有解释，本文使用的所有技术和科学术语具有如本公开内容所属技术领域的普通技术人员通常理解的相同含义。分子生物学中普通术语的定义可以见于BenjaminLewin, Genes V，由Oxford University Press出版, 1994 (ISBN 0-19-854287-9)、Kendrew等人(编辑)，The Encyclopedia of Molecular Biology，由Blackwell ScienceLtd.出版，1994 (ISBN 0-632-02182-9)、和Robert A. Meyers (编辑)，Molecular Biology and Biotechnology: a Comprehensive Desk Reference，由VCH Publishers,Inc.出版，1995 (ISBN 1-56081-569-8)。
除非上下文明确另有说明，单数术语“一个(a)”、“一个(an)”和“该(the)”包括复数指示物。类似地，除非上下文明确另有说明，词语“或”旨在包括“和”。术语“复数”指两个或更多。还应理解对核酸或多肽给出的所有碱基大小或氨基酸大小、和所有分子量(molecular weight)或分子量(molecular mass)值均是近似的，并且提供用于说明。此外，关于物质(诸如抗原)的浓度或水平给出的数值界限可以是近似的。
本专利说明书中引用的所有参考文献或专利申请以其整体通过引用并入本文。
为了本发明可以更好地理解，描述以下实施例。这些实施例仅为说明目的，而不应以任何方式解释为限定本发明的范围。
具体实施方式
实施例1：五种艰难梭菌ToxA-ToxB融合体的设计
设计包含ToxA和ToxB的C末端重复结构域的片段的融合蛋白。这些融合体包含ToxA的C末端重复结构域的片段和ToxB的C末端重复结构域的片段，和ToxA片段的C末端与ToxB片段的N末端之间的连接。设计两种策略，在第一种策略中；设计融合体从而在两条片段之间的连接处保持长螺线管结构。在第二种策略中，融合体的两条片段由接头分隔开，以允许它们独立的正确折叠。
ToxA和B的C末端部分由重复序列构成：短重复(SR)和长重复(LR) (PNAS 2005vol 102 :18373-18378)。
ToxA的C末端结构域的部分已知的3D结构(PNAS 2005 Greco等人, vol10218373-18378；Nature Structural & Molecular biology 2006 vol 13(5) :460-461;PDB代号:2F6E、2G7C和2QJ6)。
本发明人预测在ToxA和ToxB的C末端部分的残基之间存在两种重要的相互作用。第一种相互作用发生包含在LR及其在前的SR中的残基之间，并且对于维持螺线管类结构是重要的。第二类相互作用发生包含在LR和随后的SR中的残基之间，并且该相互作用介导毒素的碳水化合物结合功能。
定义新的“结构－功能”重复SR-LR-SR。在本发明人设计的融合体中该重复的结构保持完整。
图2描绘了ToxA和ToxB的C末端结构域和定义的“SR-LR-SR”盒。
ToxA和ToxB重复的短重复(SR)和长重复(LR)的位置显示于表1中。
ToxA和ToxB的C末端结构域中包含的“SR-LR-SR”盒的列表显示于表2中。
最后，两个LRs之间的SRs数目将维持在设计的融合体中，以保持长螺线管类结构。
在设计用于融合体的连接之前，定义两种工作假设：第一种假设，融合体越短，融合体在表达过程中稳定的可能性越大；第二种假设，根据“SR-LR-SR”盒的概念，必须选择起始位置以确保该之前定义的SR-LR-SR盒的第一个SR的正确折叠。因此，融合体在位于SR-LR-SR盒之前的SR的起始处起始。使用这两种假设，分析了三个起始位置：ToxA的残基2370、2234和2121。
排除了起始位置2370。由于涉及对于蛋白结构稳定性重要的相互作用的残基之一不是保守的，所以还排除了起始位置2234。因此，决定所有设计的融合体将在ToxA的残基2121处起始。
所有融合体将在ToxB的最后一个残基处结束。
设计四个融合体(F1-4)以维持在两个融合片段之间的长螺线管类结构中的完整融合。
使用相同的假设设计融合体1(F1)和2(F2)。已经使用多重比对软件比较了ToxA和ToxB的所有SR蛋白序列(ClustalW - Thompson JD等人，(1994) Nucleic Acids Res.,22, 4673-4680)。更相似的序列为ToxA的第三个SR VIII和ToxB的第三个SR II和ToxB的第三个SR III。为了在ToxB的这两个SR之间做出选择，使用部分的ToxA C末端结构域的已知3D结构(PDB代号：2QJ6)在ToxB的C末端部分上进行结构同源性建模(使用SwissModel 界面- Arnold K等人，(2006) Bioinformatics, 22, 195-201)。使用ToxA的第三个SR VIII，用ToxB的第三个SR II获得了最佳局部结构叠加(使用SwissPDBViewer进行 - Guex N等人，(1997), Electrophoresis 18, 2714-2723)。因此，设计了两个连接：第一个在ToxA的第三个SR VIII和ToxB的第四个SR II之间(F1)，并且第二个在ToxA的第二个SR VIII和ToxB的第三个SR II之间(F1)。这些连接分别显示于图3和4中。
为了设计融合体3 (F3)，在ToxA的部分C末端结构域的已知结构和ToxB的C末端结构域的预测结构之间进行整体结构叠加(使用SwissModel和SwissPDBViewer软件)。在ToxA的LR VII和ToxB的LR II之间发现最佳叠加。因此，决定在该类似的LR中产生连接。首先在其中ToxA和ToxB之间的序列是保守的区域中进行连接，随后为了保持融合体的ToxA部分，残基与在前的SR相互作用，并且最后，为了保持ToxB部分，残基与随后的SR相互作用。该连接显示于图5中。
对于融合体4 (F4)的设计，将ToxB的C末端结构域分为4个片段并且在它们上进行更精确的同源性建模(SwissModel)。实现该分裂(split)以保持“SR-LR-SR”盒完整(每一结构域在跟随LR的SR的末端处结束)。进行这些片段的预测结构和ToxA的已知3D结构之间的结构叠加，并且对于ToxB的第三个SR (SR I)和ToxA的最后一个SR (第三个SR VIII)获得了最佳结构叠加。因此，在ToxA的第二个SR VIII和ToxB的第三个SRI之间完成了连接。该设计显示于图6中。
设计最后一个融合体(F5)以允许融合体的两个片段的独立的正确折叠。在ToxA蛋白序列的最后一个残基和ToxB的第四个SR II的起始之间加入接头(始终考虑完整的“SR-LR-SR”盒的重要性)。加入仅一个外源残基(甘氨酸)作为接头并且位于两个现存的甘氨酸之间。因此，还可以将接头描述为由已知的(对于ToxA)和预测的(对于ToxB)β链环绕的3个甘氨酸构成。该最后一个设计显示于图7中。
实施例2：融合蛋白的克隆表达和纯化
表达质粒和重组菌株
使用标准方法，用NdeI/XhoI限制位点将编码ToxA和ToxB的部分C末端结构域的融合蛋白(SEQ ID NO:3、4、5、6和7)和His标签的基因克隆入pET24b(+)表达载体(Novagen)中。按照使用CaCl2处理的细胞的标准方法，用重组表达载体通过转化大肠杆菌菌株BLR (DE3)产生最终构建体(Hanahan D. « Plasmid transformation by Simanis.» In Glover, D.M. (Ed), DNA cloning.IRL Press London.(1985): 第109-135页)。
宿主菌株：
BLR(DE3)。BLR是BL21的recA衍生物。具有命名(DE3)的菌株对于包含IPTG诱导型T7RNA聚合酶的λ原噬菌体是溶原的。设计λ DE3溶原体用于来自pET载体的蛋白表达。该菌株也缺乏lon和ompT蛋白酶。
基因型：大肠杆菌BLR::DE3菌株， 。
重组蛋白的表达：
从琼脂板上刮下大肠杆菌转化子并用于接种200 ml的LBT肉汤培养基± 1% (w/v)葡萄糖+卡那霉素(50 µg/ml)，以获得0.1 -0.2之间的O.D.600nm。在37℃，250 RPM下过夜孵育培养物。
将该过夜培养物稀释在500 ml的含有卡那霉素(50 µg/ml)的LBT培养基至1:20，并且在37℃下以250 rpm的转速生长直到O.D.620达到0.5/0.6。
在O.D.600nm为0.6左右时，将培养物冷却下来，随后通过加入1 mM异丙基-β-D-1-硫代半乳糖苷(IPTG；EMD Chemicals Inc., 目录号: 5815)诱导重组蛋白的表达，并在23℃，250 RPM下过夜孵育。
过夜诱导后(16个小时左右)，评估诱导后的O.D.600nm，并且以14 000 RPM将培养物离心15分钟，并将沉淀分离地冷冻于-20℃。
纯化：
在包含500 mM NaCl和蛋白酶抑制剂的混合物(完全的，Roche)的20 mM N-二(羟乙基)甘氨酸缓冲液(pH 8.0)中重悬细菌沉淀。使用French Press系统20 000 PSI裂解细菌。通过离心例如在4℃下以20 000g 30分钟分离可溶(上清液)和不可溶(沉淀)组分。
在IMAC上在非变性条件下纯化6-His标签化的蛋白。将可溶组分装载到使用与用于细菌重悬相同的缓冲液预平衡的GE柱(15 ml例如)(Ni装载的)上。在装载上柱后，将柱用相同缓冲液洗涤。使用含有500 mM NaCl和不同浓度的咪唑(5-600 mM)的20mM N-二(羟乙基)甘氨酸缓冲液(pH 8.0)进行洗脱。凝胶分析后，选择更纯的级分，浓缩并装载到SEC层析上用于进一步的纯化步骤。
通过SDS-PAGE根据纯度选择含有融合蛋白的级分，并对N-二(羟乙基)甘氨酸缓冲液(20mM N-二(羟乙基)甘氨酸、150 mM NaCl，含有或不含有5mM EDTA pH8.0)透析，使用BioRad的DC Protein Assay测定蛋白浓度。因此将蛋白混合，在0.22 µm上无菌过滤，保存于-80℃。
或者，在IMAC纯化之前，进行使用用于装载和洗涤的2mM N-二(羟乙基)甘氨酸缓冲液(pH 8.0)的DEAE纯化步骤，并且使用相同缓冲液但加入1M NaCl的梯度洗脱。
实施例3：分离的艰难梭菌Tox A和Tox B片段的克隆表达和纯化
表达质粒和重组菌株。
使用标准方法，用NdeI/XhoI限制位点将编码ToxA和ToxB的蛋白片段(SEQ ID NO:8和SEQ ID NO:9)和His标签的基因克隆入pET24b(+)表达载体(Novagen)中。按照使用CaCl2处理的细胞的标准方法用重组表达载体通过转化大肠杆菌菌株BLR (DE3)产生最终构建体(Hanahan D. « Plasmid transformation by Simanis.» In Glover, D. M.(Ed), DNA cloning.IRL Press London.(1985): 第109-135页)。
宿主菌株：
BLR(DE3)。BLR是BL21的recA衍生物。具有命名(DE3)的菌株对于包含IPTG诱导型T7RNA聚合酶的λ原噬菌体是溶原的。设计λ DE3溶原体用于来自pET载体的蛋白表达。该菌株也缺乏lon和ompT蛋白酶。
基因型：大肠杆菌BLR::DE3菌株，
。
重组蛋白的表达：
从琼脂板上刮下大肠杆菌转化子并用于接种200 ml的LBT肉汤培养基± 1% (w/v)葡萄糖+卡那霉素(50 µg/ml)，以获得0.1 -0.2之间的O.D.600nm。在37℃，250 RPM下过夜孵育培养物。
在500 ml的含有卡那霉素(50 µg/ml)的LBT培养基中将该过夜培养物稀释至1:20，并且在37℃下以250 rpm的转速生长直到O.D.620达到0.5/0.6。
在600nm处的O.D.为0.6左右时，将培养物冷却下来，随后加入1 mM异丙基-β-D-1-硫代半乳糖苷(IPTG; EMD Chemicals Inc., 目录号: 5815)诱导重组蛋白的表达，并在23℃，250 RPM下过夜孵育。
过夜诱导后(16个小时左右)，评估诱导后的600nm处的O.D.，并且以14 000 RPM将培养物离心15分钟，并将沉淀分离地冷冻于-20℃。
纯化：
在包含500 mM NaCl并添加有蛋白酶抑制剂的混合物(完全的，但无EDTA，Roche目录11873580001)和核酸酶(benzonase)的20 mM N-二(羟乙基)甘氨酸缓冲液(pH 8.0)中重悬细菌沉淀。(Roche目录1.01695.0001)。使用French Press系统2 X 20 000 PSI裂解细菌。通过在4℃下以34 000g或48 000g离心25-30分钟分离可溶(上清液)和不可溶(沉淀)组分。收集上清液并在0.22 µm滤器上过滤。
在IMAC上在非变性条件下纯化6-His标签化的蛋白。将可溶组分装载到使用与用于细菌重悬相同的缓冲液预平衡的GE柱(例如15 ml)(Ni装载的)上。装载后，将柱用相同缓冲液洗涤。
对于ToxA：
使用含有500 mM NaCl和不同浓度的咪唑(5-100 mM)的20mM N-二(羟乙基)甘氨酸缓冲液(pH 8.0)进行洗脱。凝胶分析后，选择更纯的级分，浓缩并装载到SEC层析(SUPERDEXTM75)上，用于在无咪唑的相同缓冲液中进一步的纯化步骤。
对于ToxB：
用含有500 mM NaCl和0.5 %脱氧胆酸的20mM N-二(羟乙基)甘氨酸缓冲液(pH 8.0)或含有150 mM NaCl的相同缓冲液进行第二次洗涤。使用含有500 mM NaCl和不同浓度的咪唑(10-500 mM)的20mM N-二(羟乙基)甘氨酸缓冲液(pH 8.0)进行洗脱。凝胶分析后，选择更纯的级分，添加5 mM EDTA并装载到SEC层析(SUPERDEXTM 200)上，用于在含有5 mM EDTA的相同缓冲液中进一步的纯化步骤。
通过SDS-PAGE根据纯度选择含有ToxA或ToxB片段的级分，并对N-二(羟乙基)甘氨酸缓冲液(20mM N-二(羟乙基)甘氨酸、150 mM NaCl,pH8.0)透析，使用BioRad的RCDCProtein Assay测定蛋白浓度。因此，将蛋白混合，在0.22 µm上无菌过滤，保存于-80℃。
实施例4：五种艰难梭菌ToxA-ToxB融合体的分子量评价
使用分析超速离心，通过测定分子响应离心力而移动的速率来确定蛋白样品中不同种类在溶液中的同质性和大小分布。这基于通过沉降速度实验获得的不同种类的沉降系数的计算结果，其依赖于它们的分子形状和质量。
1. AN-60Ti转子已经被平衡至15℃后，将蛋白样品在Beckman-CoulterPROTEOMELABTM XL-1分析超速离心机中以42 000RPM旋转。
a. F1融合蛋白，500µg/ml，20mM N-二(羟乙基)甘氨酸，150mM NaCl，pH8,0
b. F2融合蛋白，500µg/ml，20mM N-二(羟乙基)甘氨酸，150mM NaCl， pH8,0
c. F3融合蛋白，500µg/ml，20mM N-二(羟乙基)甘氨酸，150mM NaCl，pH8,0
d. F4融合蛋白，500µg/ml，20mM N-二(羟乙基)甘氨酸，150mM NaCl，pH8,0
e. F5融合蛋白，500µg/ml，20mM N-二(羟乙基)甘氨酸，150mM NaCl，pH8,0。
2. 对于数据采集，在280nm处每5分钟记录160次扫描。
3. 使用程序SEDFIT进行数据分析来测定C(S)分布。用SEDNTERP软件从它们的氨基酸序列进行蛋白的微分比容的测定。SEDNTERP也用于测定缓冲液的粘度和密度。
4. 考虑到与C(M)分布(浓度相比于分子量)相比，原始数据是表征混合物的大小分布的更好的表示法，所以从C(S)分布曲线(浓度相比于沉降系数)中测定不同种类的分子量。
图8描绘通过沉降速度分析超速离心测定的ToxA-ToxB融合体的分布。
从所有五种ToxA-ToxB融合蛋白的C(S)分布检测到的主要种类的分子量对应于它们的单体形式。对五种融合体测定到的最佳拟合摩擦比率均在2-2.2之间。这可以说明蛋白以伸长形式存在于溶液中，这可以是与蛋白结构一致的。
实施例5：通过圆二色性和荧光光谱学评价艰难梭菌ToxA-ToxB融合体的二级结构 和三级结构
使用圆二色性，通过测定由于结构不对称而产生的左旋偏振光相对于右旋偏振光的吸收差异来确定蛋白的二级结构组成。蛋白展示出β折叠、α螺旋还是随机卷曲结构，在远UV区(190-250nm)中的CD光谱的形状和大小是不同的。给定蛋白样品中的每一种二级结构类型的相对丰度可以通过与参考光谱比较来计算。
蛋白样品的三级结构可以通过评价芳香族氨基酸的固定化来评定。在近UV区(250-50nm)中观察到CD信号可以归因于苯丙氨酸、酪氨酸和色氨酸残基的极化，并且是蛋白折叠为完全确定的结构的良好指示。
使用以下方案：
1. 在Jasco J-720旋光分光计上从178-250nm使用0.01cm的光程以1nm分辨率和带宽测定远UV光谱。通过Peltier恒温的RTE-111细胞模块保持细胞的温度为23℃。在测定过程中保持10L/分钟的氮气流。
2. 在Jasco J-720旋光分光计上从250-300nm使用0.01cm的光程以1nm分辨率和带宽测定近UV光谱。通过Peltier恒温的RTE-111细胞模块保持细胞的温度为23℃。在测定过程中保持6L/分钟的氮气流。
对所有五种ToxA-ToxB融合蛋白的远UV光谱(图9)的观察表明低含量的α螺旋结构和高含量的β折叠结构。而且，所有蛋白均在230nm处展示出最大值，其对于可溶性球状蛋白而言是不常见的。这已在文献中得到深入表征，并且与已知缺乏α螺旋及具有高含量的β折叠和芳香族氨基酸的一小类的蛋白相关(Zsila, Analytical Biochemistry, 391( 2009)154-156)。那些特性与对ToxA-ToxB融合蛋白预期的结构是一致的。比较了在230nm处展示出具有阳性信号的特征CD光谱的13个蛋白的晶体结构(Protein Data Bank)。那些蛋白的平均二级结构含量为42% β折叠±9%和7% α螺旋±6%。这有力地说明ToxA-ToxB融合蛋白的光谱特征是含有高β折叠和低α螺旋的蛋白的诊断结果。
对所有五种融合蛋白的近UV光谱的形状(图10)的观察说明至少某些芳香族氨基酸是固定的，其是紧密且特异的三级结构的有力说明。此外，用变性浓度的尿素处理蛋白导致近UV信号的消失，其该特征光谱是归因于蛋白折叠的另一个指示。
实施例6：用Tox A或Tox B片段和ToxA-ToxB融合体免疫小鼠
用实施例2和3中描述的构建体免疫Balb/C小鼠。
小鼠免疫
在第0、14和28天用3μg或10 µg的toxA和toxB的分离的片段(见实施例2)以及用AS03B佐剂化的ToxA-ToxB融合蛋白(见实施例3)肌内免疫15只雌性Balb/c小鼠的组。用AS03B单独接种10只小鼠的对照组。
在第42天(后III)收集的个体血清中测定抗ToxA和抗ToxB ELISA效价。
在混合的后III血清中测定血细胞凝集抑制效价。
抗ToxA和抗ToxB ELISA应答：方案
将toxA或toxB片段的样品在磷酸缓冲盐溶液(PBS)中以1 µg/ml包被在高结合微量滴定板(Nunc MAXISORPTM)上，在4℃下过夜。在室温振荡下，用PBS-BSA 1%将板封闭30分钟。将小鼠抗血清以1/500预稀释在PBS-BSA 0.2%-TWEENTM 0.05%中。并随后，在微量培养板中进行进一步的两倍稀释，并在室温振荡下孵育30分钟。洗涤后，使用以1:5000稀释在PBS-BSA0.2%-吐温0.05%中的Jackson ImmunoLaboratories Inc.过氧化物酶缀合的affiniPure山羊抗小鼠IgG (H+L) (参考:115-035-003)检测结合的鼠抗体。将检测抗体在室温(RT)振荡下孵育30分钟。在室温下，使用4 mg O-苯二胺 (OPD) + 5 µl H2O2/10 ml pH4.5 0.1M柠檬酸盐缓冲液避光显色15分钟。用50 µl HCl终止反应，并在490 nm处读取相对于620 nm的光密度(OD)。
血清中存在的抗ToxA或抗ToxB抗体的水平以中点效价表示。在每一处理组中对15个样品计算GMT(对于对照组为10个)。
血细胞凝集抑制测定：方案
在96孔U底微量培养板中进行小鼠混合抗血清(25µl)在磷酸缓冲盐溶液 (PBS)中的连续两倍稀释。
随后加入25 µl的天然的毒素A (0.2 µg/孔)并将板在室温下孵育30分钟。
孵育后，向每个孔中加入50 µl以2％稀释的纯化的兔红细胞。将板在37℃下孵育2小时。
对板进行目视分析，血细胞凝集显示为孔中弥散的红细胞，并且血细胞凝集抑制观察为孔中沉淀的红点。
将抑制效价定义为抑制血细胞凝集的血清最高稀释度的倒数。
细胞毒性测定
在37℃和5% CO2下，将IMR90成纤维细胞在EMEM + 10%胎牛血清 + 1% 谷氨酰胺 + 1%抗生素(青霉素-链霉素-两性霉素)中培养，并以5.104个细胞/孔的密度接种在96孔组织培养板中。
24小时后，从孔中去除细胞培养基。
在细胞培养基中进行小鼠混合抗血清(50µl)的连续两倍稀释。
随后加入50 µl的天然毒素B (0.5ng/ml)，并将板在37℃和5% CO2中孵育24小时。
在24小时后观察细胞，并测定圆形细胞的比例。
将抑制效价定义为抑制50％的细胞变圆的血清最高稀释度的倒数。
结果：
使用Tox A抗体的ELISA结果描述于图11中。用单独ToxA免疫后诱导了抗ToxA抗体，但使用5种融合体的每一种免疫后也诱导了抗ToxA抗体。
在血细胞凝集测定中检测这些抗体的功能特性。由于用ToxB没有观察到血细胞凝集，因此该测定仅适用于Tox A评价。
血细胞凝集(Haemagglutination)抑制效价描述于图12中。用抗Tox A片段血清或针对ToxA-ToxB融合体的每一种的血清，观察到了血细胞凝集抑制。
还进行了使用ToxB抗体的ELISA；其结果显示在图13中。用单独ToxB片段棉衣后诱导抗Tox B抗体，但使用F2、F3和F4融合体免疫后也诱导抗Tox B抗体。
细胞毒性抑制效价描述于图14中。使用来自用ToxB片段或ToxA-ToxB融合体免疫的小鼠的血清获得的抑制效价大于使用对照血清获得的抑制效价。
实施例7：4个进一步的融合蛋白的设计、克隆、表达和纯化
使用实施例1中描述的设计原则设计四个进一步的融合蛋白，将它们命名为F54 Gly(SEQ ID NO:21)、F54 New (SEQ ID NO:23)、F5 ToxB (SEQ ID NO:25)和F52 New (SEQ IDNO:27)。
按照实施例2中描述的方案表达这些融合蛋白。
实施例8：SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25和SEQ ID NO:27中描述的 艰难梭菌ToxA-ToxB融合体的分子量的评价
如实施例4中所描述测定SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25和SEQ ID NO:27中描述的融合体的分子量。
图15描绘通过沉降速度分析超速离心测定的这四个进一步的融合蛋白的分布。
从SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25和SEQ ID NO:27中描述的所有四个蛋白融合体的C(S)分布测定的主要种类的分子量对应于它们的单体形式，并且所有蛋白均展示出与F1-F5融合体类似的沉降特性。
实施例9：SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25和SEQ ID NO:27中描述的 艰难梭菌ToxA-ToxB融合体的二级结构和三级结构的评价
按照实施例5中描述的方法评价SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25和SEQ IDNO:27中描述的融合体的二级结构和三级结构。对这些融合蛋白的远UV CD可以见于图16中，并且对这些融合体的近UV光谱可以见于图17中。
SEQ ID NO:21、SEQ ID NO:23、SEQ ID NO:25和SEQ ID NO:27中描述的蛋白的近和远UV CD光谱分析显示相比F1-F5融合体，所有四个均具有同样高的β折叠结构。此外，近UV光谱观察显示，相比于F1-F5融合体，在三级结构中芳香族氨基酸的位置没有显著差异。
实施例10：用Tox A-Tox B融合体免疫小鼠
如实施例6中所述，用四种融合蛋白构建体F54 Gly (SEQ ID NO:21)、F54 New (SEQID NO:23)、F5 ToxB (SEQ ID NO:25)和F52 New (SEQ ID NO:27)免疫Balb/c小鼠。
使用实施例6中描述的抗ToxA和抗ToxB ELISA应答：方案进行ELISA，除了此处将toxA或toxB片段的样品在磷酸缓冲盐溶液中以2 µg/ml包被在高结合微量滴定板上。如实施例6中所述进行血细胞凝集抑制测定。如实施例6中所述进行toxB细胞毒性测定。如下文描述进行进一步的toxA细胞毒性测定。
ToxA细胞毒性测定
在37℃和5% CO2，将HT29细胞在DMEM +10%胎牛血清+1%谷氨酰胺+1% 抗生素(青霉素-链霉素-两性霉素)中培养，并以5.104个细胞/孔的密度接种在96孔组织培养板中。
24小时后，从孔中去除细胞培养基。
在细胞培养基中进行小鼠混合抗血清(50µl)的连续两倍稀释。
随后加入50 µl的天然毒素B (0.15ng/ml)并将板在37℃和5% CO2中孵育48小时。
在48小时后观察细胞，并测定圆形细胞的比例。
抗toxA ELISA、抗toxB ELISA、血细胞凝集抑制和细胞毒性测定的结果分别描述于图18、19、20、21和22中。
序列表
<110> GlaxoSmithKline Biologicals s.a
<120> 免疫原性组合物
<130> VB64650
<160> 35
<170> FastSEQ for Windows Version 4.0
<210> 1
<211> 2710
<212> PRT
<213> 艰难梭菌
<400> 1
Met Ser Leu Ile Ser Lys Glu Glu Leu Ile Lys Leu Ala Tyr Ser Ile
1 5 10 15
Arg Pro Arg Glu Asn Glu Tyr Lys Thr Ile Leu Thr Asn Leu Asp Glu
20 25 30
Tyr Asn Lys Leu Thr Thr Asn Asn Asn Glu Asn Lys Tyr Leu Gln Leu
35 40 45
Lys Lys Leu Asn Glu Ser Ile Asp Val Phe Met Asn Lys Tyr Lys Thr
50 55 60
Ser Ser Arg Asn Arg Ala Leu Ser Asn Leu Lys Lys Asp Ile Leu Lys
65 70 75 80
Glu Val Ile Leu Ile Lys Asn Ser Asn Thr Ser Pro Val Glu Lys Asn
85 90 95
Leu His Phe Val Trp Ile Gly Gly Glu Val Ser Asp Ile Ala Leu Glu
100 105 110
Tyr Ile Lys Gln Trp Ala Asp Ile Asn Ala Glu Tyr Asn Ile Lys Leu
115 120 125
Trp Tyr Asp Ser Glu Ala Phe Leu Val Asn Thr Leu Lys Lys Ala Ile
130 135 140
Val Glu Ser Ser Thr Thr Glu Ala Leu Gln Leu Leu Glu Glu Glu Ile
145 150 155 160
Gln Asn Pro Gln Phe Asp Asn Met Lys Phe Tyr Lys Lys Arg Met Glu
165 170 175
Phe Ile Tyr Asp Arg Gln Lys Arg Phe Ile Asn Tyr Tyr Lys Ser Gln
180 185 190
Ile Asn Lys Pro Thr Val Pro Thr Ile Asp Asp Ile Ile Lys Ser His
195 200 205
Leu Val Ser Glu Tyr Asn Arg Asp Glu Thr Val Leu Glu Ser Tyr Arg
210 215 220
Thr Asn Ser Leu Arg Lys Ile Asn Ser Asn His Gly Ile Asp Ile Arg
225 230 235 240
Ala Asn Ser Leu Phe Thr Glu Gln Glu Leu Leu Asn Ile Tyr Ser Gln
245 250 255
Glu Leu Leu Asn Arg Gly Asn Leu Ala Ala Ala Ser Asp Ile Val Arg
260 265 270
Leu Leu Ala Leu Lys Asn Phe Gly Gly Val Tyr Leu Asp Val Asp Met
275 280 285
Leu Pro Gly Ile His Ser Asp Leu Phe Lys Thr Ile Ser Arg Pro Ser
290 295 300
Ser Ile Gly Leu Asp Arg Trp Glu Met Ile Lys Leu Glu Ala Ile Met
305 310 315 320
Lys Tyr Lys Lys Tyr Ile Asn Asn Tyr Thr Ser Glu Asn Phe Asp Lys
325 330 335
Leu Asp Gln Gln Leu Lys Asp Asn Phe Lys Leu Ile Ile Glu Ser Lys
340 345 350
Ser Glu Lys Ser Glu Ile Phe Ser Lys Leu Glu Asn Leu Asn Val Ser
355 360 365
Asp Leu Glu Ile Lys Ile Ala Phe Ala Leu Gly Ser Val Ile Asn Gln
370 375 380
Ala Leu Ile Ser Lys Gln Gly Ser Tyr Leu Thr Asn Leu Val Ile Glu
385 390 395 400
Gln Val Lys Asn Arg Tyr Gln Phe Leu Asn Gln His Leu Asn Pro Ala
405 410 415
Ile Glu Ser Asp Asn Asn Phe Thr Asp Thr Thr Lys Ile Phe His Asp
420 425 430
Ser Leu Phe Asn Ser Ala Thr Ala Glu Asn Ser Met Phe Leu Thr Lys
435 440 445
Ile Ala Pro Tyr Leu Gln Val Gly Phe Met Pro Glu Ala Arg Ser Thr
450 455 460
Ile Ser Leu Ser Gly Pro Gly Ala Tyr Ala Ser Ala Tyr Tyr Asp Phe
465 470 475 480
Ile Asn Leu Gln Glu Asn Thr Ile Glu Lys Thr Leu Lys Ala Ser Asp
485 490 495
Leu Ile Glu Phe Lys Phe Pro Glu Asn Asn Leu Ser Gln Leu Thr Glu
500 505 510
Gln Glu Ile Asn Ser Leu Trp Ser Phe Asp Gln Ala Ser Ala Lys Tyr
515 520 525
Gln Phe Glu Lys Tyr Val Arg Asp Tyr Thr Gly Gly Ser Leu Ser Glu
530 535 540
Asp Asn Gly Val Asp Phe Asn Lys Asn Thr Ala Leu Asp Lys Asn Tyr
545 550 555 560
Leu Leu Asn Asn Lys Ile Pro Ser Asn Asn Val Glu Glu Ala Gly Ser
565 570 575
Lys Asn Tyr Val His Tyr Ile Ile Gln Leu Gln Gly Asp Asp Ile Ser
580 585 590
Tyr Glu Ala Thr Cys Asn Leu Phe Ser Lys Asn Pro Lys Asn Ser Ile
595 600 605
Ile Ile Gln Arg Asn Met Asn Glu Ser Ala Lys Ser Tyr Phe Leu Ser
610 615 620
Asp Asp Gly Glu Ser Ile Leu Glu Leu Asn Lys Tyr Arg Ile Pro Glu
625 630 635 640
Arg Leu Lys Asn Lys Glu Lys Val Lys Val Thr Phe Ile Gly His Gly
645 650 655
Lys Asp Glu Phe Asn Thr Ser Glu Phe Ala Arg Leu Ser Val Asp Ser
660 665 670
Leu Ser Asn Glu Ile Ser Ser Phe Leu Asp Thr Ile Lys Leu Asp Ile
675 680 685
Ser Pro Lys Asn Val Glu Val Asn Leu Leu Gly Cys Asn Met Phe Ser
690 695 700
Tyr Asp Phe Asn Val Glu Glu Thr Tyr Pro Gly Lys Leu Leu Leu Ser
705 710 715 720
Ile Met Asp Lys Ile Thr Ser Thr Leu Pro Asp Val Asn Lys Asn Ser
725 730 735
Ile Thr Ile Gly Ala Asn Gln Tyr Glu Val Arg Ile Asn Ser Glu Gly
740 745 750
Arg Lys Glu Leu Leu Ala His Ser Gly Lys Trp Ile Asn Lys Glu Glu
755 760 765
Ala Ile Met Ser Asp Leu Ser Ser Lys Glu Tyr Ile Phe Phe Asp Ser
770 775 780
Ile Asp Asn Lys Leu Lys Ala Lys Ser Lys Asn Ile Pro Gly Leu Ala
785 790 795 800
Ser Ile Ser Glu Asp Ile Lys Thr Leu Leu Leu Asp Ala Ser Val Ser
805 810 815
Pro Asp Thr Lys Phe Ile Leu Asn Asn Leu Lys Leu Asn Ile Glu Ser
820 825 830
Ser Ile Gly Asp Tyr Ile Tyr Tyr Glu Lys Leu Glu Pro Val Lys Asn
835 840 845
Ile Ile His Asn Ser Ile Asp Asp Leu Ile Asp Glu Phe Asn Leu Leu
850 855 860
Glu Asn Val Ser Asp Glu Leu Tyr Glu Leu Lys Lys Leu Asn Asn Leu
865 870 875 880
Asp Glu Lys Tyr Leu Ile Ser Phe Glu Asp Ile Ser Lys Asn Asn Ser
885 890 895
Thr Tyr Ser Val Arg Phe Ile Asn Lys Ser Asn Gly Glu Ser Val Tyr
900 905 910
Val Glu Thr Glu Lys Glu Ile Phe Ser Lys Tyr Ser Glu His Ile Thr
915 920 925
Lys Glu Ile Ser Thr Ile Lys Asn Ser Ile Ile Thr Asp Val Asn Gly
930 935 940
Asn Leu Leu Asp Asn Ile Gln Leu Asp His Thr Ser Gln Val Asn Thr
945 950 955 960
Leu Asn Ala Ala Phe Phe Ile Gln Ser Leu Ile Asp Tyr Ser Ser Asn
965 970 975
Lys Asp Val Leu Asn Asp Leu Ser Thr Ser Val Lys Val Gln Leu Tyr
980 985 990
Ala Gln Leu Phe Ser Thr Gly Leu Asn Thr Ile Tyr Asp Ser Ile Gln
995 1000 1005
Leu Val Asn Leu Ile Ser Asn Ala Val Asn Asp Thr Ile Asn Val Leu
1010 1015 1020
Pro Thr Ile Thr Glu Gly Ile Pro Ile Val Ser Thr Ile Leu Asp Gly
1025 1030 1035 1040
Ile Asn Leu Gly Ala Ala Ile Lys Glu Leu Leu Asp Glu His Asp Pro
1045 1050 1055
Leu Leu Lys Lys Glu Leu Glu Ala Lys Val Gly Val Leu Ala Ile Asn
1060 1065 1070
Met Ser Leu Ser Ile Ala Ala Thr Val Ala Ser Ile Val Gly Ile Gly
1075 1080 1085
Ala Glu Val Thr Ile Phe Leu Leu Pro Ile Ala Gly Ile Ser Ala Gly
1090 1095 1100
Ile Pro Ser Leu Val Asn Asn Glu Leu Ile Leu His Asp Lys Ala Thr
1105 1110 1115 1120
Ser Val Val Asn Tyr Phe Asn His Leu Ser Glu Ser Lys Lys Tyr Gly
1125 1130 1135
Pro Leu Lys Thr Glu Asp Asp Lys Ile Leu Val Pro Ile Asp Asp Leu
1140 1145 1150
Val Ile Ser Glu Ile Asp Phe Asn Asn Asn Ser Ile Lys Leu Gly Thr
1155 1160 1165
Cys Asn Ile Leu Ala Met Glu Gly Gly Ser Gly His Thr Val Thr Gly
1170 1175 1180
Asn Ile Asp His Phe Phe Ser Ser Pro Ser Ile Ser Ser His Ile Pro
1185 1190 1195 1200
Ser Leu Ser Ile Tyr Ser Ala Ile Gly Ile Glu Thr Glu Asn Leu Asp
1205 1210 1215
Phe Ser Lys Lys Ile Met Met Leu Pro Asn Ala Pro Ser Arg Val Phe
1220 1225 1230
Trp Trp Glu Thr Gly Ala Val Pro Gly Leu Arg Ser Leu Glu Asn Asp
1235 1240 1245
Gly Thr Arg Leu Leu Asp Ser Ile Arg Asp Leu Tyr Pro Gly Lys Phe
1250 1255 1260
Tyr Trp Arg Phe Tyr Ala Phe Phe Asp Tyr Ala Ile Thr Thr Leu Lys
1265 1270 1275 1280
Pro Val Tyr Glu Asp Thr Asn Ile Lys Ile Lys Leu Asp Lys Asp Thr
1285 1290 1295
Arg Asn Phe Ile Met Pro Thr Ile Thr Thr Asn Glu Ile Arg Asn Lys
1300 1305 1310
Leu Ser Tyr Ser Phe Asp Gly Ala Gly Gly Thr Tyr Ser Leu Leu Leu
1315 1320 1325
Ser Ser Tyr Pro Ile Ser Thr Asn Ile Asn Leu Ser Lys Asp Asp Leu
1330 1335 1340
Trp Ile Phe Asn Ile Asp Asn Glu Val Arg Glu Ile Ser Ile Glu Asn
1345 1350 1355 1360
Gly Thr Ile Lys Lys Gly Lys Leu Ile Lys Asp Val Leu Ser Lys Ile
1365 1370 1375
Asp Ile Asn Lys Asn Lys Leu Ile Ile Gly Asn Gln Thr Ile Asp Phe
1380 1385 1390
Ser Gly Asp Ile Asp Asn Lys Asp Arg Tyr Ile Phe Leu Thr Cys Glu
1395 1400 1405
Leu Asp Asp Lys Ile Ser Leu Ile Ile Glu Ile Asn Leu Val Ala Lys
1410 1415 1420
Ser Tyr Ser Leu Leu Leu Ser Gly Asp Lys Asn Tyr Leu Ile Ser Asn
1425 1430 1435 1440
Leu Ser Asn Thr Ile Glu Lys Ile Asn Thr Leu Gly Leu Asp Ser Lys
1445 1450 1455
Asn Ile Ala Tyr Asn Tyr Thr Asp Glu Ser Asn Asn Lys Tyr Phe Gly
1460 1465 1470
Ala Ile Ser Lys Thr Ser Gln Lys Ser Ile Ile His Tyr Lys Lys Asp
1475 1480 1485
Ser Lys Asn Ile Leu Glu Phe Tyr Asn Asp Ser Thr Leu Glu Phe Asn
1490 1495 1500
Ser Lys Asp Phe Ile Ala Glu Asp Ile Asn Val Phe Met Lys Asp Asp
1505 1510 1515 1520
Ile Asn Thr Ile Thr Gly Lys Tyr Tyr Val Asp Asn Asn Thr Asp Lys
1525 1530 1535
Ser Ile Asp Phe Ser Ile Ser Leu Val Ser Lys Asn Gln Val Lys Val
1540 1545 1550
Asn Gly Leu Tyr Leu Asn Glu Ser Val Tyr Ser Ser Tyr Leu Asp Phe
1555 1560 1565
Val Lys Asn Ser Asp Gly His His Asn Thr Ser Asn Phe Met Asn Leu
1570 1575 1580
Phe Leu Asp Asn Ile Ser Phe Trp Lys Leu Phe Gly Phe Glu Asn Ile
1585 1590 1595 1600
Asn Phe Val Ile Asp Lys Tyr Phe Thr Leu Val Gly Lys Thr Asn Leu
1605 1610 1615
Gly Tyr Val Glu Phe Ile Cys Asp Asn Asn Lys Asn Ile Asp Ile Tyr
1620 1625 1630
Phe Gly Glu Trp Lys Thr Ser Ser Ser Lys Ser Thr Ile Phe Ser Gly
1635 1640 1645
Asn Gly Arg Asn Val Val Val Glu Pro Ile Tyr Asn Pro Asp Thr Gly
1650 1655 1660
Glu Asp Ile Ser Thr Ser Leu Asp Phe Ser Tyr Glu Pro Leu Tyr Gly
1665 1670 1675 1680
Ile Asp Arg Tyr Ile Asn Lys Val Leu Ile Ala Pro Asp Leu Tyr Thr
1685 1690 1695
Ser Leu Ile Asn Ile Asn Thr Asn Tyr Tyr Ser Asn Glu Tyr Tyr Pro
1700 1705 1710
Glu Ile Ile Val Leu Asn Pro Asn Thr Phe His Lys Lys Val Asn Ile
1715 1720 1725
Asn Leu Asp Ser Ser Ser Phe Glu Tyr Lys Trp Ser Thr Glu Gly Ser
1730 1735 1740
Asp Phe Ile Leu Val Arg Tyr Leu Glu Glu Ser Asn Lys Lys Ile Leu
1745 1750 1755 1760
Gln Lys Ile Arg Ile Lys Gly Ile Leu Ser Asn Thr Gln Ser Phe Asn
1765 1770 1775
Lys Met Ser Ile Asp Phe Lys Asp Ile Lys Lys Leu Ser Leu Gly Tyr
1780 1785 1790
Ile Met Ser Asn Phe Lys Ser Phe Asn Ser Glu Asn Glu Leu Asp Arg
1795 1800 1805
Asp His Leu Gly Phe Lys Ile Ile Asp Asn Lys Thr Tyr Tyr Tyr Asp
1810 1815 1820
Glu Asp Ser Lys Leu Val Lys Gly Leu Ile Asn Ile Asn Asn Ser Leu
1825 1830 1835 1840
Phe Tyr Phe Asp Pro Ile Glu Phe Asn Leu Val Thr Gly Trp Gln Thr
1845 1850 1855
Ile Asn Gly Lys Lys Tyr Tyr Phe Asp Ile Asn Thr Gly Ala Ala Leu
1860 1865 1870
Thr Ser Tyr Lys Ile Ile Asn Gly Lys His Phe Tyr Phe Asn Asn Asp
1875 1880 1885
Gly Val Met Gln Leu Gly Val Phe Lys Gly Pro Asp Gly Phe Glu Tyr
1890 1895 1900
Phe Ala Pro Ala Asn Thr Gln Asn Asn Asn Ile Glu Gly Gln Ala Ile
1905 1910 1915 1920
Val Tyr Gln Ser Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe
1925 1930 1935
Asp Asn Asn Ser Lys Ala Val Thr Gly Trp Arg Ile Ile Asn Asn Glu
1940 1945 1950
Lys Tyr Tyr Phe Asn Pro Asn Asn Ala Ile Ala Ala Val Gly Leu Gln
1955 1960 1965
Val Ile Asp Asn Asn Lys Tyr Tyr Phe Asn Pro Asp Thr Ala Ile Ile
1970 1975 1980
Ser Lys Gly Trp Gln Thr Val Asn Gly Ser Arg Tyr Tyr Phe Asp Thr
1985 1990 1995 2000
Asp Thr Ala Ile Ala Phe Asn Gly Tyr Lys Thr Ile Asp Gly Lys His
2005 2010 2015
Phe Tyr Phe Asp Ser Asp Cys Val Val Lys Ile Gly Val Phe Ser Thr
2020 2025 2030
Ser Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr Tyr Asn Asn Asn
2035 2040 2045
Ile Glu Gly Gln Ala Ile Val Tyr Gln Ser Lys Phe Leu Thr Leu Asn
2050 2055 2060
Gly Lys Lys Tyr Tyr Phe Asp Asn Asn Ser Lys Ala Val Thr Gly Leu
2065 2070 2075 2080
Gln Thr Ile Asp Ser Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Ala Glu
2085 2090 2095
Ala Ala Thr Gly Trp Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe Asn
2100 2105 2110
Thr Asn Thr Ala Glu Ala Ala Thr Gly Trp Gln Thr Ile Asp Gly Lys
2115 2120 2125
Lys Tyr Tyr Phe Asn Thr Asn Thr Ala Ile Ala Ser Thr Gly Tyr Thr
2130 2135 2140
Ile Ile Asn Gly Lys His Phe Tyr Phe Asn Thr Asp Gly Ile Met Gln
2145 2150 2155 2160
Ile Gly Val Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe Ala Pro Ala
2165 2170 2175
Asn Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile Leu Tyr Gln Asn
2180 2185 2190
Glu Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Gly Ser Asp Ser
2195 2200 2205
Lys Ala Val Thr Gly Trp Arg Ile Ile Asn Asn Lys Lys Tyr Tyr Phe
2210 2215 2220
Asn Pro Asn Asn Ala Ile Ala Ala Ile His Leu Cys Thr Ile Asn Asn
2225 2230 2235 2240
Asp Lys Tyr Tyr Phe Ser Tyr Asp Gly Ile Leu Gln Asn Gly Tyr Ile
2245 2250 2255
Thr Ile Glu Arg Asn Asn Phe Tyr Phe Asp Ala Asn Asn Glu Ser Lys
2260 2265 2270
Met Val Thr Gly Val Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe Ala
2275 2280 2285
Pro Ala Asn Thr His Asn Asn Asn Ile Glu Gly Gln Ala Ile Val Tyr
2290 2295 2300
Gln Asn Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Asp Asn
2305 2310 2315 2320
Asp Ser Lys Ala Val Thr Gly Trp Gln Thr Ile Asp Gly Lys Lys Tyr
2325 2330 2335
Tyr Phe Asn Leu Asn Thr Ala Glu Ala Ala Thr Gly Trp Gln Thr Ile
2340 2345 2350
Asp Gly Lys Lys Tyr Tyr Phe Asn Leu Asn Thr Ala Glu Ala Ala Thr
2355 2360 2365
Gly Trp Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr
2370 2375 2380
Phe Ile Ala Ser Thr Gly Tyr Thr Ser Ile Asn Gly Lys His Phe Tyr
2385 2390 2395 2400
Phe Asn Thr Asp Gly Ile Met Gln Ile Gly Val Phe Lys Gly Pro Asn
2405 2410 2415
Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr Asp Ala Asn Asn Ile Glu
2420 2425 2430
Gly Gln Ala Ile Leu Tyr Gln Asn Lys Phe Leu Thr Leu Asn Gly Lys
2435 2440 2445
Lys Tyr Tyr Phe Gly Ser Asp Ser Lys Ala Val Thr Gly Leu Arg Thr
2450 2455 2460
Ile Asp Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Ala Val Ala Val
2465 2470 2475 2480
Thr Gly Trp Gln Thr Ile Asn Gly Lys Lys Tyr Tyr Phe Asn Thr Asn
2485 2490 2495
Thr Ser Ile Ala Ser Thr Gly Tyr Thr Ile Ile Ser Gly Lys His Phe
2500 2505 2510
Tyr Phe Asn Thr Asp Gly Ile Met Gln Ile Gly Val Phe Lys Gly Pro
2515 2520 2525
Asp Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr Asp Ala Asn Asn Ile
2530 2535 2540
Glu Gly Gln Ala Ile Arg Tyr Gln Asn Arg Phe Leu Tyr Leu His Asp
2545 2550 2555 2560
Asn Ile Tyr Tyr Phe Gly Asn Asn Ser Lys Ala Ala Thr Gly Trp Val
2565 2570 2575
Thr Ile Asp Gly Asn Arg Tyr Tyr Phe Glu Pro Asn Thr Ala Met Gly
2580 2585 2590
Ala Asn Gly Tyr Lys Thr Ile Asp Asn Lys Asn Phe Tyr Phe Arg Asn
2595 2600 2605
Gly Leu Pro Gln Ile Gly Val Phe Lys Gly Ser Asn Gly Phe Glu Tyr
2610 2615 2620
Phe Ala Pro Ala Asn Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile
2625 2630 2635 2640
Arg Tyr Gln Asn Arg Phe Leu His Leu Leu Gly Lys Ile Tyr Tyr Phe
2645 2650 2655
Gly Asn Asn Ser Lys Ala Val Thr Gly Trp Gln Thr Ile Asn Gly Lys
2660 2665 2670
Val Tyr Tyr Phe Met Pro Asp Thr Ala Met Ala Ala Ala Gly Gly Leu
2675 2680 2685
Phe Glu Ile Asp Gly Val Ile Tyr Phe Phe Gly Val Asp Gly Val Lys
2690 2695 2700
Ala Pro Gly Ile Tyr Gly
2705 2710
<210> 2
<211> 2366
<212> PRT
<213> 艰难梭菌
<400> 2
Met Ser Leu Val Asn Arg Lys Gln Leu Glu Lys Met Ala Asn Val Arg
1 5 10 15
Phe Arg Thr Gln Glu Asp Glu Tyr Val Ala Ile Leu Asp Ala Leu Glu
20 25 30
Glu Tyr His Asn Met Ser Glu Asn Thr Val Val Glu Lys Tyr Leu Lys
35 40 45
Leu Lys Asp Ile Asn Ser Leu Thr Asp Ile Tyr Ile Asp Thr Tyr Lys
50 55 60
Lys Ser Gly Arg Asn Lys Ala Leu Lys Lys Phe Lys Glu Tyr Leu Val
65 70 75 80
Thr Glu Val Leu Glu Leu Lys Asn Asn Asn Leu Thr Pro Val Glu Lys
85 90 95
Asn Leu His Phe Val Trp Ile Gly Gly Gln Ile Asn Asp Thr Ala Ile
100 105 110
Asn Tyr Ile Asn Gln Trp Lys Asp Val Asn Ser Asp Tyr Asn Val Asn
115 120 125
Val Phe Tyr Asp Ser Asn Ala Phe Leu Ile Asn Thr Leu Lys Lys Thr
130 135 140
Val Val Glu Ser Ala Ile Asn Asp Thr Leu Glu Ser Phe Arg Glu Asn
145 150 155 160
Leu Asn Asp Pro Arg Phe Asp Tyr Asn Lys Phe Phe Arg Lys Arg Met
165 170 175
Glu Ile Ile Tyr Asp Lys Gln Lys Asn Phe Ile Asn Tyr Tyr Lys Ala
180 185 190
Gln Arg Glu Glu Asn Pro Glu Leu Ile Ile Asp Asp Ile Val Lys Thr
195 200 205
Tyr Leu Ser Asn Glu Tyr Ser Lys Glu Ile Asp Glu Leu Asn Thr Tyr
210 215 220
Ile Glu Glu Ser Leu Asn Lys Ile Thr Gln Asn Ser Gly Asn Asp Val
225 230 235 240
Arg Asn Phe Glu Glu Phe Lys Asn Gly Glu Ser Phe Asn Leu Tyr Glu
245 250 255
Gln Glu Leu Val Glu Arg Trp Asn Leu Ala Ala Ala Ser Asp Ile Leu
260 265 270
Arg Ile Ser Ala Leu Lys Glu Ile Gly Gly Met Tyr Leu Asp Val Asp
275 280 285
Met Leu Pro Gly Ile Gln Pro Asp Leu Phe Glu Ser Ile Glu Lys Pro
290 295 300
Ser Ser Val Thr Val Asp Phe Trp Glu Met Thr Lys Leu Glu Ala Ile
305 310 315 320
Met Lys Tyr Lys Glu Tyr Ile Pro Glu Tyr Thr Ser Glu His Phe Asp
325 330 335
Met Leu Asp Glu Glu Val Gln Ser Ser Phe Glu Ser Val Leu Ala Ser
340 345 350
Lys Ser Asp Lys Ser Glu Ile Phe Ser Ser Leu Gly Asp Met Glu Ala
355 360 365
Ser Pro Leu Glu Val Lys Ile Ala Phe Asn Ser Lys Gly Ile Ile Asn
370 375 380
Gln Gly Leu Ile Ser Val Lys Asp Ser Tyr Cys Ser Asn Leu Ile Val
385 390 395 400
Lys Gln Ile Glu Asn Arg Tyr Lys Ile Leu Asn Asn Ser Leu Asn Pro
405 410 415
Ala Ile Ser Glu Asp Asn Asp Phe Asn Thr Thr Thr Asn Thr Phe Ile
420 425 430
Asp Ser Ile Met Ala Glu Ala Asn Ala Asp Asn Gly Arg Phe Met Met
435 440 445
Glu Leu Gly Lys Tyr Leu Arg Val Gly Phe Phe Pro Asp Val Lys Thr
450 455 460
Thr Ile Asn Leu Ser Gly Pro Glu Ala Tyr Ala Ala Ala Tyr Gln Asp
465 470 475 480
Leu Leu Met Phe Lys Glu Gly Ser Met Asn Ile His Leu Ile Glu Ala
485 490 495
Asp Leu Arg Asn Phe Glu Ile Ser Lys Thr Asn Ile Ser Gln Ser Thr
500 505 510
Glu Gln Glu Met Ala Ser Leu Trp Ser Phe Asp Asp Ala Arg Ala Lys
515 520 525
Ala Gln Phe Glu Glu Tyr Lys Arg Asn Tyr Phe Glu Gly Ser Leu Gly
530 535 540
Glu Asp Asp Asn Leu Asp Phe Ser Gln Asn Ile Val Val Asp Lys Glu
545 550 555 560
Tyr Leu Leu Glu Lys Ile Ser Ser Leu Ala Arg Ser Ser Glu Arg Gly
565 570 575
Tyr Ile His Tyr Ile Val Gln Leu Gln Gly Asp Lys Ile Ser Tyr Glu
580 585 590
Ala Ala Cys Asn Leu Phe Ala Lys Thr Pro Tyr Asp Ser Val Leu Phe
595 600 605
Gln Lys Asn Ile Glu Asp Ser Glu Ile Ala Tyr Tyr Tyr Asn Pro Gly
610 615 620
Asp Gly Glu Ile Gln Glu Ile Asp Lys Tyr Lys Ile Pro Ser Ile Ile
625 630 635 640
Ser Asp Arg Pro Lys Ile Lys Leu Thr Phe Ile Gly His Gly Lys Asp
645 650 655
Glu Phe Asn Thr Asp Ile Phe Ala Gly Phe Asp Val Asp Ser Leu Ser
660 665 670
Thr Glu Ile Glu Ala Ala Ile Asp Leu Ala Lys Glu Asp Ile Ser Pro
675 680 685
Lys Ser Ile Glu Ile Asn Leu Leu Gly Cys Asn Met Phe Ser Tyr Ser
690 695 700
Ile Asn Val Glu Glu Thr Tyr Pro Gly Lys Leu Leu Leu Lys Val Lys
705 710 715 720
Asp Lys Ile Ser Glu Leu Met Pro Ser Ile Ser Gln Asp Ser Ile Ile
725 730 735
Val Ser Ala Asn Gln Tyr Glu Val Arg Ile Asn Ser Glu Gly Arg Arg
740 745 750
Glu Leu Leu Asp His Ser Gly Glu Trp Ile Asn Lys Glu Glu Ser Ile
755 760 765
Ile Lys Asp Ile Ser Ser Lys Glu Tyr Ile Ser Phe Asn Pro Lys Glu
770 775 780
Asn Lys Ile Thr Val Lys Ser Lys Asn Leu Pro Glu Leu Ser Thr Leu
785 790 795 800
Leu Gln Glu Ile Arg Asn Asn Ser Asn Ser Ser Asp Ile Glu Leu Glu
805 810 815
Glu Lys Val Met Leu Thr Glu Cys Glu Ile Asn Val Ile Ser Asn Ile
820 825 830
Asp Thr Gln Ile Val Glu Glu Arg Ile Glu Glu Ala Lys Asn Leu Thr
835 840 845
Ser Asp Ser Ile Asn Tyr Ile Lys Asp Glu Phe Lys Leu Ile Glu Ser
850 855 860
Ile Ser Asp Ala Leu Cys Asp Leu Lys Gln Gln Asn Glu Leu Glu Asp
865 870 875 880
Ser His Phe Ile Ser Phe Glu Asp Ile Ser Glu Thr Asp Glu Gly Phe
885 890 895
Ser Ile Arg Phe Ile Asn Lys Glu Thr Gly Glu Ser Ile Phe Val Glu
900 905 910
Thr Glu Lys Thr Ile Phe Ser Glu Tyr Ala Asn His Ile Thr Glu Glu
915 920 925
Ile Ser Lys Ile Lys Gly Thr Ile Phe Asp Thr Val Asn Gly Lys Leu
930 935 940
Val Lys Lys Val Asn Leu Asp Thr Thr His Glu Val Asn Thr Leu Asn
945 950 955 960
Ala Ala Phe Phe Ile Gln Ser Leu Ile Glu Tyr Asn Ser Ser Lys Glu
965 970 975
Ser Leu Ser Asn Leu Ser Val Ala Met Lys Val Gln Val Tyr Ala Gln
980 985 990
Leu Phe Ser Thr Gly Leu Asn Thr Ile Thr Asp Ala Ala Lys Val Val
995 1000 1005
Glu Leu Val Ser Thr Ala Leu Asp Glu Thr Ile Asp Leu Leu Pro Thr
1010 1015 1020
Leu Ser Glu Gly Leu Pro Ile Ile Ala Thr Ile Ile Asp Gly Val Ser
1025 1030 1035 1040
Leu Gly Ala Ala Ile Lys Glu Leu Ser Glu Thr Ser Asp Pro Leu Leu
1045 1050 1055
Arg Gln Glu Ile Glu Ala Lys Ile Gly Ile Met Ala Val Asn Leu Thr
1060 1065 1070
Thr Ala Thr Thr Ala Ile Ile Thr Ser Ser Leu Gly Ile Ala Ser Gly
1075 1080 1085
Phe Ser Ile Leu Leu Val Pro Leu Ala Gly Ile Ser Ala Gly Ile Pro
1090 1095 1100
Ser Leu Val Asn Asn Glu Leu Val Leu Arg Asp Lys Ala Thr Lys Val
1105 1110 1115 1120
Val Asp Tyr Phe Lys His Val Ser Leu Val Glu Thr Glu Gly Val Phe
1125 1130 1135
Thr Leu Leu Asp Asp Lys Ile Met Met Pro Gln Asp Asp Leu Val Ile
1140 1145 1150
Ser Glu Ile Asp Phe Asn Asn Asn Ser Ile Val Leu Gly Lys Cys Glu
1155 1160 1165
Ile Trp Arg Met Glu Gly Gly Ser Gly His Thr Val Thr Asp Asp Ile
1170 1175 1180
Asp His Phe Phe Ser Ala Pro Ser Ile Thr Tyr Arg Glu Pro His Leu
1185 1190 1195 1200
Ser Ile Tyr Asp Val Leu Glu Val Gln Lys Glu Glu Leu Asp Leu Ser
1205 1210 1215
Lys Asp Leu Met Val Leu Pro Asn Ala Pro Asn Arg Val Phe Ala Trp
1220 1225 1230
Glu Thr Gly Trp Thr Pro Gly Leu Arg Ser Leu Glu Asn Asp Gly Thr
1235 1240 1245
Lys Leu Leu Asp Arg Ile Arg Asp Asn Tyr Glu Gly Glu Phe Tyr Trp
1250 1255 1260
Arg Tyr Phe Ala Phe Ile Ala Asp Ala Leu Ile Thr Thr Leu Lys Pro
1265 1270 1275 1280
Arg Tyr Glu Asp Thr Asn Ile Arg Ile Asn Leu Asp Ser Asn Thr Arg
1285 1290 1295
Ser Phe Ile Val Pro Ile Ile Thr Thr Glu Tyr Ile Arg Glu Lys Leu
1300 1305 1310
Ser Tyr Ser Phe Tyr Gly Ser Gly Gly Thr Tyr Ala Leu Ser Leu Ser
1315 1320 1325
Gln Tyr Asn Met Gly Ile Asn Ile Glu Leu Ser Glu Ser Asp Val Trp
1330 1335 1340
Ile Ile Asp Val Asp Asn Val Val Arg Asp Val Thr Ile Glu Ser Asp
1345 1350 1355 1360
Lys Ile Lys Lys Gly Asp Leu Ile Glu Gly Ile Leu Ser Thr Leu Ser
1365 1370 1375
Ile Glu Glu Asn Lys Ile Ile Leu Asn Ser His Glu Ile Asn Phe Ser
1380 1385 1390
Gly Glu Val Asn Gly Ser Asn Gly Phe Val Ser Leu Thr Phe Ser Ile
1395 1400 1405
Leu Glu Gly Ile Asn Ala Ile Ile Glu Val Asp Leu Leu Ser Lys Ser
1410 1415 1420
Tyr Lys Leu Leu Ile Ser Gly Glu Leu Lys Ile Leu Met Leu Asn Ser
1425 1430 1435 1440
Asn His Ile Gln Gln Lys Ile Asp Tyr Ile Gly Phe Asn Ser Glu Leu
1445 1450 1455
Gln Lys Asn Ile Pro Tyr Ser Phe Val Asp Ser Glu Gly Lys Glu Asn
1460 1465 1470
Gly Phe Ile Asn Gly Ser Thr Lys Glu Gly Leu Phe Val Ser Glu Leu
1475 1480 1485
Pro Asp Val Val Leu Ile Ser Lys Val Tyr Met Asp Asp Ser Lys Pro
1490 1495 1500
Ser Phe Gly Tyr Tyr Ser Asn Asn Leu Lys Asp Val Lys Val Ile Thr
1505 1510 1515 1520
Lys Asp Asn Val Asn Ile Leu Thr Gly Tyr Tyr Leu Lys Asp Asp Ile
1525 1530 1535
Lys Ile Ser Leu Ser Leu Thr Leu Gln Asp Glu Lys Thr Ile Lys Leu
1540 1545 1550
Asn Ser Val His Leu Asp Glu Ser Gly Val Ala Glu Ile Leu Lys Phe
1555 1560 1565
Met Asn Arg Lys Gly Asn Thr Asn Thr Ser Asp Ser Leu Met Ser Phe
1570 1575 1580
Leu Glu Ser Met Asn Ile Lys Ser Ile Phe Val Asn Phe Leu Gln Ser
1585 1590 1595 1600
Asn Ile Lys Phe Ile Leu Asp Ala Asn Phe Ile Ile Ser Gly Thr Thr
1605 1610 1615
Ser Ile Gly Gln Phe Glu Phe Ile Cys Asp Glu Asn Asp Asn Ile Gln
1620 1625 1630
Pro Tyr Phe Ile Lys Phe Asn Thr Leu Glu Thr Asn Tyr Thr Leu Tyr
1635 1640 1645
Val Gly Asn Arg Gln Asn Met Ile Val Glu Pro Asn Tyr Asp Leu Asp
1650 1655 1660
Asp Ser Gly Asp Ile Ser Ser Thr Val Ile Asn Phe Ser Gln Lys Tyr
1665 1670 1675 1680
Leu Tyr Gly Ile Asp Ser Cys Val Asn Lys Val Val Ile Ser Pro Asn
1685 1690 1695
Ile Tyr Thr Asp Glu Ile Asn Ile Thr Pro Val Tyr Glu Thr Asn Asn
1700 1705 1710
Thr Tyr Pro Glu Val Ile Val Leu Asp Ala Asn Tyr Ile Asn Glu Lys
1715 1720 1725
Ile Asn Val Asn Ile Asn Asp Leu Ser Ile Arg Tyr Val Trp Ser Asn
1730 1735 1740
Asp Gly Asn Asp Phe Ile Leu Met Ser Thr Ser Glu Glu Asn Lys Val
1745 1750 1755 1760
Ser Gln Val Lys Ile Arg Phe Val Asn Val Phe Lys Asp Lys Thr Leu
1765 1770 1775
Ala Asn Lys Leu Ser Phe Asn Phe Ser Asp Lys Gln Asp Val Pro Val
1780 1785 1790
Ser Glu Ile Ile Leu Ser Phe Thr Pro Ser Tyr Tyr Glu Asp Gly Leu
1795 1800 1805
Ile Gly Tyr Asp Leu Gly Leu Val Ser Leu Tyr Asn Glu Lys Phe Tyr
1810 1815 1820
Ile Asn Asn Phe Gly Met Met Val Ser Gly Leu Ile Tyr Ile Asn Asp
1825 1830 1835 1840
Ser Leu Tyr Tyr Phe Lys Pro Pro Val Asn Asn Leu Ile Thr Gly Phe
1845 1850 1855
Val Thr Val Gly Asp Asp Lys Tyr Tyr Phe Asn Pro Ile Asn Gly Gly
1860 1865 1870
Ala Ala Ser Ile Gly Glu Thr Ile Ile Asp Asp Lys Asn Tyr Tyr Phe
1875 1880 1885
Asn Gln Ser Gly Val Leu Gln Thr Gly Val Phe Ser Thr Glu Asp Gly
1890 1895 1900
Phe Lys Tyr Phe Ala Pro Ala Asn Thr Leu Asp Glu Asn Leu Glu Gly
1905 1910 1915 1920
Glu Ala Ile Asp Phe Thr Gly Lys Leu Ile Ile Asp Glu Asn Ile Tyr
1925 1930 1935
Tyr Phe Asp Asp Asn Tyr Arg Gly Ala Val Glu Trp Lys Glu Leu Asp
1940 1945 1950
Gly Glu Met His Tyr Phe Ser Pro Glu Thr Gly Lys Ala Phe Lys Gly
1955 1960 1965
Leu Asn Gln Ile Gly Asp Tyr Lys Tyr Tyr Phe Asn Ser Asp Gly Val
1970 1975 1980
Met Gln Lys Gly Phe Val Ser Ile Asn Asp Asn Lys His Tyr Phe Asp
1985 1990 1995 2000
Asp Ser Gly Val Met Lys Val Gly Tyr Thr Glu Ile Asp Gly Lys His
2005 2010 2015
Phe Tyr Phe Ala Glu Asn Gly Glu Met Gln Ile Gly Val Phe Asn Thr
2020 2025 2030
Glu Asp Gly Phe Lys Tyr Phe Ala His His Asn Glu Asp Leu Gly Asn
2035 2040 2045
Glu Glu Gly Glu Glu Ile Ser Tyr Ser Gly Ile Leu Asn Phe Asn Asn
2050 2055 2060
Lys Ile Tyr Tyr Phe Asp Asp Ser Phe Thr Ala Val Val Gly Trp Lys
2065 2070 2075 2080
Asp Leu Glu Asp Gly Ser Lys Tyr Tyr Phe Asp Glu Asp Thr Ala Glu
2085 2090 2095
Ala Tyr Ile Gly Leu Ser Leu Ile Asn Asp Gly Gln Tyr Tyr Phe Asn
2100 2105 2110
Asp Asp Gly Ile Met Gln Val Gly Phe Val Thr Ile Asn Asp Lys Val
2115 2120 2125
Phe Tyr Phe Ser Asp Ser Gly Ile Ile Glu Ser Gly Val Gln Asn Ile
2130 2135 2140
Asp Asp Asn Tyr Phe Tyr Ile Asp Asp Asn Gly Ile Val Gln Ile Gly
2145 2150 2155 2160
Val Phe Asp Thr Ser Asp Gly Tyr Lys Tyr Phe Ala Pro Ala Asn Thr
2165 2170 2175
Val Asn Asp Asn Ile Tyr Gly Gln Ala Val Glu Tyr Ser Gly Leu Val
2180 2185 2190
Arg Val Gly Glu Asp Val Tyr Tyr Phe Gly Glu Thr Tyr Thr Ile Glu
2195 2200 2205
Thr Gly Trp Ile Tyr Asp Met Glu Asn Glu Ser Asp Lys Tyr Tyr Phe
2210 2215 2220
Asn Pro Glu Thr Lys Lys Ala Cys Lys Gly Ile Asn Leu Ile Asp Asp
2225 2230 2235 2240
Ile Lys Tyr Tyr Phe Asp Glu Lys Gly Ile Met Arg Thr Gly Leu Ile
2245 2250 2255
Ser Phe Glu Asn Asn Asn Tyr Tyr Phe Asn Glu Asn Gly Glu Met Gln
2260 2265 2270
Phe Gly Tyr Ile Asn Ile Glu Asp Lys Met Phe Tyr Phe Gly Glu Asp
2275 2280 2285
Gly Val Met Gln Ile Gly Val Phe Asn Thr Pro Asp Gly Phe Lys Tyr
2290 2295 2300
Phe Ala His Gln Asn Thr Leu Asp Glu Asn Phe Glu Gly Glu Ser Ile
2305 2310 2315 2320
Asn Tyr Thr Gly Trp Leu Asp Leu Asp Glu Lys Arg Tyr Tyr Phe Thr
2325 2330 2335
Asp Glu Tyr Ile Ala Ala Thr Gly Ser Val Ile Ile Asp Gly Glu Glu
2340 2345 2350
Tyr Tyr Phe Asp Pro Asp Thr Ala Gln Leu Val Ile Ser Glu
2355 2360 2365
<210> 3
<211> 966
<212> PRT
<213> 艰难梭菌
<400> 3
Met Gly Trp Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe Asn Thr Asn
1 5 10 15
Thr Ala Ile Ala Ser Thr Gly Tyr Thr Ile Ile Asn Gly Lys His Phe
20 25 30
Tyr Phe Asn Thr Asp Gly Ile Met Gln Ile Gly Val Phe Lys Gly Pro
35 40 45
Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr Asp Ala Asn Asn Ile
50 55 60
Glu Gly Gln Ala Ile Leu Tyr Gln Asn Glu Phe Leu Thr Leu Asn Gly
65 70 75 80
Lys Lys Tyr Tyr Phe Gly Ser Asp Ser Lys Ala Val Thr Gly Trp Arg
85 90 95
Ile Ile Asn Asn Lys Lys Tyr Tyr Phe Asn Pro Asn Asn Ala Ile Ala
100 105 110
Ala Ile His Leu Cys Thr Ile Asn Asn Asp Lys Tyr Tyr Phe Ser Tyr
115 120 125
Asp Gly Ile Leu Gln Asn Gly Tyr Ile Thr Ile Glu Arg Asn Asn Phe
130 135 140
Tyr Phe Asp Ala Asn Asn Glu Ser Lys Met Val Thr Gly Val Phe Lys
145 150 155 160
Gly Pro Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr His Asn Asn
165 170 175
Asn Ile Glu Gly Gln Ala Ile Val Tyr Gln Asn Lys Phe Leu Thr Leu
180 185 190
Asn Gly Lys Lys Tyr Tyr Phe Asp Asn Asp Ser Lys Ala Val Thr Gly
195 200 205
Trp Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe Asn Leu Asn Thr Ala
210 215 220
Glu Ala Ala Thr Gly Trp Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe
225 230 235 240
Asn Leu Asn Thr Ala Glu Ala Ala Thr Gly Trp Gln Thr Ile Asp Gly
245 250 255
Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Phe Ile Ala Ser Thr Gly Tyr
260 265 270
Thr Ser Ile Asn Gly Lys His Phe Tyr Phe Asn Thr Asp Gly Ile Met
275 280 285
Gln Ile Gly Val Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe Ala Pro
290 295 300
Ala Asn Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile Leu Tyr Gln
305 310 315 320
Asn Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Gly Ser Asp
325 330 335
Ser Lys Ala Val Thr Gly Leu Arg Thr Ile Asp Gly Lys Lys Tyr Tyr
340 345 350
Phe Asn Thr Asn Thr Ala Val Ala Val Thr Gly Trp Gln Thr Ile Asn
355 360 365
Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Ser Ile Ala Ser Thr Gly
370 375 380
Tyr Thr Ile Ile Ser Gly Lys His Phe Tyr Phe Asn Thr Asp Gly Ile
385 390 395 400
Met Gln Ile Gly Val Phe Lys Gly Pro Asp Gly Phe Glu Tyr Phe Ala
405 410 415
Pro Ala Asn Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile Arg Tyr
420 425 430
Gln Asn Arg Phe Leu Tyr Leu His Asp Asn Ile Tyr Tyr Phe Gly Asn
435 440 445
Asn Ser Lys Ala Ala Thr Gly Trp Val Thr Ile Asp Gly Asn Arg Tyr
450 455 460
Tyr Phe Glu Pro Asn Thr Ala Met Gly Ala Asn Gly Tyr Lys Thr Ile
465 470 475 480
Asp Asn Lys Asn Phe Tyr Phe Arg Asn Gly Leu Pro Gln Ile Gly Val
485 490 495
Phe Lys Gly Ser Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr Asp
500 505 510
Ala Asn Asn Ile Glu Gly Gln Ala Ile Arg Tyr Gln Asn Arg Phe Leu
515 520 525
His Leu Leu Gly Lys Ile Tyr Tyr Phe Gly Asn Asn Ser Lys Ala Val
530 535 540
Thr Gly Trp Gln Thr Ile Asn Gly Lys Val Tyr Tyr Phe Met Pro Asp
545 550 555 560
Thr Ala Met Ala Ala Ala Gly Gly Leu Phe Glu Ile Asp Gly Val Ile
565 570 575
Tyr Phe Phe Gly Val Asp Gly Val Lys Ala Pro Gly Phe Val Ser Ile
580 585 590
Asn Asp Asn Lys His Tyr Phe Asp Asp Ser Gly Val Met Lys Val Gly
595 600 605
Tyr Thr Glu Ile Asp Gly Lys His Phe Tyr Phe Ala Glu Asn Gly Glu
610 615 620
Met Gln Ile Gly Val Phe Asn Thr Glu Asp Gly Phe Lys Tyr Phe Ala
625 630 635 640
His His Asn Glu Asp Leu Gly Asn Glu Glu Gly Glu Glu Ile Ser Tyr
645 650 655
Ser Gly Ile Leu Asn Phe Asn Asn Lys Ile Tyr Tyr Phe Asp Asp Ser
660 665 670
Phe Thr Ala Val Val Gly Trp Lys Asp Leu Glu Asp Gly Ser Lys Tyr
675 680 685
Tyr Phe Asp Glu Asp Thr Ala Glu Ala Tyr Ile Gly Leu Ser Leu Ile
690 695 700
Asn Asp Gly Gln Tyr Tyr Phe Asn Asp Asp Gly Ile Met Gln Val Gly
705 710 715 720
Phe Val Thr Ile Asn Asp Lys Val Phe Tyr Phe Ser Asp Ser Gly Ile
725 730 735
Ile Glu Ser Gly Val Gln Asn Ile Asp Asp Asn Tyr Phe Tyr Ile Asp
740 745 750
Asp Asn Gly Ile Val Gln Ile Gly Val Phe Asp Thr Ser Asp Gly Tyr
755 760 765
Lys Tyr Phe Ala Pro Ala Asn Thr Val Asn Asp Asn Ile Tyr Gly Gln
770 775 780
Ala Val Glu Tyr Ser Gly Leu Val Arg Val Gly Glu Asp Val Tyr Tyr
785 790 795 800
Phe Gly Glu Thr Tyr Thr Ile Glu Thr Gly Trp Ile Tyr Asp Met Glu
805 810 815
Asn Glu Ser Asp Lys Tyr Tyr Phe Asn Pro Glu Thr Lys Lys Ala Cys
820 825 830
Lys Gly Ile Asn Leu Ile Asp Asp Ile Lys Tyr Tyr Phe Asp Glu Lys
835 840 845
Gly Ile Met Arg Thr Gly Leu Ile Ser Phe Glu Asn Asn Asn Tyr Tyr
850 855 860
Phe Asn Glu Asn Gly Glu Met Gln Phe Gly Tyr Ile Asn Ile Glu Asp
865 870 875 880
Lys Met Phe Tyr Phe Gly Glu Asp Gly Val Met Gln Ile Gly Val Phe
885 890 895
Asn Thr Pro Asp Gly Phe Lys Tyr Phe Ala His Gln Asn Thr Leu Asp
900 905 910
Glu Asn Phe Glu Gly Glu Ser Ile Asn Tyr Thr Gly Trp Leu Asp Leu
915 920 925
Asp Glu Lys Arg Tyr Tyr Phe Thr Asp Glu Tyr Ile Ala Ala Thr Gly
930 935 940
Ser Val Ile Ile Asp Gly Glu Glu Tyr Tyr Phe Asp Pro Asp Thr Ala
945 950 955 960
Gln Leu Val Ile Ser Glu
965
<210> 4
<211> 966
<212> PRT
<213> 艰难梭菌
<400> 4
Met Gly Trp Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe Asn Thr Asn
1 5 10 15
Thr Ala Ile Ala Ser Thr Gly Tyr Thr Ile Ile Asn Gly Lys His Phe
20 25 30
Tyr Phe Asn Thr Asp Gly Ile Met Gln Ile Gly Val Phe Lys Gly Pro
35 40 45
Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr Asp Ala Asn Asn Ile
50 55 60
Glu Gly Gln Ala Ile Leu Tyr Gln Asn Glu Phe Leu Thr Leu Asn Gly
65 70 75 80
Lys Lys Tyr Tyr Phe Gly Ser Asp Ser Lys Ala Val Thr Gly Trp Arg
85 90 95
Ile Ile Asn Asn Lys Lys Tyr Tyr Phe Asn Pro Asn Asn Ala Ile Ala
100 105 110
Ala Ile His Leu Cys Thr Ile Asn Asn Asp Lys Tyr Tyr Phe Ser Tyr
115 120 125
Asp Gly Ile Leu Gln Asn Gly Tyr Ile Thr Ile Glu Arg Asn Asn Phe
130 135 140
Tyr Phe Asp Ala Asn Asn Glu Ser Lys Met Val Thr Gly Val Phe Lys
145 150 155 160
Gly Pro Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr His Asn Asn
165 170 175
Asn Ile Glu Gly Gln Ala Ile Val Tyr Gln Asn Lys Phe Leu Thr Leu
180 185 190
Asn Gly Lys Lys Tyr Tyr Phe Asp Asn Asp Ser Lys Ala Val Thr Gly
195 200 205
Trp Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe Asn Leu Asn Thr Ala
210 215 220
Glu Ala Ala Thr Gly Trp Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe
225 230 235 240
Asn Leu Asn Thr Ala Glu Ala Ala Thr Gly Trp Gln Thr Ile Asp Gly
245 250 255
Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Phe Ile Ala Ser Thr Gly Tyr
260 265 270
Thr Ser Ile Asn Gly Lys His Phe Tyr Phe Asn Thr Asp Gly Ile Met
275 280 285
Gln Ile Gly Val Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe Ala Pro
290 295 300
Ala Asn Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile Leu Tyr Gln
305 310 315 320
Asn Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Gly Ser Asp
325 330 335
Ser Lys Ala Val Thr Gly Leu Arg Thr Ile Asp Gly Lys Lys Tyr Tyr
340 345 350
Phe Asn Thr Asn Thr Ala Val Ala Val Thr Gly Trp Gln Thr Ile Asn
355 360 365
Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Ser Ile Ala Ser Thr Gly
370 375 380
Tyr Thr Ile Ile Ser Gly Lys His Phe Tyr Phe Asn Thr Asp Gly Ile
385 390 395 400
Met Gln Ile Gly Val Phe Lys Gly Pro Asp Gly Phe Glu Tyr Phe Ala
405 410 415
Pro Ala Asn Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile Arg Tyr
420 425 430
Gln Asn Arg Phe Leu Tyr Leu His Asp Asn Ile Tyr Tyr Phe Gly Asn
435 440 445
Asn Ser Lys Ala Ala Thr Gly Trp Val Thr Ile Asp Gly Asn Arg Tyr
450 455 460
Tyr Phe Glu Pro Asn Thr Ala Met Gly Ala Asn Gly Tyr Lys Thr Ile
465 470 475 480
Asp Asn Lys Asn Phe Tyr Phe Arg Asn Gly Leu Pro Gln Ile Gly Val
485 490 495
Phe Lys Gly Ser Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr Asp
500 505 510
Ala Asn Asn Ile Glu Gly Gln Ala Ile Arg Tyr Gln Asn Arg Phe Leu
515 520 525
His Leu Leu Gly Lys Ile Tyr Tyr Phe Gly Asn Asn Ser Lys Ala Val
530 535 540
Thr Gly Trp Gln Thr Ile Asn Gly Lys Val Tyr Tyr Phe Met Pro Asp
545 550 555 560
Thr Ala Met Ala Ala Ala Gly Gly Leu Asn Gln Ile Gly Asp Tyr Lys
565 570 575
Tyr Tyr Phe Asn Ser Asp Gly Val Met Gln Lys Gly Phe Val Ser Ile
580 585 590
Asn Asp Asn Lys His Tyr Phe Asp Asp Ser Gly Val Met Lys Val Gly
595 600 605
Tyr Thr Glu Ile Asp Gly Lys His Phe Tyr Phe Ala Glu Asn Gly Glu
610 615 620
Met Gln Ile Gly Val Phe Asn Thr Glu Asp Gly Phe Lys Tyr Phe Ala
625 630 635 640
His His Asn Glu Asp Leu Gly Asn Glu Glu Gly Glu Glu Ile Ser Tyr
645 650 655
Ser Gly Ile Leu Asn Phe Asn Asn Lys Ile Tyr Tyr Phe Asp Asp Ser
660 665 670
Phe Thr Ala Val Val Gly Trp Lys Asp Leu Glu Asp Gly Ser Lys Tyr
675 680 685
Tyr Phe Asp Glu Asp Thr Ala Glu Ala Tyr Ile Gly Leu Ser Leu Ile
690 695 700
Asn Asp Gly Gln Tyr Tyr Phe Asn Asp Asp Gly Ile Met Gln Val Gly
705 710 715 720
Phe Val Thr Ile Asn Asp Lys Val Phe Tyr Phe Ser Asp Ser Gly Ile
725 730 735
Ile Glu Ser Gly Val Gln Asn Ile Asp Asp Asn Tyr Phe Tyr Ile Asp
740 745 750
Asp Asn Gly Ile Val Gln Ile Gly Val Phe Asp Thr Ser Asp Gly Tyr
755 760 765
Lys Tyr Phe Ala Pro Ala Asn Thr Val Asn Asp Asn Ile Tyr Gly Gln
770 775 780
Ala Val Glu Tyr Ser Gly Leu Val Arg Val Gly Glu Asp Val Tyr Tyr
785 790 795 800
Phe Gly Glu Thr Tyr Thr Ile Glu Thr Gly Trp Ile Tyr Asp Met Glu
805 810 815
Asn Glu Ser Asp Lys Tyr Tyr Phe Asn Pro Glu Thr Lys Lys Ala Cys
820 825 830
Lys Gly Ile Asn Leu Ile Asp Asp Ile Lys Tyr Tyr Phe Asp Glu Lys
835 840 845
Gly Ile Met Arg Thr Gly Leu Ile Ser Phe Glu Asn Asn Asn Tyr Tyr
850 855 860
Phe Asn Glu Asn Gly Glu Met Gln Phe Gly Tyr Ile Asn Ile Glu Asp
865 870 875 880
Lys Met Phe Tyr Phe Gly Glu Asp Gly Val Met Gln Ile Gly Val Phe
885 890 895
Asn Thr Pro Asp Gly Phe Lys Tyr Phe Ala His Gln Asn Thr Leu Asp
900 905 910
Glu Asn Phe Glu Gly Glu Ser Ile Asn Tyr Thr Gly Trp Leu Asp Leu
915 920 925
Asp Glu Lys Arg Tyr Tyr Phe Thr Asp Glu Tyr Ile Ala Ala Thr Gly
930 935 940
Ser Val Ile Ile Asp Gly Glu Glu Tyr Tyr Phe Asp Pro Asp Thr Ala
945 950 955 960
Gln Leu Val Ile Ser Glu
965
<210> 5
<211> 833
<212> PRT
<213> 艰难梭菌
<400> 5
Met Gly Trp Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe Asn Thr Asn
1 5 10 15
Thr Ala Ile Ala Ser Thr Gly Tyr Thr Ile Ile Asn Gly Lys His Phe
20 25 30
Tyr Phe Asn Thr Asp Gly Ile Met Gln Ile Gly Val Phe Lys Gly Pro
35 40 45
Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr Asp Ala Asn Asn Ile
50 55 60
Glu Gly Gln Ala Ile Leu Tyr Gln Asn Glu Phe Leu Thr Leu Asn Gly
65 70 75 80
Lys Lys Tyr Tyr Phe Gly Ser Asp Ser Lys Ala Val Thr Gly Trp Arg
85 90 95
Ile Ile Asn Asn Lys Lys Tyr Tyr Phe Asn Pro Asn Asn Ala Ile Ala
100 105 110
Ala Ile His Leu Cys Thr Ile Asn Asn Asp Lys Tyr Tyr Phe Ser Tyr
115 120 125
Asp Gly Ile Leu Gln Asn Gly Tyr Ile Thr Ile Glu Arg Asn Asn Phe
130 135 140
Tyr Phe Asp Ala Asn Asn Glu Ser Lys Met Val Thr Gly Val Phe Lys
145 150 155 160
Gly Pro Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr His Asn Asn
165 170 175
Asn Ile Glu Gly Gln Ala Ile Val Tyr Gln Asn Lys Phe Leu Thr Leu
180 185 190
Asn Gly Lys Lys Tyr Tyr Phe Asp Asn Asp Ser Lys Ala Val Thr Gly
195 200 205
Trp Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe Asn Leu Asn Thr Ala
210 215 220
Glu Ala Ala Thr Gly Trp Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe
225 230 235 240
Asn Leu Asn Thr Ala Glu Ala Ala Thr Gly Trp Gln Thr Ile Asp Gly
245 250 255
Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Phe Ile Ala Ser Thr Gly Tyr
260 265 270
Thr Ser Ile Asn Gly Lys His Phe Tyr Phe Asn Thr Asp Gly Ile Met
275 280 285
Gln Ile Gly Val Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe Ala Pro
290 295 300
Ala Asn Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile Leu Tyr Gln
305 310 315 320
Asn Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Gly Ser Asp
325 330 335
Ser Lys Ala Val Thr Gly Leu Arg Thr Ile Asp Gly Lys Lys Tyr Tyr
340 345 350
Phe Asn Thr Asn Thr Ala Val Ala Val Thr Gly Trp Gln Thr Ile Asn
355 360 365
Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Ser Ile Ala Ser Thr Gly
370 375 380
Tyr Thr Ile Ile Ser Gly Lys His Phe Tyr Phe Asn Thr Asp Gly Ile
385 390 395 400
Met Gln Ile Gly Val Phe Lys Gly Pro Asp Gly Phe Glu Tyr Phe Ala
405 410 415
Pro Ala Asn Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile Arg Tyr
420 425 430
Gln Asn Arg Phe Leu Tyr Leu His Asp Asn Ile Tyr Tyr Phe Gly Asn
435 440 445
Asn Ser Lys Ala Ala Thr Gly Trp Val Thr Ile Asp Gly Asn Arg Tyr
450 455 460
Tyr Phe Glu Pro Asn Thr Ala Met Gly Ala Asn Gly Tyr Lys Thr Ile
465 470 475 480
Asp Asn Lys Asn Phe Tyr Phe Arg Asn Gly Leu Pro Gln Ile Gly Val
485 490 495
Phe Lys Gly Ser Asn Gly Phe Glu Tyr Phe Ala His His Asn Glu Asp
500 505 510
Leu Gly Asn Glu Glu Gly Glu Glu Ile Ser Tyr Ser Gly Ile Leu Asn
515 520 525
Phe Asn Asn Lys Ile Tyr Tyr Phe Asp Asp Ser Phe Thr Ala Val Val
530 535 540
Gly Trp Lys Asp Leu Glu Asp Gly Ser Lys Tyr Tyr Phe Asp Glu Asp
545 550 555 560
Thr Ala Glu Ala Tyr Ile Gly Leu Ser Leu Ile Asn Asp Gly Gln Tyr
565 570 575
Tyr Phe Asn Asp Asp Gly Ile Met Gln Val Gly Phe Val Thr Ile Asn
580 585 590
Asp Lys Val Phe Tyr Phe Ser Asp Ser Gly Ile Ile Glu Ser Gly Val
595 600 605
Gln Asn Ile Asp Asp Asn Tyr Phe Tyr Ile Asp Asp Asn Gly Ile Val
610 615 620
Gln Ile Gly Val Phe Asp Thr Ser Asp Gly Tyr Lys Tyr Phe Ala Pro
625 630 635 640
Ala Asn Thr Val Asn Asp Asn Ile Tyr Gly Gln Ala Val Glu Tyr Ser
645 650 655
Gly Leu Val Arg Val Gly Glu Asp Val Tyr Tyr Phe Gly Glu Thr Tyr
660 665 670
Thr Ile Glu Thr Gly Trp Ile Tyr Asp Met Glu Asn Glu Ser Asp Lys
675 680 685
Tyr Tyr Phe Asn Pro Glu Thr Lys Lys Ala Cys Lys Gly Ile Asn Leu
690 695 700
Ile Asp Asp Ile Lys Tyr Tyr Phe Asp Glu Lys Gly Ile Met Arg Thr
705 710 715 720
Gly Leu Ile Ser Phe Glu Asn Asn Asn Tyr Tyr Phe Asn Glu Asn Gly
725 730 735
Glu Met Gln Phe Gly Tyr Ile Asn Ile Glu Asp Lys Met Phe Tyr Phe
740 745 750
Gly Glu Asp Gly Val Met Gln Ile Gly Val Phe Asn Thr Pro Asp Gly
755 760 765
Phe Lys Tyr Phe Ala His Gln Asn Thr Leu Asp Glu Asn Phe Glu Gly
770 775 780
Glu Ser Ile Asn Tyr Thr Gly Trp Leu Asp Leu Asp Glu Lys Arg Tyr
785 790 795 800
Tyr Phe Thr Asp Glu Tyr Ile Ala Ala Thr Gly Ser Val Ile Ile Asp
805 810 815
Gly Glu Glu Tyr Tyr Phe Asp Pro Asp Thr Ala Gln Leu Val Ile Ser
820 825 830
Glu
<210> 6
<211> 1057
<212> PRT
<213> 艰难梭菌
<400> 6
Met Gly Trp Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe Asn Thr Asn
1 5 10 15
Thr Ala Ile Ala Ser Thr Gly Tyr Thr Ile Ile Asn Gly Lys His Phe
20 25 30
Tyr Phe Asn Thr Asp Gly Ile Met Gln Ile Gly Val Phe Lys Gly Pro
35 40 45
Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr Asp Ala Asn Asn Ile
50 55 60
Glu Gly Gln Ala Ile Leu Tyr Gln Asn Glu Phe Leu Thr Leu Asn Gly
65 70 75 80
Lys Lys Tyr Tyr Phe Gly Ser Asp Ser Lys Ala Val Thr Gly Trp Arg
85 90 95
Ile Ile Asn Asn Lys Lys Tyr Tyr Phe Asn Pro Asn Asn Ala Ile Ala
100 105 110
Ala Ile His Leu Cys Thr Ile Asn Asn Asp Lys Tyr Tyr Phe Ser Tyr
115 120 125
Asp Gly Ile Leu Gln Asn Gly Tyr Ile Thr Ile Glu Arg Asn Asn Phe
130 135 140
Tyr Phe Asp Ala Asn Asn Glu Ser Lys Met Val Thr Gly Val Phe Lys
145 150 155 160
Gly Pro Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr His Asn Asn
165 170 175
Asn Ile Glu Gly Gln Ala Ile Val Tyr Gln Asn Lys Phe Leu Thr Leu
180 185 190
Asn Gly Lys Lys Tyr Tyr Phe Asp Asn Asp Ser Lys Ala Val Thr Gly
195 200 205
Trp Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe Asn Leu Asn Thr Ala
210 215 220
Glu Ala Ala Thr Gly Trp Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe
225 230 235 240
Asn Leu Asn Thr Ala Glu Ala Ala Thr Gly Trp Gln Thr Ile Asp Gly
245 250 255
Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Phe Ile Ala Ser Thr Gly Tyr
260 265 270
Thr Ser Ile Asn Gly Lys His Phe Tyr Phe Asn Thr Asp Gly Ile Met
275 280 285
Gln Ile Gly Val Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe Ala Pro
290 295 300
Ala Asn Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile Leu Tyr Gln
305 310 315 320
Asn Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Gly Ser Asp
325 330 335
Ser Lys Ala Val Thr Gly Leu Arg Thr Ile Asp Gly Lys Lys Tyr Tyr
340 345 350
Phe Asn Thr Asn Thr Ala Val Ala Val Thr Gly Trp Gln Thr Ile Asn
355 360 365
Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Ser Ile Ala Ser Thr Gly
370 375 380
Tyr Thr Ile Ile Ser Gly Lys His Phe Tyr Phe Asn Thr Asp Gly Ile
385 390 395 400
Met Gln Ile Gly Val Phe Lys Gly Pro Asp Gly Phe Glu Tyr Phe Ala
405 410 415
Pro Ala Asn Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile Arg Tyr
420 425 430
Gln Asn Arg Phe Leu Tyr Leu His Asp Asn Ile Tyr Tyr Phe Gly Asn
435 440 445
Asn Ser Lys Ala Ala Thr Gly Trp Val Thr Ile Asp Gly Asn Arg Tyr
450 455 460
Tyr Phe Glu Pro Asn Thr Ala Met Gly Ala Asn Gly Tyr Lys Thr Ile
465 470 475 480
Asp Asn Lys Asn Phe Tyr Phe Arg Asn Gly Leu Pro Gln Ile Gly Val
485 490 495
Phe Lys Gly Ser Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr Asp
500 505 510
Ala Asn Asn Ile Glu Gly Gln Ala Ile Arg Tyr Gln Asn Arg Phe Leu
515 520 525
His Leu Leu Gly Lys Ile Tyr Tyr Phe Gly Asn Asn Ser Lys Ala Val
530 535 540
Thr Gly Trp Gln Thr Ile Asn Gly Lys Val Tyr Tyr Phe Met Pro Asp
545 550 555 560
Thr Ala Met Ala Ala Ala Gly Gly Glu Thr Ile Ile Asp Asp Lys Asn
565 570 575
Tyr Tyr Phe Asn Gln Ser Gly Val Leu Gln Thr Gly Val Phe Ser Thr
580 585 590
Glu Asp Gly Phe Lys Tyr Phe Ala Pro Ala Asn Thr Leu Asp Glu Asn
595 600 605
Leu Glu Gly Glu Ala Ile Asp Phe Thr Gly Lys Leu Ile Ile Asp Glu
610 615 620
Asn Ile Tyr Tyr Phe Asp Asp Asn Tyr Arg Gly Ala Val Glu Trp Lys
625 630 635 640
Glu Leu Asp Gly Glu Met His Tyr Phe Ser Pro Glu Thr Gly Lys Ala
645 650 655
Phe Lys Gly Leu Asn Gln Ile Gly Asp Tyr Lys Tyr Tyr Phe Asn Ser
660 665 670
Asp Gly Val Met Gln Lys Gly Phe Val Ser Ile Asn Asp Asn Lys His
675 680 685
Tyr Phe Asp Asp Ser Gly Val Met Lys Val Gly Tyr Thr Glu Ile Asp
690 695 700
Gly Lys His Phe Tyr Phe Ala Glu Asn Gly Glu Met Gln Ile Gly Val
705 710 715 720
Phe Asn Thr Glu Asp Gly Phe Lys Tyr Phe Ala His His Asn Glu Asp
725 730 735
Leu Gly Asn Glu Glu Gly Glu Glu Ile Ser Tyr Ser Gly Ile Leu Asn
740 745 750
Phe Asn Asn Lys Ile Tyr Tyr Phe Asp Asp Ser Phe Thr Ala Val Val
755 760 765
Gly Trp Lys Asp Leu Glu Asp Gly Ser Lys Tyr Tyr Phe Asp Glu Asp
770 775 780
Thr Ala Glu Ala Tyr Ile Gly Leu Ser Leu Ile Asn Asp Gly Gln Tyr
785 790 795 800
Tyr Phe Asn Asp Asp Gly Ile Met Gln Val Gly Phe Val Thr Ile Asn
805 810 815
Asp Lys Val Phe Tyr Phe Ser Asp Ser Gly Ile Ile Glu Ser Gly Val
820 825 830
Gln Asn Ile Asp Asp Asn Tyr Phe Tyr Ile Asp Asp Asn Gly Ile Val
835 840 845
Gln Ile Gly Val Phe Asp Thr Ser Asp Gly Tyr Lys Tyr Phe Ala Pro
850 855 860
Ala Asn Thr Val Asn Asp Asn Ile Tyr Gly Gln Ala Val Glu Tyr Ser
865 870 875 880
Gly Leu Val Arg Val Gly Glu Asp Val Tyr Tyr Phe Gly Glu Thr Tyr
885 890 895
Thr Ile Glu Thr Gly Trp Ile Tyr Asp Met Glu Asn Glu Ser Asp Lys
900 905 910
Tyr Tyr Phe Asn Pro Glu Thr Lys Lys Ala Cys Lys Gly Ile Asn Leu
915 920 925
Ile Asp Asp Ile Lys Tyr Tyr Phe Asp Glu Lys Gly Ile Met Arg Thr
930 935 940
Gly Leu Ile Ser Phe Glu Asn Asn Asn Tyr Tyr Phe Asn Glu Asn Gly
945 950 955 960
Glu Met Gln Phe Gly Tyr Ile Asn Ile Glu Asp Lys Met Phe Tyr Phe
965 970 975
Gly Glu Asp Gly Val Met Gln Ile Gly Val Phe Asn Thr Pro Asp Gly
980 985 990
Phe Lys Tyr Phe Ala His Gln Asn Thr Leu Asp Glu Asn Phe Glu Gly
995 1000 1005
Glu Ser Ile Asn Tyr Thr Gly Trp Leu Asp Leu Asp Glu Lys Arg Tyr
1010 1015 1020
Tyr Phe Thr Asp Glu Tyr Ile Ala Ala Thr Gly Ser Val Ile Ile Asp
1025 1030 1035 1040
Gly Glu Glu Tyr Tyr Phe Asp Pro Asp Thr Ala Gln Leu Val Ile Ser
1045 1050 1055
Glu
<210> 7
<211> 971
<212> PRT
<213> 艰难梭菌
<400> 7
Met Gly Trp Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe Asn Thr Asn
1 5 10 15
Thr Ala Ile Ala Ser Thr Gly Tyr Thr Ile Ile Asn Gly Lys His Phe
20 25 30
Tyr Phe Asn Thr Asp Gly Ile Met Gln Ile Gly Val Phe Lys Gly Pro
35 40 45
Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr Asp Ala Asn Asn Ile
50 55 60
Glu Gly Gln Ala Ile Leu Tyr Gln Asn Glu Phe Leu Thr Leu Asn Gly
65 70 75 80
Lys Lys Tyr Tyr Phe Gly Ser Asp Ser Lys Ala Val Thr Gly Trp Arg
85 90 95
Ile Ile Asn Asn Lys Lys Tyr Tyr Phe Asn Pro Asn Asn Ala Ile Ala
100 105 110
Ala Ile His Leu Cys Thr Ile Asn Asn Asp Lys Tyr Tyr Phe Ser Tyr
115 120 125
Asp Gly Ile Leu Gln Asn Gly Tyr Ile Thr Ile Glu Arg Asn Asn Phe
130 135 140
Tyr Phe Asp Ala Asn Asn Glu Ser Lys Met Val Thr Gly Val Phe Lys
145 150 155 160
Gly Pro Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr His Asn Asn
165 170 175
Asn Ile Glu Gly Gln Ala Ile Val Tyr Gln Asn Lys Phe Leu Thr Leu
180 185 190
Asn Gly Lys Lys Tyr Tyr Phe Asp Asn Asp Ser Lys Ala Val Thr Gly
195 200 205
Trp Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe Asn Leu Asn Thr Ala
210 215 220
Glu Ala Ala Thr Gly Trp Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe
225 230 235 240
Asn Leu Asn Thr Ala Glu Ala Ala Thr Gly Trp Gln Thr Ile Asp Gly
245 250 255
Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Phe Ile Ala Ser Thr Gly Tyr
260 265 270
Thr Ser Ile Asn Gly Lys His Phe Tyr Phe Asn Thr Asp Gly Ile Met
275 280 285
Gln Ile Gly Val Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe Ala Pro
290 295 300
Ala Asn Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile Leu Tyr Gln
305 310 315 320
Asn Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Gly Ser Asp
325 330 335
Ser Lys Ala Val Thr Gly Leu Arg Thr Ile Asp Gly Lys Lys Tyr Tyr
340 345 350
Phe Asn Thr Asn Thr Ala Val Ala Val Thr Gly Trp Gln Thr Ile Asn
355 360 365
Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Ser Ile Ala Ser Thr Gly
370 375 380
Tyr Thr Ile Ile Ser Gly Lys His Phe Tyr Phe Asn Thr Asp Gly Ile
385 390 395 400
Met Gln Ile Gly Val Phe Lys Gly Pro Asp Gly Phe Glu Tyr Phe Ala
405 410 415
Pro Ala Asn Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile Arg Tyr
420 425 430
Gln Asn Arg Phe Leu Tyr Leu His Asp Asn Ile Tyr Tyr Phe Gly Asn
435 440 445
Asn Ser Lys Ala Ala Thr Gly Trp Val Thr Ile Asp Gly Asn Arg Tyr
450 455 460
Tyr Phe Glu Pro Asn Thr Ala Met Gly Ala Asn Gly Tyr Lys Thr Ile
465 470 475 480
Asp Asn Lys Asn Phe Tyr Phe Arg Asn Gly Leu Pro Gln Ile Gly Val
485 490 495
Phe Lys Gly Ser Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr Asp
500 505 510
Ala Asn Asn Ile Glu Gly Gln Ala Ile Arg Tyr Gln Asn Arg Phe Leu
515 520 525
His Leu Leu Gly Lys Ile Tyr Tyr Phe Gly Asn Asn Ser Lys Ala Val
530 535 540
Thr Gly Trp Gln Thr Ile Asn Gly Lys Val Tyr Tyr Phe Met Pro Asp
545 550 555 560
Thr Ala Met Ala Ala Ala Gly Gly Leu Phe Glu Ile Asp Gly Val Ile
565 570 575
Tyr Phe Phe Gly Val Asp Gly Val Lys Ala Pro Gly Ile Tyr Gly Gly
580 585 590
Gly Phe Val Ser Ile Asn Asp Asn Lys His Tyr Phe Asp Asp Ser Gly
595 600 605
Val Met Lys Val Gly Tyr Thr Glu Ile Asp Gly Lys His Phe Tyr Phe
610 615 620
Ala Glu Asn Gly Glu Met Gln Ile Gly Val Phe Asn Thr Glu Asp Gly
625 630 635 640
Phe Lys Tyr Phe Ala His His Asn Glu Asp Leu Gly Asn Glu Glu Gly
645 650 655
Glu Glu Ile Ser Tyr Ser Gly Ile Leu Asn Phe Asn Asn Lys Ile Tyr
660 665 670
Tyr Phe Asp Asp Ser Phe Thr Ala Val Val Gly Trp Lys Asp Leu Glu
675 680 685
Asp Gly Ser Lys Tyr Tyr Phe Asp Glu Asp Thr Ala Glu Ala Tyr Ile
690 695 700
Gly Leu Ser Leu Ile Asn Asp Gly Gln Tyr Tyr Phe Asn Asp Asp Gly
705 710 715 720
Ile Met Gln Val Gly Phe Val Thr Ile Asn Asp Lys Val Phe Tyr Phe
725 730 735
Ser Asp Ser Gly Ile Ile Glu Ser Gly Val Gln Asn Ile Asp Asp Asn
740 745 750
Tyr Phe Tyr Ile Asp Asp Asn Gly Ile Val Gln Ile Gly Val Phe Asp
755 760 765
Thr Ser Asp Gly Tyr Lys Tyr Phe Ala Pro Ala Asn Thr Val Asn Asp
770 775 780
Asn Ile Tyr Gly Gln Ala Val Glu Tyr Ser Gly Leu Val Arg Val Gly
785 790 795 800
Glu Asp Val Tyr Tyr Phe Gly Glu Thr Tyr Thr Ile Glu Thr Gly Trp
805 810 815
Ile Tyr Asp Met Glu Asn Glu Ser Asp Lys Tyr Tyr Phe Asn Pro Glu
820 825 830
Thr Lys Lys Ala Cys Lys Gly Ile Asn Leu Ile Asp Asp Ile Lys Tyr
835 840 845
Tyr Phe Asp Glu Lys Gly Ile Met Arg Thr Gly Leu Ile Ser Phe Glu
850 855 860
Asn Asn Asn Tyr Tyr Phe Asn Glu Asn Gly Glu Met Gln Phe Gly Tyr
865 870 875 880
Ile Asn Ile Glu Asp Lys Met Phe Tyr Phe Gly Glu Asp Gly Val Met
885 890 895
Gln Ile Gly Val Phe Asn Thr Pro Asp Gly Phe Lys Tyr Phe Ala His
900 905 910
Gln Asn Thr Leu Asp Glu Asn Phe Glu Gly Glu Ser Ile Asn Tyr Thr
915 920 925
Gly Trp Leu Asp Leu Asp Glu Lys Arg Tyr Tyr Phe Thr Asp Glu Tyr
930 935 940
Ile Ala Ala Thr Gly Ser Val Ile Ile Asp Gly Glu Glu Tyr Tyr Phe
945 950 955 960
Asp Pro Asp Thr Ala Gln Leu Val Ile Ser Glu
965 970
<210> 8
<211> 321
<212> PRT
<213> 艰难梭菌
<400> 8
Met Ala Ser Thr Gly Tyr Thr Ser Ile Asn Gly Lys His Phe Tyr Phe
1 5 10 15
Asn Thr Asp Gly Ile Met Gln Ile Gly Val Phe Lys Gly Pro Asn Gly
20 25 30
Phe Glu Tyr Phe Ala Pro Ala Asn Thr Asp Ala Asn Asn Ile Glu Gly
35 40 45
Gln Ala Ile Leu Tyr Gln Asn Lys Phe Leu Thr Leu Asn Gly Lys Lys
50 55 60
Tyr Tyr Phe Gly Ser Asp Ser Lys Ala Val Thr Gly Leu Arg Thr Ile
65 70 75 80
Asp Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Ala Val Ala Val Thr
85 90 95
Gly Trp Gln Thr Ile Asn Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr
100 105 110
Ser Ile Ala Ser Thr Gly Tyr Thr Ile Ile Ser Gly Lys His Phe Tyr
115 120 125
Phe Asn Thr Asp Gly Ile Met Gln Ile Gly Val Phe Lys Gly Pro Asp
130 135 140
Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr Asp Ala Asn Asn Ile Glu
145 150 155 160
Gly Gln Ala Ile Arg Tyr Gln Asn Arg Phe Leu Tyr Leu His Asp Asn
165 170 175
Ile Tyr Tyr Phe Gly Asn Asn Ser Lys Ala Ala Thr Gly Trp Val Thr
180 185 190
Ile Asp Gly Asn Arg Tyr Tyr Phe Glu Pro Asn Thr Ala Met Gly Ala
195 200 205
Asn Gly Tyr Lys Thr Ile Asp Asn Lys Asn Phe Tyr Phe Arg Asn Gly
210 215 220
Leu Pro Gln Ile Gly Val Phe Lys Gly Ser Asn Gly Phe Glu Tyr Phe
225 230 235 240
Ala Pro Ala Asn Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile Arg
245 250 255
Tyr Gln Asn Arg Phe Leu His Leu Leu Gly Lys Ile Tyr Tyr Phe Gly
260 265 270
Asn Asn Ser Lys Ala Val Thr Gly Trp Gln Thr Ile Asn Gly Lys Val
275 280 285
Tyr Tyr Phe Met Pro Asp Thr Ala Met Ala Ala Ala Gly Gly Leu Phe
290 295 300
Glu Ile Asp Gly Val Ile Tyr Phe Phe Gly Val Asp Gly Val Lys Ala
305 310 315 320
Pro
<210> 9
<211> 612
<212> PRT
<213> 艰难梭菌
<400> 9
Met Ile Leu Met Ser Thr Ser Glu Glu Asn Lys Val Ser Gln Val Lys
1 5 10 15
Ile Arg Phe Val Asn Val Phe Lys Asp Lys Thr Leu Ala Asn Lys Leu
20 25 30
Ser Phe Asn Phe Ser Asp Lys Gln Asp Val Pro Val Ser Glu Ile Ile
35 40 45
Leu Ser Phe Thr Pro Ser Tyr Tyr Glu Asp Gly Leu Ile Gly Tyr Asp
50 55 60
Leu Gly Leu Val Ser Leu Tyr Asn Glu Lys Phe Tyr Ile Asn Asn Phe
65 70 75 80
Gly Met Met Val Ser Gly Leu Ile Tyr Ile Asn Asp Ser Leu Tyr Tyr
85 90 95
Phe Lys Pro Pro Val Asn Asn Leu Ile Thr Gly Phe Val Thr Val Gly
100 105 110
Asp Asp Lys Tyr Tyr Phe Asn Pro Ile Asn Gly Gly Ala Ala Ser Ile
115 120 125
Gly Glu Thr Ile Ile Asp Asp Lys Asn Tyr Tyr Phe Asn Gln Ser Gly
130 135 140
Val Leu Gln Thr Gly Val Phe Ser Thr Glu Asp Gly Phe Lys Tyr Phe
145 150 155 160
Ala Pro Ala Asn Thr Leu Asp Glu Asn Leu Glu Gly Glu Ala Ile Asp
165 170 175
Phe Thr Gly Lys Leu Ile Ile Asp Glu Asn Ile Tyr Tyr Phe Asp Asp
180 185 190
Asn Tyr Arg Gly Ala Val Glu Trp Lys Glu Leu Asp Gly Glu Met His
195 200 205
Tyr Phe Ser Pro Glu Thr Gly Lys Ala Phe Lys Gly Leu Asn Gln Ile
210 215 220
Gly Asp Tyr Lys Tyr Tyr Phe Asn Ser Asp Gly Val Met Gln Lys Gly
225 230 235 240
Phe Val Ser Ile Asn Asp Asn Lys His Tyr Phe Asp Asp Ser Gly Val
245 250 255
Met Lys Val Gly Tyr Thr Glu Ile Asp Gly Lys His Phe Tyr Phe Ala
260 265 270
Glu Asn Gly Glu Met Gln Ile Gly Val Phe Asn Thr Glu Asp Gly Phe
275 280 285
Lys Tyr Phe Ala His His Asn Glu Asp Leu Gly Asn Glu Glu Gly Glu
290 295 300
Glu Ile Ser Tyr Ser Gly Ile Leu Asn Phe Asn Asn Lys Ile Tyr Tyr
305 310 315 320
Phe Asp Asp Ser Phe Thr Ala Val Val Gly Trp Lys Asp Leu Glu Asp
325 330 335
Gly Ser Lys Tyr Tyr Phe Asp Glu Asp Thr Ala Glu Ala Tyr Ile Gly
340 345 350
Leu Ser Leu Ile Asn Asp Gly Gln Tyr Tyr Phe Asn Asp Asp Gly Ile
355 360 365
Met Gln Val Gly Phe Val Thr Ile Asn Asp Lys Val Phe Tyr Phe Ser
370 375 380
Asp Ser Gly Ile Ile Glu Ser Gly Val Gln Asn Ile Asp Asp Asn Tyr
385 390 395 400
Phe Tyr Ile Asp Asp Asn Gly Ile Val Gln Ile Gly Val Phe Asp Thr
405 410 415
Ser Asp Gly Tyr Lys Tyr Phe Ala Pro Ala Asn Thr Val Asn Asp Asn
420 425 430
Ile Tyr Gly Gln Ala Val Glu Tyr Ser Gly Leu Val Arg Val Gly Glu
435 440 445
Asp Val Tyr Tyr Phe Gly Glu Thr Tyr Thr Ile Glu Thr Gly Trp Ile
450 455 460
Tyr Asp Met Glu Asn Glu Ser Asp Lys Tyr Tyr Phe Asn Pro Glu Thr
465 470 475 480
Lys Lys Ala Cys Lys Gly Ile Asn Leu Ile Asp Asp Ile Lys Tyr Tyr
485 490 495
Phe Asp Glu Lys Gly Ile Met Arg Thr Gly Leu Ile Ser Phe Glu Asn
500 505 510
Asn Asn Tyr Tyr Phe Asn Glu Asn Gly Glu Met Gln Phe Gly Tyr Ile
515 520 525
Asn Ile Glu Asp Lys Met Phe Tyr Phe Gly Glu Asp Gly Val Met Gln
530 535 540
Ile Gly Val Phe Asn Thr Pro Asp Gly Phe Lys Tyr Phe Ala His Gln
545 550 555 560
Asn Thr Leu Asp Glu Asn Phe Glu Gly Glu Ser Ile Asn Tyr Thr Gly
565 570 575
Trp Leu Asp Leu Asp Glu Lys Arg Tyr Tyr Phe Thr Asp Glu Tyr Ile
580 585 590
Ala Ala Thr Gly Ser Val Ile Ile Asp Gly Glu Glu Tyr Tyr Phe Asp
595 600 605
Pro Asp Thr Ala
610
<210> 10
<211> 587
<212> PRT
<213> 艰难梭菌
<400> 10
Met Gly Trp Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe Asn Thr Asn
1 5 10 15
Thr Ala Ile Ala Ser Thr Gly Tyr Thr Ile Ile Asn Gly Lys His Phe
20 25 30
Tyr Phe Asn Thr Asp Gly Ile Met Gln Ile Gly Val Phe Lys Gly Pro
35 40 45
Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr Asp Ala Asn Asn Ile
50 55 60
Glu Gly Gln Ala Ile Leu Tyr Gln Asn Glu Phe Leu Thr Leu Asn Gly
65 70 75 80
Lys Lys Tyr Tyr Phe Gly Ser Asp Ser Lys Ala Val Thr Gly Trp Arg
85 90 95
Ile Ile Asn Asn Lys Lys Tyr Tyr Phe Asn Pro Asn Asn Ala Ile Ala
100 105 110
Ala Ile His Leu Cys Thr Ile Asn Asn Asp Lys Tyr Tyr Phe Ser Tyr
115 120 125
Asp Gly Ile Leu Gln Asn Gly Tyr Ile Thr Ile Glu Arg Asn Asn Phe
130 135 140
Tyr Phe Asp Ala Asn Asn Glu Ser Lys Met Val Thr Gly Val Phe Lys
145 150 155 160
Gly Pro Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr His Asn Asn
165 170 175
Asn Ile Glu Gly Gln Ala Ile Val Tyr Gln Asn Lys Phe Leu Thr Leu
180 185 190
Asn Gly Lys Lys Tyr Tyr Phe Asp Asn Asp Ser Lys Ala Val Thr Gly
195 200 205
Trp Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe Asn Leu Asn Thr Ala
210 215 220
Glu Ala Ala Thr Gly Trp Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe
225 230 235 240
Asn Leu Asn Thr Ala Glu Ala Ala Thr Gly Trp Gln Thr Ile Asp Gly
245 250 255
Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Phe Ile Ala Ser Thr Gly Tyr
260 265 270
Thr Ser Ile Asn Gly Lys His Phe Tyr Phe Asn Thr Asp Gly Ile Met
275 280 285
Gln Ile Gly Val Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe Ala Pro
290 295 300
Ala Asn Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile Leu Tyr Gln
305 310 315 320
Asn Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Gly Ser Asp
325 330 335
Ser Lys Ala Val Thr Gly Leu Arg Thr Ile Asp Gly Lys Lys Tyr Tyr
340 345 350
Phe Asn Thr Asn Thr Ala Val Ala Val Thr Gly Trp Gln Thr Ile Asn
355 360 365
Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Ser Ile Ala Ser Thr Gly
370 375 380
Tyr Thr Ile Ile Ser Gly Lys His Phe Tyr Phe Asn Thr Asp Gly Ile
385 390 395 400
Met Gln Ile Gly Val Phe Lys Gly Pro Asp Gly Phe Glu Tyr Phe Ala
405 410 415
Pro Ala Asn Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile Arg Tyr
420 425 430
Gln Asn Arg Phe Leu Tyr Leu His Asp Asn Ile Tyr Tyr Phe Gly Asn
435 440 445
Asn Ser Lys Ala Ala Thr Gly Trp Val Thr Ile Asp Gly Asn Arg Tyr
450 455 460
Tyr Phe Glu Pro Asn Thr Ala Met Gly Ala Asn Gly Tyr Lys Thr Ile
465 470 475 480
Asp Asn Lys Asn Phe Tyr Phe Arg Asn Gly Leu Pro Gln Ile Gly Val
485 490 495
Phe Lys Gly Ser Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr Asp
500 505 510
Ala Asn Asn Ile Glu Gly Gln Ala Ile Arg Tyr Gln Asn Arg Phe Leu
515 520 525
His Leu Leu Gly Lys Ile Tyr Tyr Phe Gly Asn Asn Ser Lys Ala Val
530 535 540
Thr Gly Trp Gln Thr Ile Asn Gly Lys Val Tyr Tyr Phe Met Pro Asp
545 550 555 560
Thr Ala Met Ala Ala Ala Gly Gly Leu Phe Glu Ile Asp Gly Val Ile
565 570 575
Tyr Phe Phe Gly Val Asp Gly Val Lys Ala Pro
580 585
<210> 11
<211> 567
<212> PRT
<213> 艰难梭菌
<400> 11
Met Gly Trp Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe Asn Thr Asn
1 5 10 15
Thr Ala Ile Ala Ser Thr Gly Tyr Thr Ile Ile Asn Gly Lys His Phe
20 25 30
Tyr Phe Asn Thr Asp Gly Ile Met Gln Ile Gly Val Phe Lys Gly Pro
35 40 45
Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr Asp Ala Asn Asn Ile
50 55 60
Glu Gly Gln Ala Ile Leu Tyr Gln Asn Glu Phe Leu Thr Leu Asn Gly
65 70 75 80
Lys Lys Tyr Tyr Phe Gly Ser Asp Ser Lys Ala Val Thr Gly Trp Arg
85 90 95
Ile Ile Asn Asn Lys Lys Tyr Tyr Phe Asn Pro Asn Asn Ala Ile Ala
100 105 110
Ala Ile His Leu Cys Thr Ile Asn Asn Asp Lys Tyr Tyr Phe Ser Tyr
115 120 125
Asp Gly Ile Leu Gln Asn Gly Tyr Ile Thr Ile Glu Arg Asn Asn Phe
130 135 140
Tyr Phe Asp Ala Asn Asn Glu Ser Lys Met Val Thr Gly Val Phe Lys
145 150 155 160
Gly Pro Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr His Asn Asn
165 170 175
Asn Ile Glu Gly Gln Ala Ile Val Tyr Gln Asn Lys Phe Leu Thr Leu
180 185 190
Asn Gly Lys Lys Tyr Tyr Phe Asp Asn Asp Ser Lys Ala Val Thr Gly
195 200 205
Trp Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe Asn Leu Asn Thr Ala
210 215 220
Glu Ala Ala Thr Gly Trp Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe
225 230 235 240
Asn Leu Asn Thr Ala Glu Ala Ala Thr Gly Trp Gln Thr Ile Asp Gly
245 250 255
Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Phe Ile Ala Ser Thr Gly Tyr
260 265 270
Thr Ser Ile Asn Gly Lys His Phe Tyr Phe Asn Thr Asp Gly Ile Met
275 280 285
Gln Ile Gly Val Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe Ala Pro
290 295 300
Ala Asn Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile Leu Tyr Gln
305 310 315 320
Asn Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Gly Ser Asp
325 330 335
Ser Lys Ala Val Thr Gly Leu Arg Thr Ile Asp Gly Lys Lys Tyr Tyr
340 345 350
Phe Asn Thr Asn Thr Ala Val Ala Val Thr Gly Trp Gln Thr Ile Asn
355 360 365
Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Ser Ile Ala Ser Thr Gly
370 375 380
Tyr Thr Ile Ile Ser Gly Lys His Phe Tyr Phe Asn Thr Asp Gly Ile
385 390 395 400
Met Gln Ile Gly Val Phe Lys Gly Pro Asp Gly Phe Glu Tyr Phe Ala
405 410 415
Pro Ala Asn Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile Arg Tyr
420 425 430
Gln Asn Arg Phe Leu Tyr Leu His Asp Asn Ile Tyr Tyr Phe Gly Asn
435 440 445
Asn Ser Lys Ala Ala Thr Gly Trp Val Thr Ile Asp Gly Asn Arg Tyr
450 455 460
Tyr Phe Glu Pro Asn Thr Ala Met Gly Ala Asn Gly Tyr Lys Thr Ile
465 470 475 480
Asp Asn Lys Asn Phe Tyr Phe Arg Asn Gly Leu Pro Gln Ile Gly Val
485 490 495
Phe Lys Gly Ser Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr Asp
500 505 510
Ala Asn Asn Ile Glu Gly Gln Ala Ile Arg Tyr Gln Asn Arg Phe Leu
515 520 525
His Leu Leu Gly Lys Ile Tyr Tyr Phe Gly Asn Asn Ser Lys Ala Val
530 535 540
Thr Gly Trp Gln Thr Ile Asn Gly Lys Val Tyr Tyr Phe Met Pro Asp
545 550 555 560
Thr Ala Met Ala Ala Ala Gly
565
<210> 12
<211> 505
<212> PRT
<213> 艰难梭菌
<400> 12
Met Gly Trp Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe Asn Thr Asn
1 5 10 15
Thr Ala Ile Ala Ser Thr Gly Tyr Thr Ile Ile Asn Gly Lys His Phe
20 25 30
Tyr Phe Asn Thr Asp Gly Ile Met Gln Ile Gly Val Phe Lys Gly Pro
35 40 45
Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr Asp Ala Asn Asn Ile
50 55 60
Glu Gly Gln Ala Ile Leu Tyr Gln Asn Glu Phe Leu Thr Leu Asn Gly
65 70 75 80
Lys Lys Tyr Tyr Phe Gly Ser Asp Ser Lys Ala Val Thr Gly Trp Arg
85 90 95
Ile Ile Asn Asn Lys Lys Tyr Tyr Phe Asn Pro Asn Asn Ala Ile Ala
100 105 110
Ala Ile His Leu Cys Thr Ile Asn Asn Asp Lys Tyr Tyr Phe Ser Tyr
115 120 125
Asp Gly Ile Leu Gln Asn Gly Tyr Ile Thr Ile Glu Arg Asn Asn Phe
130 135 140
Tyr Phe Asp Ala Asn Asn Glu Ser Lys Met Val Thr Gly Val Phe Lys
145 150 155 160
Gly Pro Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr His Asn Asn
165 170 175
Asn Ile Glu Gly Gln Ala Ile Val Tyr Gln Asn Lys Phe Leu Thr Leu
180 185 190
Asn Gly Lys Lys Tyr Tyr Phe Asp Asn Asp Ser Lys Ala Val Thr Gly
195 200 205
Trp Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe Asn Leu Asn Thr Ala
210 215 220
Glu Ala Ala Thr Gly Trp Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe
225 230 235 240
Asn Leu Asn Thr Ala Glu Ala Ala Thr Gly Trp Gln Thr Ile Asp Gly
245 250 255
Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Phe Ile Ala Ser Thr Gly Tyr
260 265 270
Thr Ser Ile Asn Gly Lys His Phe Tyr Phe Asn Thr Asp Gly Ile Met
275 280 285
Gln Ile Gly Val Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe Ala Pro
290 295 300
Ala Asn Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile Leu Tyr Gln
305 310 315 320
Asn Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Gly Ser Asp
325 330 335
Ser Lys Ala Val Thr Gly Leu Arg Thr Ile Asp Gly Lys Lys Tyr Tyr
340 345 350
Phe Asn Thr Asn Thr Ala Val Ala Val Thr Gly Trp Gln Thr Ile Asn
355 360 365
Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Ser Ile Ala Ser Thr Gly
370 375 380
Tyr Thr Ile Ile Ser Gly Lys His Phe Tyr Phe Asn Thr Asp Gly Ile
385 390 395 400
Met Gln Ile Gly Val Phe Lys Gly Pro Asp Gly Phe Glu Tyr Phe Ala
405 410 415
Pro Ala Asn Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile Arg Tyr
420 425 430
Gln Asn Arg Phe Leu Tyr Leu His Asp Asn Ile Tyr Tyr Phe Gly Asn
435 440 445
Asn Ser Lys Ala Ala Thr Gly Trp Val Thr Ile Asp Gly Asn Arg Tyr
450 455 460
Tyr Phe Glu Pro Asn Thr Ala Met Gly Ala Asn Gly Tyr Lys Thr Ile
465 470 475 480
Asp Asn Lys Asn Phe Tyr Phe Arg Asn Gly Leu Pro Gln Ile Gly Val
485 490 495
Phe Lys Gly Ser Asn Gly Phe Glu Tyr
500 505
<210> 13
<211> 567
<212> PRT
<213> 艰难梭菌
<400> 13
Met Gly Trp Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe Asn Thr Asn
1 5 10 15
Thr Ala Ile Ala Ser Thr Gly Tyr Thr Ile Ile Asn Gly Lys His Phe
20 25 30
Tyr Phe Asn Thr Asp Gly Ile Met Gln Ile Gly Val Phe Lys Gly Pro
35 40 45
Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr Asp Ala Asn Asn Ile
50 55 60
Glu Gly Gln Ala Ile Leu Tyr Gln Asn Glu Phe Leu Thr Leu Asn Gly
65 70 75 80
Lys Lys Tyr Tyr Phe Gly Ser Asp Ser Lys Ala Val Thr Gly Trp Arg
85 90 95
Ile Ile Asn Asn Lys Lys Tyr Tyr Phe Asn Pro Asn Asn Ala Ile Ala
100 105 110
Ala Ile His Leu Cys Thr Ile Asn Asn Asp Lys Tyr Tyr Phe Ser Tyr
115 120 125
Asp Gly Ile Leu Gln Asn Gly Tyr Ile Thr Ile Glu Arg Asn Asn Phe
130 135 140
Tyr Phe Asp Ala Asn Asn Glu Ser Lys Met Val Thr Gly Val Phe Lys
145 150 155 160
Gly Pro Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr His Asn Asn
165 170 175
Asn Ile Glu Gly Gln Ala Ile Val Tyr Gln Asn Lys Phe Leu Thr Leu
180 185 190
Asn Gly Lys Lys Tyr Tyr Phe Asp Asn Asp Ser Lys Ala Val Thr Gly
195 200 205
Trp Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe Asn Leu Asn Thr Ala
210 215 220
Glu Ala Ala Thr Gly Trp Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe
225 230 235 240
Asn Leu Asn Thr Ala Glu Ala Ala Thr Gly Trp Gln Thr Ile Asp Gly
245 250 255
Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Phe Ile Ala Ser Thr Gly Tyr
260 265 270
Thr Ser Ile Asn Gly Lys His Phe Tyr Phe Asn Thr Asp Gly Ile Met
275 280 285
Gln Ile Gly Val Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe Ala Pro
290 295 300
Ala Asn Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile Leu Tyr Gln
305 310 315 320
Asn Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Gly Ser Asp
325 330 335
Ser Lys Ala Val Thr Gly Leu Arg Thr Ile Asp Gly Lys Lys Tyr Tyr
340 345 350
Phe Asn Thr Asn Thr Ala Val Ala Val Thr Gly Trp Gln Thr Ile Asn
355 360 365
Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Ser Ile Ala Ser Thr Gly
370 375 380
Tyr Thr Ile Ile Ser Gly Lys His Phe Tyr Phe Asn Thr Asp Gly Ile
385 390 395 400
Met Gln Ile Gly Val Phe Lys Gly Pro Asp Gly Phe Glu Tyr Phe Ala
405 410 415
Pro Ala Asn Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile Arg Tyr
420 425 430
Gln Asn Arg Phe Leu Tyr Leu His Asp Asn Ile Tyr Tyr Phe Gly Asn
435 440 445
Asn Ser Lys Ala Ala Thr Gly Trp Val Thr Ile Asp Gly Asn Arg Tyr
450 455 460
Tyr Phe Glu Pro Asn Thr Ala Met Gly Ala Asn Gly Tyr Lys Thr Ile
465 470 475 480
Asp Asn Lys Asn Phe Tyr Phe Arg Asn Gly Leu Pro Gln Ile Gly Val
485 490 495
Phe Lys Gly Ser Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr Asp
500 505 510
Ala Asn Asn Ile Glu Gly Gln Ala Ile Arg Tyr Gln Asn Arg Phe Leu
515 520 525
His Leu Leu Gly Lys Ile Tyr Tyr Phe Gly Asn Asn Ser Lys Ala Val
530 535 540
Thr Gly Trp Gln Thr Ile Asn Gly Lys Val Tyr Tyr Phe Met Pro Asp
545 550 555 560
Thr Ala Met Ala Ala Ala Gly
565
<210> 14
<211> 591
<212> PRT
<213> 艰难梭菌
<400> 14
Met Gly Trp Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe Asn Thr Asn
1 5 10 15
Thr Ala Ile Ala Ser Thr Gly Tyr Thr Ile Ile Asn Gly Lys His Phe
20 25 30
Tyr Phe Asn Thr Asp Gly Ile Met Gln Ile Gly Val Phe Lys Gly Pro
35 40 45
Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr Asp Ala Asn Asn Ile
50 55 60
Glu Gly Gln Ala Ile Leu Tyr Gln Asn Glu Phe Leu Thr Leu Asn Gly
65 70 75 80
Lys Lys Tyr Tyr Phe Gly Ser Asp Ser Lys Ala Val Thr Gly Trp Arg
85 90 95
Ile Ile Asn Asn Lys Lys Tyr Tyr Phe Asn Pro Asn Asn Ala Ile Ala
100 105 110
Ala Ile His Leu Cys Thr Ile Asn Asn Asp Lys Tyr Tyr Phe Ser Tyr
115 120 125
Asp Gly Ile Leu Gln Asn Gly Tyr Ile Thr Ile Glu Arg Asn Asn Phe
130 135 140
Tyr Phe Asp Ala Asn Asn Glu Ser Lys Met Val Thr Gly Val Phe Lys
145 150 155 160
Gly Pro Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr His Asn Asn
165 170 175
Asn Ile Glu Gly Gln Ala Ile Val Tyr Gln Asn Lys Phe Leu Thr Leu
180 185 190
Asn Gly Lys Lys Tyr Tyr Phe Asp Asn Asp Ser Lys Ala Val Thr Gly
195 200 205
Trp Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe Asn Leu Asn Thr Ala
210 215 220
Glu Ala Ala Thr Gly Trp Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe
225 230 235 240
Asn Leu Asn Thr Ala Glu Ala Ala Thr Gly Trp Gln Thr Ile Asp Gly
245 250 255
Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Phe Ile Ala Ser Thr Gly Tyr
260 265 270
Thr Ser Ile Asn Gly Lys His Phe Tyr Phe Asn Thr Asp Gly Ile Met
275 280 285
Gln Ile Gly Val Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe Ala Pro
290 295 300
Ala Asn Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile Leu Tyr Gln
305 310 315 320
Asn Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Gly Ser Asp
325 330 335
Ser Lys Ala Val Thr Gly Leu Arg Thr Ile Asp Gly Lys Lys Tyr Tyr
340 345 350
Phe Asn Thr Asn Thr Ala Val Ala Val Thr Gly Trp Gln Thr Ile Asn
355 360 365
Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Ser Ile Ala Ser Thr Gly
370 375 380
Tyr Thr Ile Ile Ser Gly Lys His Phe Tyr Phe Asn Thr Asp Gly Ile
385 390 395 400
Met Gln Ile Gly Val Phe Lys Gly Pro Asp Gly Phe Glu Tyr Phe Ala
405 410 415
Pro Ala Asn Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile Arg Tyr
420 425 430
Gln Asn Arg Phe Leu Tyr Leu His Asp Asn Ile Tyr Tyr Phe Gly Asn
435 440 445
Asn Ser Lys Ala Ala Thr Gly Trp Val Thr Ile Asp Gly Asn Arg Tyr
450 455 460
Tyr Phe Glu Pro Asn Thr Ala Met Gly Ala Asn Gly Tyr Lys Thr Ile
465 470 475 480
Asp Asn Lys Asn Phe Tyr Phe Arg Asn Gly Leu Pro Gln Ile Gly Val
485 490 495
Phe Lys Gly Ser Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr Asp
500 505 510
Ala Asn Asn Ile Glu Gly Gln Ala Ile Arg Tyr Gln Asn Arg Phe Leu
515 520 525
His Leu Leu Gly Lys Ile Tyr Tyr Phe Gly Asn Asn Ser Lys Ala Val
530 535 540
Thr Gly Trp Gln Thr Ile Asn Gly Lys Val Tyr Tyr Phe Met Pro Asp
545 550 555 560
Thr Ala Met Ala Ala Ala Gly Gly Leu Phe Glu Ile Asp Gly Val Ile
565 570 575
Tyr Phe Phe Gly Val Asp Gly Val Lys Ala Pro Gly Ile Tyr Gly
580 585 590
<210> 15
<211> 379
<212> PRT
<213> 艰难梭菌
<400> 15
Gly Phe Val Ser Ile Asn Asp Asn Lys His Tyr Phe Asp Asp Ser Gly
1 5 10 15
Val Met Lys Val Gly Tyr Thr Glu Ile Asp Gly Lys His Phe Tyr Phe
20 25 30
Ala Glu Asn Gly Glu Met Gln Ile Gly Val Phe Asn Thr Glu Asp Gly
35 40 45
Phe Lys Tyr Phe Ala His His Asn Glu Asp Leu Gly Asn Glu Glu Gly
50 55 60
Glu Glu Ile Ser Tyr Ser Gly Ile Leu Asn Phe Asn Asn Lys Ile Tyr
65 70 75 80
Tyr Phe Asp Asp Ser Phe Thr Ala Val Val Gly Trp Lys Asp Leu Glu
85 90 95
Asp Gly Ser Lys Tyr Tyr Phe Asp Glu Asp Thr Ala Glu Ala Tyr Ile
100 105 110
Gly Leu Ser Leu Ile Asn Asp Gly Gln Tyr Tyr Phe Asn Asp Asp Gly
115 120 125
Ile Met Gln Val Gly Phe Val Thr Ile Asn Asp Lys Val Phe Tyr Phe
130 135 140
Ser Asp Ser Gly Ile Ile Glu Ser Gly Val Gln Asn Ile Asp Asp Asn
145 150 155 160
Tyr Phe Tyr Ile Asp Asp Asn Gly Ile Val Gln Ile Gly Val Phe Asp
165 170 175
Thr Ser Asp Gly Tyr Lys Tyr Phe Ala Pro Ala Asn Thr Val Asn Asp
180 185 190
Asn Ile Tyr Gly Gln Ala Val Glu Tyr Ser Gly Leu Val Arg Val Gly
195 200 205
Glu Asp Val Tyr Tyr Phe Gly Glu Thr Tyr Thr Ile Glu Thr Gly Trp
210 215 220
Ile Tyr Asp Met Glu Asn Glu Ser Asp Lys Tyr Tyr Phe Asn Pro Glu
225 230 235 240
Thr Lys Lys Ala Cys Lys Gly Ile Asn Leu Ile Asp Asp Ile Lys Tyr
245 250 255
Tyr Phe Asp Glu Lys Gly Ile Met Arg Thr Gly Leu Ile Ser Phe Glu
260 265 270
Asn Asn Asn Tyr Tyr Phe Asn Glu Asn Gly Glu Met Gln Phe Gly Tyr
275 280 285
Ile Asn Ile Glu Asp Lys Met Phe Tyr Phe Gly Glu Asp Gly Val Met
290 295 300
Gln Ile Gly Val Phe Asn Thr Pro Asp Gly Phe Lys Tyr Phe Ala His
305 310 315 320
Gln Asn Thr Leu Asp Glu Asn Phe Glu Gly Glu Ser Ile Asn Tyr Thr
325 330 335
Gly Trp Leu Asp Leu Asp Glu Lys Arg Tyr Tyr Phe Thr Asp Glu Tyr
340 345 350
Ile Ala Ala Thr Gly Ser Val Ile Ile Asp Gly Glu Glu Tyr Tyr Phe
355 360 365
Asp Pro Asp Thr Ala Gln Leu Val Ile Ser Glu
370 375
<210> 16
<211> 399
<212> PRT
<213> 艰难梭菌
<400> 16
Gly Leu Asn Gln Ile Gly Asp Tyr Lys Tyr Tyr Phe Asn Ser Asp Gly
1 5 10 15
Val Met Gln Lys Gly Phe Val Ser Ile Asn Asp Asn Lys His Tyr Phe
20 25 30
Asp Asp Ser Gly Val Met Lys Val Gly Tyr Thr Glu Ile Asp Gly Lys
35 40 45
His Phe Tyr Phe Ala Glu Asn Gly Glu Met Gln Ile Gly Val Phe Asn
50 55 60
Thr Glu Asp Gly Phe Lys Tyr Phe Ala His His Asn Glu Asp Leu Gly
65 70 75 80
Asn Glu Glu Gly Glu Glu Ile Ser Tyr Ser Gly Ile Leu Asn Phe Asn
85 90 95
Asn Lys Ile Tyr Tyr Phe Asp Asp Ser Phe Thr Ala Val Val Gly Trp
100 105 110
Lys Asp Leu Glu Asp Gly Ser Lys Tyr Tyr Phe Asp Glu Asp Thr Ala
115 120 125
Glu Ala Tyr Ile Gly Leu Ser Leu Ile Asn Asp Gly Gln Tyr Tyr Phe
130 135 140
Asn Asp Asp Gly Ile Met Gln Val Gly Phe Val Thr Ile Asn Asp Lys
145 150 155 160
Val Phe Tyr Phe Ser Asp Ser Gly Ile Ile Glu Ser Gly Val Gln Asn
165 170 175
Ile Asp Asp Asn Tyr Phe Tyr Ile Asp Asp Asn Gly Ile Val Gln Ile
180 185 190
Gly Val Phe Asp Thr Ser Asp Gly Tyr Lys Tyr Phe Ala Pro Ala Asn
195 200 205
Thr Val Asn Asp Asn Ile Tyr Gly Gln Ala Val Glu Tyr Ser Gly Leu
210 215 220
Val Arg Val Gly Glu Asp Val Tyr Tyr Phe Gly Glu Thr Tyr Thr Ile
225 230 235 240
Glu Thr Gly Trp Ile Tyr Asp Met Glu Asn Glu Ser Asp Lys Tyr Tyr
245 250 255
Phe Asn Pro Glu Thr Lys Lys Ala Cys Lys Gly Ile Asn Leu Ile Asp
260 265 270
Asp Ile Lys Tyr Tyr Phe Asp Glu Lys Gly Ile Met Arg Thr Gly Leu
275 280 285
Ile Ser Phe Glu Asn Asn Asn Tyr Tyr Phe Asn Glu Asn Gly Glu Met
290 295 300
Gln Phe Gly Tyr Ile Asn Ile Glu Asp Lys Met Phe Tyr Phe Gly Glu
305 310 315 320
Asp Gly Val Met Gln Ile Gly Val Phe Asn Thr Pro Asp Gly Phe Lys
325 330 335
Tyr Phe Ala His Gln Asn Thr Leu Asp Glu Asn Phe Glu Gly Glu Ser
340 345 350
Ile Asn Tyr Thr Gly Trp Leu Asp Leu Asp Glu Lys Arg Tyr Tyr Phe
355 360 365
Thr Asp Glu Tyr Ile Ala Ala Thr Gly Ser Val Ile Ile Asp Gly Glu
370 375 380
Glu Tyr Tyr Phe Asp Pro Asp Thr Ala Gln Leu Val Ile Ser Glu
385 390 395
<210> 17
<211> 328
<212> PRT
<213> 艰难梭菌
<400> 17
Phe Ala His His Asn Glu Asp Leu Gly Asn Glu Glu Gly Glu Glu Ile
1 5 10 15
Ser Tyr Ser Gly Ile Leu Asn Phe Asn Asn Lys Ile Tyr Tyr Phe Asp
20 25 30
Asp Ser Phe Thr Ala Val Val Gly Trp Lys Asp Leu Glu Asp Gly Ser
35 40 45
Lys Tyr Tyr Phe Asp Glu Asp Thr Ala Glu Ala Tyr Ile Gly Leu Ser
50 55 60
Leu Ile Asn Asp Gly Gln Tyr Tyr Phe Asn Asp Asp Gly Ile Met Gln
65 70 75 80
Val Gly Phe Val Thr Ile Asn Asp Lys Val Phe Tyr Phe Ser Asp Ser
85 90 95
Gly Ile Ile Glu Ser Gly Val Gln Asn Ile Asp Asp Asn Tyr Phe Tyr
100 105 110
Ile Asp Asp Asn Gly Ile Val Gln Ile Gly Val Phe Asp Thr Ser Asp
115 120 125
Gly Tyr Lys Tyr Phe Ala Pro Ala Asn Thr Val Asn Asp Asn Ile Tyr
130 135 140
Gly Gln Ala Val Glu Tyr Ser Gly Leu Val Arg Val Gly Glu Asp Val
145 150 155 160
Tyr Tyr Phe Gly Glu Thr Tyr Thr Ile Glu Thr Gly Trp Ile Tyr Asp
165 170 175
Met Glu Asn Glu Ser Asp Lys Tyr Tyr Phe Asn Pro Glu Thr Lys Lys
180 185 190
Ala Cys Lys Gly Ile Asn Leu Ile Asp Asp Ile Lys Tyr Tyr Phe Asp
195 200 205
Glu Lys Gly Ile Met Arg Thr Gly Leu Ile Ser Phe Glu Asn Asn Asn
210 215 220
Tyr Tyr Phe Asn Glu Asn Gly Glu Met Gln Phe Gly Tyr Ile Asn Ile
225 230 235 240
Glu Asp Lys Met Phe Tyr Phe Gly Glu Asp Gly Val Met Gln Ile Gly
245 250 255
Val Phe Asn Thr Pro Asp Gly Phe Lys Tyr Phe Ala His Gln Asn Thr
260 265 270
Leu Asp Glu Asn Phe Glu Gly Glu Ser Ile Asn Tyr Thr Gly Trp Leu
275 280 285
Asp Leu Asp Glu Lys Arg Tyr Tyr Phe Thr Asp Glu Tyr Ile Ala Ala
290 295 300
Thr Gly Ser Val Ile Ile Asp Gly Glu Glu Tyr Tyr Phe Asp Pro Asp
305 310 315 320
Thr Ala Gln Leu Val Ile Ser Glu
325
<210> 18
<211> 490
<212> PRT
<213> 艰难梭菌
<400> 18
Gly Glu Thr Ile Ile Asp Asp Lys Asn Tyr Tyr Phe Asn Gln Ser Gly
1 5 10 15
Val Leu Gln Thr Gly Val Phe Ser Thr Glu Asp Gly Phe Lys Tyr Phe
20 25 30
Ala Pro Ala Asn Thr Leu Asp Glu Asn Leu Glu Gly Glu Ala Ile Asp
35 40 45
Phe Thr Gly Lys Leu Ile Ile Asp Glu Asn Ile Tyr Tyr Phe Asp Asp
50 55 60
Asn Tyr Arg Gly Ala Val Glu Trp Lys Glu Leu Asp Gly Glu Met His
65 70 75 80
Tyr Phe Ser Pro Glu Thr Gly Lys Ala Phe Lys Gly Leu Asn Gln Ile
85 90 95
Gly Asp Tyr Lys Tyr Tyr Phe Asn Ser Asp Gly Val Met Gln Lys Gly
100 105 110
Phe Val Ser Ile Asn Asp Asn Lys His Tyr Phe Asp Asp Ser Gly Val
115 120 125
Met Lys Val Gly Tyr Thr Glu Ile Asp Gly Lys His Phe Tyr Phe Ala
130 135 140
Glu Asn Gly Glu Met Gln Ile Gly Val Phe Asn Thr Glu Asp Gly Phe
145 150 155 160
Lys Tyr Phe Ala His His Asn Glu Asp Leu Gly Asn Glu Glu Gly Glu
165 170 175
Glu Ile Ser Tyr Ser Gly Ile Leu Asn Phe Asn Asn Lys Ile Tyr Tyr
180 185 190
Phe Asp Asp Ser Phe Thr Ala Val Val Gly Trp Lys Asp Leu Glu Asp
195 200 205
Gly Ser Lys Tyr Tyr Phe Asp Glu Asp Thr Ala Glu Ala Tyr Ile Gly
210 215 220
Leu Ser Leu Ile Asn Asp Gly Gln Tyr Tyr Phe Asn Asp Asp Gly Ile
225 230 235 240
Met Gln Val Gly Phe Val Thr Ile Asn Asp Lys Val Phe Tyr Phe Ser
245 250 255
Asp Ser Gly Ile Ile Glu Ser Gly Val Gln Asn Ile Asp Asp Asn Tyr
260 265 270
Phe Tyr Ile Asp Asp Asn Gly Ile Val Gln Ile Gly Val Phe Asp Thr
275 280 285
Ser Asp Gly Tyr Lys Tyr Phe Ala Pro Ala Asn Thr Val Asn Asp Asn
290 295 300
Ile Tyr Gly Gln Ala Val Glu Tyr Ser Gly Leu Val Arg Val Gly Glu
305 310 315 320
Asp Val Tyr Tyr Phe Gly Glu Thr Tyr Thr Ile Glu Thr Gly Trp Ile
325 330 335
Tyr Asp Met Glu Asn Glu Ser Asp Lys Tyr Tyr Phe Asn Pro Glu Thr
340 345 350
Lys Lys Ala Cys Lys Gly Ile Asn Leu Ile Asp Asp Ile Lys Tyr Tyr
355 360 365
Phe Asp Glu Lys Gly Ile Met Arg Thr Gly Leu Ile Ser Phe Glu Asn
370 375 380
Asn Asn Tyr Tyr Phe Asn Glu Asn Gly Glu Met Gln Phe Gly Tyr Ile
385 390 395 400
Asn Ile Glu Asp Lys Met Phe Tyr Phe Gly Glu Asp Gly Val Met Gln
405 410 415
Ile Gly Val Phe Asn Thr Pro Asp Gly Phe Lys Tyr Phe Ala His Gln
420 425 430
Asn Thr Leu Asp Glu Asn Phe Glu Gly Glu Ser Ile Asn Tyr Thr Gly
435 440 445
Trp Leu Asp Leu Asp Glu Lys Arg Tyr Tyr Phe Thr Asp Glu Tyr Ile
450 455 460
Ala Ala Thr Gly Ser Val Ile Ile Asp Gly Glu Glu Tyr Tyr Phe Asp
465 470 475 480
Pro Asp Thr Ala Gln Leu Val Ile Ser Glu
485 490
<210> 19
<211> 379
<212> PRT
<213> 艰难梭菌
<400> 19
Gly Phe Val Ser Ile Asn Asp Asn Lys His Tyr Phe Asp Asp Ser Gly
1 5 10 15
Val Met Lys Val Gly Tyr Thr Glu Ile Asp Gly Lys His Phe Tyr Phe
20 25 30
Ala Glu Asn Gly Glu Met Gln Ile Gly Val Phe Asn Thr Glu Asp Gly
35 40 45
Phe Lys Tyr Phe Ala His His Asn Glu Asp Leu Gly Asn Glu Glu Gly
50 55 60
Glu Glu Ile Ser Tyr Ser Gly Ile Leu Asn Phe Asn Asn Lys Ile Tyr
65 70 75 80
Tyr Phe Asp Asp Ser Phe Thr Ala Val Val Gly Trp Lys Asp Leu Glu
85 90 95
Asp Gly Ser Lys Tyr Tyr Phe Asp Glu Asp Thr Ala Glu Ala Tyr Ile
100 105 110
Gly Leu Ser Leu Ile Asn Asp Gly Gln Tyr Tyr Phe Asn Asp Asp Gly
115 120 125
Ile Met Gln Val Gly Phe Val Thr Ile Asn Asp Lys Val Phe Tyr Phe
130 135 140
Ser Asp Ser Gly Ile Ile Glu Ser Gly Val Gln Asn Ile Asp Asp Asn
145 150 155 160
Tyr Phe Tyr Ile Asp Asp Asn Gly Ile Val Gln Ile Gly Val Phe Asp
165 170 175
Thr Ser Asp Gly Tyr Lys Tyr Phe Ala Pro Ala Asn Thr Val Asn Asp
180 185 190
Asn Ile Tyr Gly Gln Ala Val Glu Tyr Ser Gly Leu Val Arg Val Gly
195 200 205
Glu Asp Val Tyr Tyr Phe Gly Glu Thr Tyr Thr Ile Glu Thr Gly Trp
210 215 220
Ile Tyr Asp Met Glu Asn Glu Ser Asp Lys Tyr Tyr Phe Asn Pro Glu
225 230 235 240
Thr Lys Lys Ala Cys Lys Gly Ile Asn Leu Ile Asp Asp Ile Lys Tyr
245 250 255
Tyr Phe Asp Glu Lys Gly Ile Met Arg Thr Gly Leu Ile Ser Phe Glu
260 265 270
Asn Asn Asn Tyr Tyr Phe Asn Glu Asn Gly Glu Met Gln Phe Gly Tyr
275 280 285
Ile Asn Ile Glu Asp Lys Met Phe Tyr Phe Gly Glu Asp Gly Val Met
290 295 300
Gln Ile Gly Val Phe Asn Thr Pro Asp Gly Phe Lys Tyr Phe Ala His
305 310 315 320
Gln Asn Thr Leu Asp Glu Asn Phe Glu Gly Glu Ser Ile Asn Tyr Thr
325 330 335
Gly Trp Leu Asp Leu Asp Glu Lys Arg Tyr Tyr Phe Thr Asp Glu Tyr
340 345 350
Ile Ala Ala Thr Gly Ser Val Ile Ile Asp Gly Glu Glu Tyr Tyr Phe
355 360 365
Asp Pro Asp Thr Ala Gln Leu Val Ile Ser Glu
370 375
<210> 20
<211> 3339
<212> DNA
<213> 艰难梭菌
<400> 20
atggcaaccg gttggcagac catcgatggc aaaaaatatt attttaatac caacaccgca 60
attgcaagca ccggctatac cattatcaac ggcaaacact tttattttaa caccgacggc 120
attatgcaga ttggtgtgtt taaaggtccg aacggctttg aatactttgc accggcaaat 180
accgatgcca ataatattga aggccaggcc attctgtatc agaatgaatt tctgaccctg 240
aacggcaaaa aatactactt tggcagcgat agcaaagcag ttaccggttg gcgcatcatc 300
aacaataaga aatattactt caacccgaat aatgcaattg cagcaattca tctgtgcacc 360
attaacaacg acaaatatta tttcagctat gacggtattc tgcagaatgg ctacattacc 420
atcgaacgca acaactttta tttcgatgcc aacaacgaaa gcaaaatggt gaccggtgtt 480
ttcaaaggcc ctaatggttt tgagtatttc gctccggcaa acacccataa taacaacatt 540
gaaggtcagg cgatcgttta tcagaacaaa ttcctgacgc tgaatggtaa gaaatactat 600
ttcgataatg acagcaaagc cgtgaccggc tggcagacaa ttgacgggaa gaaatattac 660
tttaatctga ataccgcaga agcagcaacc ggttggcaaa cgatcgacgg taaaaagtac 720
tacttcaacc tgaacacagc cgaagcagcc acaggatggc agactattga tggaaaaaaa 780
tactatttca acaccaacac ctttattgca tctaccggtt ataccagcat taacggtaaa 840
catttctact tcaacaccga tggtatcatg cagatcggcg ttttcaaagg tccaaatggt 900
ttcgaatact ttgcccctgc caatacagat gcaaataaca tcgagggtca ggcaatcctg 960
taccaaaaca aatttctgac cctgaatggg aaaaaatatt actttggtag cgattctaaa 1020
gccgttaccg gtctgcgtac cattgatggt aaaaaatact actttaatac gaatacagcc 1080
gttgcggtta caggctggca gaccattaac gggaaaaaat actattttaa cacaaatacc 1140
agcattgcct caacgggtta taccattatt tcgggtaaac acttctactt taataccgat 1200
ggtattatgc aaatcggagt ctttaaagga cctgatgggt tcgaatattt tgcgcctgcg 1260
aacactgatg cgaacaatat cgaaggacag gcaatccgct atcagaatcg ctttctgtat 1320
ctgcacgaca acatctatta ttttggcaac aattcaaaag cagccaccgg ctgggttaca 1380
attgatggca accgctacta tttcgaaccg aataccgcaa tgggtgcaaa tggctacaaa 1440
accatcgata ataaaaattt ctattttcgc aacggtctgc cgcagatcgg ggtatttaaa 1500
ggtagcaacg gcttcgaata cttcgctcca gcgaatacgg acgcgaacaa tattgagggt 1560
caagcgattc gttatcaaaa ccgttttctg catctgctgg gcaaaatcta ctactttggc 1620
aataacagta aagcagttac tggatggcag acaatcaatg gtaaagtgta ctattttatg 1680
ccggataccg ccatggcagc agccggtggt ctgtttgaaa ttgatggcgt gatctatttt 1740
tttggtgtgg atggtgttaa agcaccggga atatacggtg gtaccggctt tgtgaccgtg 1800
ggtgatgata aatactattt caatccgatt aacggtggtg cagcgagcat tggcgaaacc 1860
atcatcgatg acaaaaacta ttatttcaac cagagcggtg tgctgcagac cggtgtgttt 1920
agcaccgaag atggctttaa atattttgcg ccagcgaaca ccctggatga aaacctggaa 1980
ggcgaagcga ttgattttac cggcaaactg atcatcgatg aaaacatcta ttacttcgat 2040
gataactatc gtggtgcggt ggaatggaaa gaactggatg gcgaaatgca ttatttttct 2100
ccggaaaccg gtaaagcgtt taaaggcctg aaccagatcg gcgattacaa atactacttc 2160
aacagcgatg gcgtgatgca gaaaggcttt gtgagcatca acgataacaa acactatttc 2220
gatgatagcg gtgtgatgaa agtgggctat accgaaattg atggcaaaca tttctacttc 2280
gcggaaaacg gcgaaatgca gattggcgtg ttcaataccg aagatggttt caaatacttc 2340
gcgcaccata acgaagatct gggtaacgaa gaaggcgaag aaattagcta tagcggcatc 2400
ctgaacttca acaacaaaat ctactacttt gatgatagct ttaccgcggt ggtgggctgg 2460
aaagatctgg aagatggcag caaatattat ttcgatgaag ataccgcgga agcgtatatt 2520
ggcctgagcc tgattaacga tggccagtac tattttaacg atgatggcat tatgcaggtg 2580
ggtttcgtga ccattaatga taaagtgttc tatttcagcg atagcggcat tattgaaagc 2640
ggcgtgcaga acattgatga taactacttc tacatcgatg ataacggcat tgtgcagatc 2700
ggcgtttttg ataccagcga tggctacaaa tatttcgcac cggccaatac cgtgaacgat 2760
aacatttatg gccaggcggt ggaatatagc ggtctggtgc gtgtgggcga agatgtgtat 2820
tatttcggcg aaacctatac catcgaaacc ggctggattt atgatatgga aaacgaaagc 2880
gataaatatt actttaatcc ggaaacgaaa aaagcgtgca aaggcattaa cctgatcgat 2940
gatatcaaat actattttga tgaaaaaggc attatgcgta ccggtctgat tagcttcgaa 3000
aacaacaact attacttcaa cgaaaacggt gaaatgcagt tcggctacat caacatcgaa 3060
gataaaatgt tctacttcgg cgaagatggt gttatgcaga ttggtgtttt taacaccccg 3120
gatggcttca aatactttgc ccatcagaat accctggatg aaaatttcga aggtgaaagc 3180
attaactata ccggctggct ggatctggat gaaaaacgct actacttcac cgatgaatac 3240
attgcggcga ccggcagcgt gattattgat ggcgaagaat actacttcga tccggatacc 3300
gcgcagctgg tgattagcga acatcatcat catcaccat 3339
<210> 21
<211> 1113
<212> PRT
<213> 艰难梭菌
<400> 21
Met Ala Thr Gly Trp Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe Asn
1 5 10 15
Thr Asn Thr Ala Ile Ala Ser Thr Gly Tyr Thr Ile Ile Asn Gly Lys
20 25 30
His Phe Tyr Phe Asn Thr Asp Gly Ile Met Gln Ile Gly Val Phe Lys
35 40 45
Gly Pro Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr Asp Ala Asn
50 55 60
Asn Ile Glu Gly Gln Ala Ile Leu Tyr Gln Asn Glu Phe Leu Thr Leu
65 70 75 80
Asn Gly Lys Lys Tyr Tyr Phe Gly Ser Asp Ser Lys Ala Val Thr Gly
85 90 95
Trp Arg Ile Ile Asn Asn Lys Lys Tyr Tyr Phe Asn Pro Asn Asn Ala
100 105 110
Ile Ala Ala Ile His Leu Cys Thr Ile Asn Asn Asp Lys Tyr Tyr Phe
115 120 125
Ser Tyr Asp Gly Ile Leu Gln Asn Gly Tyr Ile Thr Ile Glu Arg Asn
130 135 140
Asn Phe Tyr Phe Asp Ala Asn Asn Glu Ser Lys Met Val Thr Gly Val
145 150 155 160
Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr His
165 170 175
Asn Asn Asn Ile Glu Gly Gln Ala Ile Val Tyr Gln Asn Lys Phe Leu
180 185 190
Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Asp Asn Asp Ser Lys Ala Val
195 200 205
Thr Gly Trp Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe Asn Leu Asn
210 215 220
Thr Ala Glu Ala Ala Thr Gly Trp Gln Thr Ile Asp Gly Lys Lys Tyr
225 230 235 240
Tyr Phe Asn Leu Asn Thr Ala Glu Ala Ala Thr Gly Trp Gln Thr Ile
245 250 255
Asp Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Phe Ile Ala Ser Thr
260 265 270
Gly Tyr Thr Ser Ile Asn Gly Lys His Phe Tyr Phe Asn Thr Asp Gly
275 280 285
Ile Met Gln Ile Gly Val Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe
290 295 300
Ala Pro Ala Asn Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile Leu
305 310 315 320
Tyr Gln Asn Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Gly
325 330 335
Ser Asp Ser Lys Ala Val Thr Gly Leu Arg Thr Ile Asp Gly Lys Lys
340 345 350
Tyr Tyr Phe Asn Thr Asn Thr Ala Val Ala Val Thr Gly Trp Gln Thr
355 360 365
Ile Asn Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Ser Ile Ala Ser
370 375 380
Thr Gly Tyr Thr Ile Ile Ser Gly Lys His Phe Tyr Phe Asn Thr Asp
385 390 395 400
Gly Ile Met Gln Ile Gly Val Phe Lys Gly Pro Asp Gly Phe Glu Tyr
405 410 415
Phe Ala Pro Ala Asn Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile
420 425 430
Arg Tyr Gln Asn Arg Phe Leu Tyr Leu His Asp Asn Ile Tyr Tyr Phe
435 440 445
Gly Asn Asn Ser Lys Ala Ala Thr Gly Trp Val Thr Ile Asp Gly Asn
450 455 460
Arg Tyr Tyr Phe Glu Pro Asn Thr Ala Met Gly Ala Asn Gly Tyr Lys
465 470 475 480
Thr Ile Asp Asn Lys Asn Phe Tyr Phe Arg Asn Gly Leu Pro Gln Ile
485 490 495
Gly Val Phe Lys Gly Ser Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn
500 505 510
Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile Arg Tyr Gln Asn Arg
515 520 525
Phe Leu His Leu Leu Gly Lys Ile Tyr Tyr Phe Gly Asn Asn Ser Lys
530 535 540
Ala Val Thr Gly Trp Gln Thr Ile Asn Gly Lys Val Tyr Tyr Phe Met
545 550 555 560
Pro Asp Thr Ala Met Ala Ala Ala Gly Gly Leu Phe Glu Ile Asp Gly
565 570 575
Val Ile Tyr Phe Phe Gly Val Asp Gly Val Lys Ala Pro Gly Ile Tyr
580 585 590
Gly Gly Thr Gly Phe Val Thr Val Gly Asp Asp Lys Tyr Tyr Phe Asn
595 600 605
Pro Ile Asn Gly Gly Ala Ala Ser Ile Gly Glu Thr Ile Ile Asp Asp
610 615 620
Lys Asn Tyr Tyr Phe Asn Gln Ser Gly Val Leu Gln Thr Gly Val Phe
625 630 635 640
Ser Thr Glu Asp Gly Phe Lys Tyr Phe Ala Pro Ala Asn Thr Leu Asp
645 650 655
Glu Asn Leu Glu Gly Glu Ala Ile Asp Phe Thr Gly Lys Leu Ile Ile
660 665 670
Asp Glu Asn Ile Tyr Tyr Phe Asp Asp Asn Tyr Arg Gly Ala Val Glu
675 680 685
Trp Lys Glu Leu Asp Gly Glu Met His Tyr Phe Ser Pro Glu Thr Gly
690 695 700
Lys Ala Phe Lys Gly Leu Asn Gln Ile Gly Asp Tyr Lys Tyr Tyr Phe
705 710 715 720
Asn Ser Asp Gly Val Met Gln Lys Gly Phe Val Ser Ile Asn Asp Asn
725 730 735
Lys His Tyr Phe Asp Asp Ser Gly Val Met Lys Val Gly Tyr Thr Glu
740 745 750
Ile Asp Gly Lys His Phe Tyr Phe Ala Glu Asn Gly Glu Met Gln Ile
755 760 765
Gly Val Phe Asn Thr Glu Asp Gly Phe Lys Tyr Phe Ala His His Asn
770 775 780
Glu Asp Leu Gly Asn Glu Glu Gly Glu Glu Ile Ser Tyr Ser Gly Ile
785 790 795 800
Leu Asn Phe Asn Asn Lys Ile Tyr Tyr Phe Asp Asp Ser Phe Thr Ala
805 810 815
Val Val Gly Trp Lys Asp Leu Glu Asp Gly Ser Lys Tyr Tyr Phe Asp
820 825 830
Glu Asp Thr Ala Glu Ala Tyr Ile Gly Leu Ser Leu Ile Asn Asp Gly
835 840 845
Gln Tyr Tyr Phe Asn Asp Asp Gly Ile Met Gln Val Gly Phe Val Thr
850 855 860
Ile Asn Asp Lys Val Phe Tyr Phe Ser Asp Ser Gly Ile Ile Glu Ser
865 870 875 880
Gly Val Gln Asn Ile Asp Asp Asn Tyr Phe Tyr Ile Asp Asp Asn Gly
885 890 895
Ile Val Gln Ile Gly Val Phe Asp Thr Ser Asp Gly Tyr Lys Tyr Phe
900 905 910
Ala Pro Ala Asn Thr Val Asn Asp Asn Ile Tyr Gly Gln Ala Val Glu
915 920 925
Tyr Ser Gly Leu Val Arg Val Gly Glu Asp Val Tyr Tyr Phe Gly Glu
930 935 940
Thr Tyr Thr Ile Glu Thr Gly Trp Ile Tyr Asp Met Glu Asn Glu Ser
945 950 955 960
Asp Lys Tyr Tyr Phe Asn Pro Glu Thr Lys Lys Ala Cys Lys Gly Ile
965 970 975
Asn Leu Ile Asp Asp Ile Lys Tyr Tyr Phe Asp Glu Lys Gly Ile Met
980 985 990
Arg Thr Gly Leu Ile Ser Phe Glu Asn Asn Asn Tyr Tyr Phe Asn Glu
995 1000 1005
Asn Gly Glu Met Gln Phe Gly Tyr Ile Asn Ile Glu Asp Lys Met Phe
1010 1015 1020
Tyr Phe Gly Glu Asp Gly Val Met Gln Ile Gly Val Phe Asn Thr Pro
1025 1030 1035 1040
Asp Gly Phe Lys Tyr Phe Ala His Gln Asn Thr Leu Asp Glu Asn Phe
1045 1050 1055
Glu Gly Glu Ser Ile Asn Tyr Thr Gly Trp Leu Asp Leu Asp Glu Lys
1060 1065 1070
Arg Tyr Tyr Phe Thr Asp Glu Tyr Ile Ala Ala Thr Gly Ser Val Ile
1075 1080 1085
Ile Asp Gly Glu Glu Tyr Tyr Phe Asp Pro Asp Thr Ala Gln Leu Val
1090 1095 1100
Ile Ser Glu His His His His His His
1105 1110
<210> 22
<211> 3324
<212> DNA
<213> 艰难梭菌
<400> 22
atggcaaccg gttggcagac catcgatggc aaaaaatatt attttaatac caacaccgca 60
attgcaagca ccggctatac cattatcaac ggcaaacact tttattttaa caccgacggc 120
attatgcaga ttggtgtgtt taaaggtccg aacggctttg aatactttgc accggcaaat 180
accgatgcca ataatattga aggccaggcc attctgtatc agaatgaatt tctgaccctg 240
aacggcaaaa aatactactt tggcagcgat agcaaagcag ttaccggttg gcgcatcatc 300
aacaataaga aatattactt caacccgaat aatgcaattg cagcaattca tctgtgcacc 360
attaacaacg acaaatatta tttcagctat gacggtattc tgcagaatgg ctacattacc 420
atcgaacgca acaactttta tttcgatgcc aacaacgaaa gcaaaatggt gaccggtgtt 480
ttcaaaggcc ctaatggttt tgagtatttc gctccggcaa acacccataa taacaacatt 540
gaaggtcagg cgatcgttta tcagaacaaa ttcctgacgc tgaatggtaa gaaatactat 600
ttcgataatg acagcaaagc cgtgaccggc tggcagacaa ttgacgggaa gaaatattac 660
tttaatctga ataccgcaga agcagcaacc ggttggcaaa cgatcgacgg taaaaagtac 720
tacttcaacc tgaacacagc cgaagcagcc acaggatggc agactattga tggaaaaaaa 780
tactatttca acaccaacac ctttattgca tctaccggtt ataccagcat taacggtaaa 840
catttctact tcaacaccga tggtatcatg cagatcggcg ttttcaaagg tccaaatggt 900
ttcgaatact ttgcccctgc caatacagat gcaaataaca tcgagggtca ggcaatcctg 960
taccaaaaca aatttctgac cctgaatggg aaaaaatatt actttggtag cgattctaaa 1020
gccgttaccg gtctgcgtac cattgatggt aaaaaatact actttaatac gaatacagcc 1080
gttgcggtta caggctggca gaccattaac gggaaaaaat actattttaa cacaaatacc 1140
agcattgcct caacgggtta taccattatt tcgggtaaac acttctactt taataccgat 1200
ggtattatgc aaatcggagt ctttaaagga cctgatgggt tcgaatattt tgcgcctgcg 1260
aacactgatg cgaacaatat cgaaggacag gcaatccgct atcagaatcg ctttctgtat 1320
ctgcacgaca acatctatta ttttggcaac aattcaaaag cagccaccgg ctgggttaca 1380
attgatggca accgctacta tttcgaaccg aataccgcaa tgggtgcaaa tggctacaaa 1440
accatcgata ataaaaattt ctattttcgc aacggtctgc cgcagatcgg ggtatttaaa 1500
ggtagcaacg gcttcgaata cttcgctcca gcgaatacgg acgcgaacaa tattgagggt 1560
caagcgattc gttatcaaaa ccgttttctg catctgctgg gcaaaatcta ctactttggc 1620
aataacagta aagcagttac tggatggcag acaatcaatg gtaaagtgta ctattttatg 1680
ccggataccg ccatggcagc agccggtggt ctgtttgaaa ttgatggcgt gatctatttt 1740
tttggtgtgg atggtgttaa agcagttacc ggctttgtga ccgtgggtga tgataaatac 1800
tatttcaatc cgattaacgg tggtgcagcg agcattggcg aaaccatcat cgatgacaaa 1860
aactattatt tcaaccagag cggtgtgctg cagaccggtg tgtttagcac cgaagatggc 1920
tttaaatatt ttgcgccagc gaacaccctg gatgaaaacc tggaaggcga agcgattgat 1980
tttaccggca aactgatcat cgatgaaaac atctattact tcgatgataa ctatcgtggt 2040
gcggtggaat ggaaagaact ggatggcgaa atgcattatt tttctccgga aaccggtaaa 2100
gcgtttaaag gcctgaacca gatcggcgat tacaaatact acttcaacag cgatggcgtg 2160
atgcagaaag gctttgtgag catcaacgat aacaaacact atttcgatga tagcggtgtg 2220
atgaaagtgg gctataccga aattgatggc aaacatttct acttcgcgga aaacggcgaa 2280
atgcagattg gcgtgttcaa taccgaagat ggtttcaaat acttcgcgca ccataacgaa 2340
gatctgggta acgaagaagg cgaagaaatt agctatagcg gcatcctgaa cttcaacaac 2400
aaaatctact actttgatga tagctttacc gcggtggtgg gctggaaaga tctggaagat 2460
ggcagcaaat attatttcga tgaagatacc gcggaagcgt atattggcct gagcctgatt 2520
aacgatggcc agtactattt taacgatgat ggcattatgc aggtgggttt cgtgaccatt 2580
aatgataaag tgttctattt cagcgatagc ggcattattg aaagcggcgt gcagaacatt 2640
gatgataact acttctacat cgatgataac ggcattgtgc agatcggcgt ttttgatacc 2700
agcgatggct acaaatattt cgcaccggcc aataccgtga acgataacat ttatggccag 2760
gcggtggaat atagcggtct ggtgcgtgtg ggcgaagatg tgtattattt cggcgaaacc 2820
tataccatcg aaaccggctg gatttatgat atggaaaacg aaagcgataa atattacttt 2880
aatccggaaa cgaaaaaagc gtgcaaaggc attaacctga tcgatgatat caaatactat 2940
tttgatgaaa aaggcattat gcgtaccggt ctgattagct tcgaaaacaa caactattac 3000
ttcaacgaaa acggtgaaat gcagttcggc tacatcaaca tcgaagataa aatgttctac 3060
ttcggcgaag atggtgttat gcagattggt gtttttaaca ccccggatgg cttcaaatac 3120
tttgcccatc agaataccct ggatgaaaat ttcgaaggtg aaagcattaa ctataccggc 3180
tggctggatc tggatgaaaa acgctactac ttcaccgatg aatacattgc ggcgaccggc 3240
agcgtgatta ttgatggcga agaatactac ttcgatccgg ataccgcgca gctggtgatt 3300
agcgaacatc atcatcatca ccat 3324
<210> 23
<211> 1108
<212> PRT
<213> 艰难梭菌
<400> 23
Met Ala Thr Gly Trp Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe Asn
1 5 10 15
Thr Asn Thr Ala Ile Ala Ser Thr Gly Tyr Thr Ile Ile Asn Gly Lys
20 25 30
His Phe Tyr Phe Asn Thr Asp Gly Ile Met Gln Ile Gly Val Phe Lys
35 40 45
Gly Pro Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr Asp Ala Asn
50 55 60
Asn Ile Glu Gly Gln Ala Ile Leu Tyr Gln Asn Glu Phe Leu Thr Leu
65 70 75 80
Asn Gly Lys Lys Tyr Tyr Phe Gly Ser Asp Ser Lys Ala Val Thr Gly
85 90 95
Trp Arg Ile Ile Asn Asn Lys Lys Tyr Tyr Phe Asn Pro Asn Asn Ala
100 105 110
Ile Ala Ala Ile His Leu Cys Thr Ile Asn Asn Asp Lys Tyr Tyr Phe
115 120 125
Ser Tyr Asp Gly Ile Leu Gln Asn Gly Tyr Ile Thr Ile Glu Arg Asn
130 135 140
Asn Phe Tyr Phe Asp Ala Asn Asn Glu Ser Lys Met Val Thr Gly Val
145 150 155 160
Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr His
165 170 175
Asn Asn Asn Ile Glu Gly Gln Ala Ile Val Tyr Gln Asn Lys Phe Leu
180 185 190
Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Asp Asn Asp Ser Lys Ala Val
195 200 205
Thr Gly Trp Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe Asn Leu Asn
210 215 220
Thr Ala Glu Ala Ala Thr Gly Trp Gln Thr Ile Asp Gly Lys Lys Tyr
225 230 235 240
Tyr Phe Asn Leu Asn Thr Ala Glu Ala Ala Thr Gly Trp Gln Thr Ile
245 250 255
Asp Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Phe Ile Ala Ser Thr
260 265 270
Gly Tyr Thr Ser Ile Asn Gly Lys His Phe Tyr Phe Asn Thr Asp Gly
275 280 285
Ile Met Gln Ile Gly Val Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe
290 295 300
Ala Pro Ala Asn Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile Leu
305 310 315 320
Tyr Gln Asn Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Gly
325 330 335
Ser Asp Ser Lys Ala Val Thr Gly Leu Arg Thr Ile Asp Gly Lys Lys
340 345 350
Tyr Tyr Phe Asn Thr Asn Thr Ala Val Ala Val Thr Gly Trp Gln Thr
355 360 365
Ile Asn Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Ser Ile Ala Ser
370 375 380
Thr Gly Tyr Thr Ile Ile Ser Gly Lys His Phe Tyr Phe Asn Thr Asp
385 390 395 400
Gly Ile Met Gln Ile Gly Val Phe Lys Gly Pro Asp Gly Phe Glu Tyr
405 410 415
Phe Ala Pro Ala Asn Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile
420 425 430
Arg Tyr Gln Asn Arg Phe Leu Tyr Leu His Asp Asn Ile Tyr Tyr Phe
435 440 445
Gly Asn Asn Ser Lys Ala Ala Thr Gly Trp Val Thr Ile Asp Gly Asn
450 455 460
Arg Tyr Tyr Phe Glu Pro Asn Thr Ala Met Gly Ala Asn Gly Tyr Lys
465 470 475 480
Thr Ile Asp Asn Lys Asn Phe Tyr Phe Arg Asn Gly Leu Pro Gln Ile
485 490 495
Gly Val Phe Lys Gly Ser Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn
500 505 510
Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile Arg Tyr Gln Asn Arg
515 520 525
Phe Leu His Leu Leu Gly Lys Ile Tyr Tyr Phe Gly Asn Asn Ser Lys
530 535 540
Ala Val Thr Gly Trp Gln Thr Ile Asn Gly Lys Val Tyr Tyr Phe Met
545 550 555 560
Pro Asp Thr Ala Met Ala Ala Ala Gly Gly Leu Phe Glu Ile Asp Gly
565 570 575
Val Ile Tyr Phe Phe Gly Val Asp Gly Val Lys Ala Val Thr Gly Phe
580 585 590
Val Thr Val Gly Asp Asp Lys Tyr Tyr Phe Asn Pro Ile Asn Gly Gly
595 600 605
Ala Ala Ser Ile Gly Glu Thr Ile Ile Asp Asp Lys Asn Tyr Tyr Phe
610 615 620
Asn Gln Ser Gly Val Leu Gln Thr Gly Val Phe Ser Thr Glu Asp Gly
625 630 635 640
Phe Lys Tyr Phe Ala Pro Ala Asn Thr Leu Asp Glu Asn Leu Glu Gly
645 650 655
Glu Ala Ile Asp Phe Thr Gly Lys Leu Ile Ile Asp Glu Asn Ile Tyr
660 665 670
Tyr Phe Asp Asp Asn Tyr Arg Gly Ala Val Glu Trp Lys Glu Leu Asp
675 680 685
Gly Glu Met His Tyr Phe Ser Pro Glu Thr Gly Lys Ala Phe Lys Gly
690 695 700
Leu Asn Gln Ile Gly Asp Tyr Lys Tyr Tyr Phe Asn Ser Asp Gly Val
705 710 715 720
Met Gln Lys Gly Phe Val Ser Ile Asn Asp Asn Lys His Tyr Phe Asp
725 730 735
Asp Ser Gly Val Met Lys Val Gly Tyr Thr Glu Ile Asp Gly Lys His
740 745 750
Phe Tyr Phe Ala Glu Asn Gly Glu Met Gln Ile Gly Val Phe Asn Thr
755 760 765
Glu Asp Gly Phe Lys Tyr Phe Ala His His Asn Glu Asp Leu Gly Asn
770 775 780
Glu Glu Gly Glu Glu Ile Ser Tyr Ser Gly Ile Leu Asn Phe Asn Asn
785 790 795 800
Lys Ile Tyr Tyr Phe Asp Asp Ser Phe Thr Ala Val Val Gly Trp Lys
805 810 815
Asp Leu Glu Asp Gly Ser Lys Tyr Tyr Phe Asp Glu Asp Thr Ala Glu
820 825 830
Ala Tyr Ile Gly Leu Ser Leu Ile Asn Asp Gly Gln Tyr Tyr Phe Asn
835 840 845
Asp Asp Gly Ile Met Gln Val Gly Phe Val Thr Ile Asn Asp Lys Val
850 855 860
Phe Tyr Phe Ser Asp Ser Gly Ile Ile Glu Ser Gly Val Gln Asn Ile
865 870 875 880
Asp Asp Asn Tyr Phe Tyr Ile Asp Asp Asn Gly Ile Val Gln Ile Gly
885 890 895
Val Phe Asp Thr Ser Asp Gly Tyr Lys Tyr Phe Ala Pro Ala Asn Thr
900 905 910
Val Asn Asp Asn Ile Tyr Gly Gln Ala Val Glu Tyr Ser Gly Leu Val
915 920 925
Arg Val Gly Glu Asp Val Tyr Tyr Phe Gly Glu Thr Tyr Thr Ile Glu
930 935 940
Thr Gly Trp Ile Tyr Asp Met Glu Asn Glu Ser Asp Lys Tyr Tyr Phe
945 950 955 960
Asn Pro Glu Thr Lys Lys Ala Cys Lys Gly Ile Asn Leu Ile Asp Asp
965 970 975
Ile Lys Tyr Tyr Phe Asp Glu Lys Gly Ile Met Arg Thr Gly Leu Ile
980 985 990
Ser Phe Glu Asn Asn Asn Tyr Tyr Phe Asn Glu Asn Gly Glu Met Gln
995 1000 1005
Phe Gly Tyr Ile Asn Ile Glu Asp Lys Met Phe Tyr Phe Gly Glu Asp
1010 1015 1020
Gly Val Met Gln Ile Gly Val Phe Asn Thr Pro Asp Gly Phe Lys Tyr
1025 1030 1035 1040
Phe Ala His Gln Asn Thr Leu Asp Glu Asn Phe Glu Gly Glu Ser Ile
1045 1050 1055
Asn Tyr Thr Gly Trp Leu Asp Leu Asp Glu Lys Arg Tyr Tyr Phe Thr
1060 1065 1070
Asp Glu Tyr Ile Ala Ala Thr Gly Ser Val Ile Ile Asp Gly Glu Glu
1075 1080 1085
Tyr Tyr Phe Asp Pro Asp Thr Ala Gln Leu Val Ile Ser Glu His His
1090 1095 1100
His His His His
1105
<210> 24
<211> 3387
<212> DNA
<213> 艰难梭菌
<400> 24
atggcaaccg gttggcagac catcgatggc aaaaaatatt attttaatac caacaccgca 60
attgcaagca ccggctatac cattatcaac ggcaaacact tttattttaa caccgacggc 120
attatgcaga ttggtgtgtt taaaggtccg aacggctttg aatactttgc accggcaaat 180
accgatgcca ataatattga aggccaggcc attctgtatc agaatgaatt tctgaccctg 240
aacggcaaaa aatactactt tggcagcgat agcaaagcag ttaccggttg gcgcatcatc 300
aacaataaga aatattactt caacccgaat aatgcaattg cagcaattca tctgtgcacc 360
attaacaacg acaaatatta tttcagctat gacggtattc tgcagaatgg ctacattacc 420
atcgaacgca acaactttta tttcgatgcc aacaacgaaa gcaaaatggt gaccggtgtt 480
ttcaaaggcc ctaatggttt tgagtatttc gctccggcaa acacccataa taacaacatt 540
gaaggtcagg cgatcgttta tcagaacaaa ttcctgacgc tgaatggtaa gaaatactat 600
ttcgataatg acagcaaagc cgtgaccggc tggcagacaa ttgacgggaa gaaatattac 660
tttaatctga ataccgcaga agcagcaacc ggttggcaaa cgatcgacgg taaaaagtac 720
tacttcaacc tgaacacagc cgaagcagcc acaggatggc agactattga tggaaaaaaa 780
tactatttca acaccaacac ctttattgca tctaccggtt ataccagcat taacggtaaa 840
catttctact tcaacaccga tggtatcatg cagatcggcg ttttcaaagg tccaaatggt 900
ttcgaatact ttgcccctgc caatacagat gcaaataaca tcgagggtca ggcaatcctg 960
taccaaaaca aatttctgac cctgaatggg aaaaaatatt actttggtag cgattctaaa 1020
gccgttaccg gtctgcgtac cattgatggt aaaaaatact actttaatac gaatacagcc 1080
gttgcggtta caggctggca gaccattaac gggaaaaaat actattttaa cacaaatacc 1140
agcattgcct caacgggtta taccattatt tcgggtaaac acttctactt taataccgat 1200
ggtattatgc aaatcggagt ctttaaagga cctgatgggt tcgaatattt tgcgcctgcg 1260
aacactgatg cgaacaatat cgaaggacag gcaatccgct atcagaatcg ctttctgtat 1320
ctgcacgaca acatctatta ttttggcaac aattcaaaag cagccaccgg ctgggttaca 1380
attgatggca accgctacta tttcgaaccg aataccgcaa tgggtgcaaa tggctacaaa 1440
accatcgata ataaaaattt ctattttcgc aacggtctgc cgcagatcgg ggtatttaaa 1500
ggtagcaacg gcttcgaata cttcgctcca gcgaatacgg acgcgaacaa tattgagggt 1560
caagcgattc gttatcaaaa ccgttttctg catctgctgg gcaaaatcta ctactttggc 1620
aataacagta aagcagttac tggatggcag acaatcaatg gtaaagtgta ctattttatg 1680
ccggataccg ccatggcagc agccggtggt ctgtttgaaa ttgatggcgt gatctatttt 1740
tttggtgtgg atggtgttaa agcagtgagc ggtctgattt atattaacga tagcctgtat 1800
tactttaaac caccggtgaa taacctgatt accggctttg tgaccgtggg tgatgataaa 1860
tactatttca atccgattaa cggtggtgca gcgagcattg gcgaaaccat catcgatgac 1920
aaaaactatt atttcaacca gagcggtgtg ctgcagaccg gtgtgtttag caccgaagat 1980
ggctttaaat attttgcgcc agcgaacacc ctggatgaaa acctggaagg cgaagcgatt 2040
gattttaccg gcaaactgat catcgatgaa aacatctatt acttcgatga taactatcgt 2100
ggtgcggtgg aatggaaaga actggatggc gaaatgcatt atttttctcc ggaaaccggt 2160
aaagcgttta aaggcctgaa ccagatcggc gattacaaat actacttcaa cagcgatggc 2220
gtgatgcaga aaggctttgt gagcatcaac gataacaaac actatttcga tgatagcggt 2280
gtgatgaaag tgggctatac cgaaattgat ggcaaacatt tctacttcgc ggaaaacggc 2340
gaaatgcaga ttggcgtgtt caataccgaa gatggtttca aatacttcgc gcaccataac 2400
gaagatctgg gtaacgaaga aggcgaagaa attagctata gcggcatcct gaacttcaac 2460
aacaaaatct actactttga tgatagcttt accgcggtgg tgggctggaa agatctggaa 2520
gatggcagca aatattattt cgatgaagat accgcggaag cgtatattgg cctgagcctg 2580
attaacgatg gccagtacta ttttaacgat gatggcatta tgcaggtggg tttcgtgacc 2640
attaatgata aagtgttcta tttcagcgat agcggcatta ttgaaagcgg cgtgcagaac 2700
attgatgata actacttcta catcgatgat aacggcattg tgcagatcgg cgtttttgat 2760
accagcgatg gctacaaata tttcgcaccg gccaataccg tgaacgataa catttatggc 2820
caggcggtgg aatatagcgg tctggtgcgt gtgggcgaag atgtgtatta tttcggcgaa 2880
acctatacca tcgaaaccgg ctggatttat gatatggaaa acgaaagcga taaatattac 2940
tttaatccgg aaacgaaaaa agcgtgcaaa ggcattaacc tgatcgatga tatcaaatac 3000
tattttgatg aaaaaggcat tatgcgtacc ggtctgatta gcttcgaaaa caacaactat 3060
tacttcaacg aaaacggtga aatgcagttc ggctacatca acatcgaaga taaaatgttc 3120
tacttcggcg aagatggtgt tatgcagatt ggtgttttta acaccccgga tggcttcaaa 3180
tactttgccc atcagaatac cctggatgaa aatttcgaag gtgaaagcat taactatacc 3240
ggctggctgg atctggatga aaaacgctac tacttcaccg atgaatacat tgcggcgacc 3300
ggcagcgtga ttattgatgg cgaagaatac tacttcgatc cggataccgc gcagctggtg 3360
attagcgaac atcatcatca tcaccat 3387
<210> 25
<211> 1129
<212> PRT
<213> 艰难梭菌
<400> 25
Met Ala Thr Gly Trp Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe Asn
1 5 10 15
Thr Asn Thr Ala Ile Ala Ser Thr Gly Tyr Thr Ile Ile Asn Gly Lys
20 25 30
His Phe Tyr Phe Asn Thr Asp Gly Ile Met Gln Ile Gly Val Phe Lys
35 40 45
Gly Pro Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr Asp Ala Asn
50 55 60
Asn Ile Glu Gly Gln Ala Ile Leu Tyr Gln Asn Glu Phe Leu Thr Leu
65 70 75 80
Asn Gly Lys Lys Tyr Tyr Phe Gly Ser Asp Ser Lys Ala Val Thr Gly
85 90 95
Trp Arg Ile Ile Asn Asn Lys Lys Tyr Tyr Phe Asn Pro Asn Asn Ala
100 105 110
Ile Ala Ala Ile His Leu Cys Thr Ile Asn Asn Asp Lys Tyr Tyr Phe
115 120 125
Ser Tyr Asp Gly Ile Leu Gln Asn Gly Tyr Ile Thr Ile Glu Arg Asn
130 135 140
Asn Phe Tyr Phe Asp Ala Asn Asn Glu Ser Lys Met Val Thr Gly Val
145 150 155 160
Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr His
165 170 175
Asn Asn Asn Ile Glu Gly Gln Ala Ile Val Tyr Gln Asn Lys Phe Leu
180 185 190
Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Asp Asn Asp Ser Lys Ala Val
195 200 205
Thr Gly Trp Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe Asn Leu Asn
210 215 220
Thr Ala Glu Ala Ala Thr Gly Trp Gln Thr Ile Asp Gly Lys Lys Tyr
225 230 235 240
Tyr Phe Asn Leu Asn Thr Ala Glu Ala Ala Thr Gly Trp Gln Thr Ile
245 250 255
Asp Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Phe Ile Ala Ser Thr
260 265 270
Gly Tyr Thr Ser Ile Asn Gly Lys His Phe Tyr Phe Asn Thr Asp Gly
275 280 285
Ile Met Gln Ile Gly Val Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe
290 295 300
Ala Pro Ala Asn Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile Leu
305 310 315 320
Tyr Gln Asn Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Gly
325 330 335
Ser Asp Ser Lys Ala Val Thr Gly Leu Arg Thr Ile Asp Gly Lys Lys
340 345 350
Tyr Tyr Phe Asn Thr Asn Thr Ala Val Ala Val Thr Gly Trp Gln Thr
355 360 365
Ile Asn Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Ser Ile Ala Ser
370 375 380
Thr Gly Tyr Thr Ile Ile Ser Gly Lys His Phe Tyr Phe Asn Thr Asp
385 390 395 400
Gly Ile Met Gln Ile Gly Val Phe Lys Gly Pro Asp Gly Phe Glu Tyr
405 410 415
Phe Ala Pro Ala Asn Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile
420 425 430
Arg Tyr Gln Asn Arg Phe Leu Tyr Leu His Asp Asn Ile Tyr Tyr Phe
435 440 445
Gly Asn Asn Ser Lys Ala Ala Thr Gly Trp Val Thr Ile Asp Gly Asn
450 455 460
Arg Tyr Tyr Phe Glu Pro Asn Thr Ala Met Gly Ala Asn Gly Tyr Lys
465 470 475 480
Thr Ile Asp Asn Lys Asn Phe Tyr Phe Arg Asn Gly Leu Pro Gln Ile
485 490 495
Gly Val Phe Lys Gly Ser Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn
500 505 510
Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile Arg Tyr Gln Asn Arg
515 520 525
Phe Leu His Leu Leu Gly Lys Ile Tyr Tyr Phe Gly Asn Asn Ser Lys
530 535 540
Ala Val Thr Gly Trp Gln Thr Ile Asn Gly Lys Val Tyr Tyr Phe Met
545 550 555 560
Pro Asp Thr Ala Met Ala Ala Ala Gly Gly Leu Phe Glu Ile Asp Gly
565 570 575
Val Ile Tyr Phe Phe Gly Val Asp Gly Val Lys Ala Val Ser Gly Leu
580 585 590
Ile Tyr Ile Asn Asp Ser Leu Tyr Tyr Phe Lys Pro Pro Val Asn Asn
595 600 605
Leu Ile Thr Gly Phe Val Thr Val Gly Asp Asp Lys Tyr Tyr Phe Asn
610 615 620
Pro Ile Asn Gly Gly Ala Ala Ser Ile Gly Glu Thr Ile Ile Asp Asp
625 630 635 640
Lys Asn Tyr Tyr Phe Asn Gln Ser Gly Val Leu Gln Thr Gly Val Phe
645 650 655
Ser Thr Glu Asp Gly Phe Lys Tyr Phe Ala Pro Ala Asn Thr Leu Asp
660 665 670
Glu Asn Leu Glu Gly Glu Ala Ile Asp Phe Thr Gly Lys Leu Ile Ile
675 680 685
Asp Glu Asn Ile Tyr Tyr Phe Asp Asp Asn Tyr Arg Gly Ala Val Glu
690 695 700
Trp Lys Glu Leu Asp Gly Glu Met His Tyr Phe Ser Pro Glu Thr Gly
705 710 715 720
Lys Ala Phe Lys Gly Leu Asn Gln Ile Gly Asp Tyr Lys Tyr Tyr Phe
725 730 735
Asn Ser Asp Gly Val Met Gln Lys Gly Phe Val Ser Ile Asn Asp Asn
740 745 750
Lys His Tyr Phe Asp Asp Ser Gly Val Met Lys Val Gly Tyr Thr Glu
755 760 765
Ile Asp Gly Lys His Phe Tyr Phe Ala Glu Asn Gly Glu Met Gln Ile
770 775 780
Gly Val Phe Asn Thr Glu Asp Gly Phe Lys Tyr Phe Ala His His Asn
785 790 795 800
Glu Asp Leu Gly Asn Glu Glu Gly Glu Glu Ile Ser Tyr Ser Gly Ile
805 810 815
Leu Asn Phe Asn Asn Lys Ile Tyr Tyr Phe Asp Asp Ser Phe Thr Ala
820 825 830
Val Val Gly Trp Lys Asp Leu Glu Asp Gly Ser Lys Tyr Tyr Phe Asp
835 840 845
Glu Asp Thr Ala Glu Ala Tyr Ile Gly Leu Ser Leu Ile Asn Asp Gly
850 855 860
Gln Tyr Tyr Phe Asn Asp Asp Gly Ile Met Gln Val Gly Phe Val Thr
865 870 875 880
Ile Asn Asp Lys Val Phe Tyr Phe Ser Asp Ser Gly Ile Ile Glu Ser
885 890 895
Gly Val Gln Asn Ile Asp Asp Asn Tyr Phe Tyr Ile Asp Asp Asn Gly
900 905 910
Ile Val Gln Ile Gly Val Phe Asp Thr Ser Asp Gly Tyr Lys Tyr Phe
915 920 925
Ala Pro Ala Asn Thr Val Asn Asp Asn Ile Tyr Gly Gln Ala Val Glu
930 935 940
Tyr Ser Gly Leu Val Arg Val Gly Glu Asp Val Tyr Tyr Phe Gly Glu
945 950 955 960
Thr Tyr Thr Ile Glu Thr Gly Trp Ile Tyr Asp Met Glu Asn Glu Ser
965 970 975
Asp Lys Tyr Tyr Phe Asn Pro Glu Thr Lys Lys Ala Cys Lys Gly Ile
980 985 990
Asn Leu Ile Asp Asp Ile Lys Tyr Tyr Phe Asp Glu Lys Gly Ile Met
995 1000 1005
Arg Thr Gly Leu Ile Ser Phe Glu Asn Asn Asn Tyr Tyr Phe Asn Glu
1010 1015 1020
Asn Gly Glu Met Gln Phe Gly Tyr Ile Asn Ile Glu Asp Lys Met Phe
1025 1030 1035 1040
Tyr Phe Gly Glu Asp Gly Val Met Gln Ile Gly Val Phe Asn Thr Pro
1045 1050 1055
Asp Gly Phe Lys Tyr Phe Ala His Gln Asn Thr Leu Asp Glu Asn Phe
1060 1065 1070
Glu Gly Glu Ser Ile Asn Tyr Thr Gly Trp Leu Asp Leu Asp Glu Lys
1075 1080 1085
Arg Tyr Tyr Phe Thr Asp Glu Tyr Ile Ala Ala Thr Gly Ser Val Ile
1090 1095 1100
Ile Asp Gly Glu Glu Tyr Tyr Phe Asp Pro Asp Thr Ala Gln Leu Val
1105 1110 1115 1120
Ile Ser Glu His His His His His His
1125
<210> 26
<211> 2985
<212> DNA
<213> 艰难梭菌
<400> 26
atggcaaccg gttggcagac catcgatggc aaaaaatatt attttaatac caacaccgca 60
attgcaagca ccggctatac cattatcaac ggcaaacact tttattttaa caccgacggc 120
attatgcaga ttggtgtgtt taaaggtccg aacggctttg aatactttgc accggcaaat 180
accgatgcca ataatattga aggccaggcc attctgtatc agaatgaatt tctgaccctg 240
aacggcaaaa aatactactt tggcagcgat agcaaagcag ttaccggttg gcgcatcatc 300
aacaataaga aatattactt caacccgaat aatgcaattg cagcaattca tctgtgcacc 360
attaacaacg acaaatatta tttcagctat gacggtattc tgcagaatgg ctacattacc 420
atcgaacgca acaactttta tttcgatgcc aacaacgaaa gcaaaatggt gaccggtgtt 480
ttcaaaggcc ctaatggttt tgagtatttc gctccggcaa acacccataa taacaacatt 540
gaaggtcagg cgatcgttta tcagaacaaa ttcctgacgc tgaatggtaa gaaatactat 600
ttcgataatg acagcaaagc cgtgaccggc tggcagacaa ttgacgggaa gaaatattac 660
tttaatctga ataccgcaga agcagcaacc ggttggcaaa cgatcgacgg taaaaagtac 720
tacttcaacc tgaacacagc cgaagcagcc acaggatggc agactattga tggaaaaaaa 780
tactatttca acaccaacac ctttattgca tctaccggtt ataccagcat taacggtaaa 840
catttctact tcaacaccga tggtatcatg cagatcggcg ttttcaaagg tccaaatggt 900
ttcgaatact ttgcccctgc caatacagat gcaaataaca tcgagggtca ggcaatcctg 960
taccaaaaca aatttctgac cctgaatggg aaaaaatatt actttggtag cgattctaaa 1020
gccgttaccg gtctgcgtac cattgatggt aaaaaatact actttaatac gaatacagcc 1080
gttgcggtta caggctggca gaccattaac gggaaaaaat actattttaa cacaaatacc 1140
agcattgcct caacgggtta taccattatt tcgggtaaac acttctactt taataccgat 1200
ggtattatgc aaatcggagt ctttaaagga cctgatgggt tcgaatattt tgcgcctgcg 1260
aacactgatg cgaacaatat cgaaggacag gcaatccgct atcagaatcg ctttctgtat 1320
ctgcacgaca acatctatta ttttggcaac aattcaaaag cagccaccgg ctgggttaca 1380
attgatggca accgctacta tttcgaaccg aataccgcaa tgggtgcaaa tggctacaaa 1440
accatcgata ataaaaattt ctattttcgc aacggtctgc cgcagatcgg ggtatttaaa 1500
ggtagcaacg gcttcgaata cttcgctcca gcgaatacgg acgcgaacaa tattgagggt 1560
caagcgattc gttatcaaaa ccgttttctg catctgctgg gcaaaatcta ctactttggc 1620
aataacagta aagcagttac tggatggcag acaatcaatg gtaaagtgta ctattttatg 1680
ccggataccg ccatggcagc agccggtggt ctgtttgaaa ttgatggcgt gatctatttt 1740
tttggtgtgg atggtgttaa agcagtgaaa ggcctgaacc agatcggcga ttacaaatac 1800
tacttcaaca gcgatggcgt gatgcagaaa ggctttgtga gcatcaacga taacaaacac 1860
tatttcgatg atagcggtgt gatgaaagtg ggctataccg aaattgatgg caaacatttc 1920
tacttcgcgg aaaacggcga aatgcagatt ggcgtgttca ataccgaaga tggtttcaaa 1980
tacttcgcgc accataacga agatctgggt aacgaagaag gcgaagaaat tagctatagc 2040
ggcatcctga acttcaacaa caaaatctac tactttgatg atagctttac cgcggtggtg 2100
ggctggaaag atctggaaga tggcagcaaa tattatttcg atgaagatac cgcggaagcg 2160
tatattggcc tgagcctgat taacgatggc cagtactatt ttaacgatga tggcattatg 2220
caggtgggtt tcgtgaccat taatgataaa gtgttctatt tcagcgatag cggcattatt 2280
gaaagcggcg tgcagaacat tgatgataac tacttctaca tcgatgataa cggcattgtg 2340
cagatcggcg tttttgatac cagcgatggc tacaaatatt tcgcaccggc caataccgtg 2400
aacgataaca tttatggcca ggcggtggaa tatagcggtc tggtgcgtgt gggcgaagat 2460
gtgtattatt tcggcgaaac ctataccatc gaaaccggct ggatttatga tatggaaaac 2520
gaaagcgata aatattactt taatccggaa acgaaaaaag cgtgcaaagg cattaacctg 2580
atcgatgata tcaaatacta ttttgatgaa aaaggcatta tgcgtaccgg tctgattagc 2640
ttcgaaaaca acaactatta cttcaacgaa aacggtgaaa tgcagttcgg ctacatcaac 2700
atcgaagata aaatgttcta cttcggcgaa gatggtgtta tgcagattgg tgtttttaac 2760
accccggatg gcttcaaata ctttgcccat cagaataccc tggatgaaaa tttcgaaggt 2820
gaaagcatta actataccgg ctggctggat ctggatgaaa aacgctacta cttcaccgat 2880
gaatacattg cggcgaccgg cagcgtgatt attgatggcg aagaatacta cttcgatccg 2940
gataccgcgc agctggtgat tagcgaacat catcatcatc accat 2985
<210> 27
<211> 995
<212> PRT
<213> 艰难梭菌
<400> 27
Met Ala Thr Gly Trp Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe Asn
1 5 10 15
Thr Asn Thr Ala Ile Ala Ser Thr Gly Tyr Thr Ile Ile Asn Gly Lys
20 25 30
His Phe Tyr Phe Asn Thr Asp Gly Ile Met Gln Ile Gly Val Phe Lys
35 40 45
Gly Pro Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr Asp Ala Asn
50 55 60
Asn Ile Glu Gly Gln Ala Ile Leu Tyr Gln Asn Glu Phe Leu Thr Leu
65 70 75 80
Asn Gly Lys Lys Tyr Tyr Phe Gly Ser Asp Ser Lys Ala Val Thr Gly
85 90 95
Trp Arg Ile Ile Asn Asn Lys Lys Tyr Tyr Phe Asn Pro Asn Asn Ala
100 105 110
Ile Ala Ala Ile His Leu Cys Thr Ile Asn Asn Asp Lys Tyr Tyr Phe
115 120 125
Ser Tyr Asp Gly Ile Leu Gln Asn Gly Tyr Ile Thr Ile Glu Arg Asn
130 135 140
Asn Phe Tyr Phe Asp Ala Asn Asn Glu Ser Lys Met Val Thr Gly Val
145 150 155 160
Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr His
165 170 175
Asn Asn Asn Ile Glu Gly Gln Ala Ile Val Tyr Gln Asn Lys Phe Leu
180 185 190
Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Asp Asn Asp Ser Lys Ala Val
195 200 205
Thr Gly Trp Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe Asn Leu Asn
210 215 220
Thr Ala Glu Ala Ala Thr Gly Trp Gln Thr Ile Asp Gly Lys Lys Tyr
225 230 235 240
Tyr Phe Asn Leu Asn Thr Ala Glu Ala Ala Thr Gly Trp Gln Thr Ile
245 250 255
Asp Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Phe Ile Ala Ser Thr
260 265 270
Gly Tyr Thr Ser Ile Asn Gly Lys His Phe Tyr Phe Asn Thr Asp Gly
275 280 285
Ile Met Gln Ile Gly Val Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe
290 295 300
Ala Pro Ala Asn Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile Leu
305 310 315 320
Tyr Gln Asn Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Gly
325 330 335
Ser Asp Ser Lys Ala Val Thr Gly Leu Arg Thr Ile Asp Gly Lys Lys
340 345 350
Tyr Tyr Phe Asn Thr Asn Thr Ala Val Ala Val Thr Gly Trp Gln Thr
355 360 365
Ile Asn Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Ser Ile Ala Ser
370 375 380
Thr Gly Tyr Thr Ile Ile Ser Gly Lys His Phe Tyr Phe Asn Thr Asp
385 390 395 400
Gly Ile Met Gln Ile Gly Val Phe Lys Gly Pro Asp Gly Phe Glu Tyr
405 410 415
Phe Ala Pro Ala Asn Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile
420 425 430
Arg Tyr Gln Asn Arg Phe Leu Tyr Leu His Asp Asn Ile Tyr Tyr Phe
435 440 445
Gly Asn Asn Ser Lys Ala Ala Thr Gly Trp Val Thr Ile Asp Gly Asn
450 455 460
Arg Tyr Tyr Phe Glu Pro Asn Thr Ala Met Gly Ala Asn Gly Tyr Lys
465 470 475 480
Thr Ile Asp Asn Lys Asn Phe Tyr Phe Arg Asn Gly Leu Pro Gln Ile
485 490 495
Gly Val Phe Lys Gly Ser Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn
500 505 510
Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile Arg Tyr Gln Asn Arg
515 520 525
Phe Leu His Leu Leu Gly Lys Ile Tyr Tyr Phe Gly Asn Asn Ser Lys
530 535 540
Ala Val Thr Gly Trp Gln Thr Ile Asn Gly Lys Val Tyr Tyr Phe Met
545 550 555 560
Pro Asp Thr Ala Met Ala Ala Ala Gly Gly Leu Phe Glu Ile Asp Gly
565 570 575
Val Ile Tyr Phe Phe Gly Val Asp Gly Val Lys Ala Val Lys Gly Leu
580 585 590
Asn Gln Ile Gly Asp Tyr Lys Tyr Tyr Phe Asn Ser Asp Gly Val Met
595 600 605
Gln Lys Gly Phe Val Ser Ile Asn Asp Asn Lys His Tyr Phe Asp Asp
610 615 620
Ser Gly Val Met Lys Val Gly Tyr Thr Glu Ile Asp Gly Lys His Phe
625 630 635 640
Tyr Phe Ala Glu Asn Gly Glu Met Gln Ile Gly Val Phe Asn Thr Glu
645 650 655
Asp Gly Phe Lys Tyr Phe Ala His His Asn Glu Asp Leu Gly Asn Glu
660 665 670
Glu Gly Glu Glu Ile Ser Tyr Ser Gly Ile Leu Asn Phe Asn Asn Lys
675 680 685
Ile Tyr Tyr Phe Asp Asp Ser Phe Thr Ala Val Val Gly Trp Lys Asp
690 695 700
Leu Glu Asp Gly Ser Lys Tyr Tyr Phe Asp Glu Asp Thr Ala Glu Ala
705 710 715 720
Tyr Ile Gly Leu Ser Leu Ile Asn Asp Gly Gln Tyr Tyr Phe Asn Asp
725 730 735
Asp Gly Ile Met Gln Val Gly Phe Val Thr Ile Asn Asp Lys Val Phe
740 745 750
Tyr Phe Ser Asp Ser Gly Ile Ile Glu Ser Gly Val Gln Asn Ile Asp
755 760 765
Asp Asn Tyr Phe Tyr Ile Asp Asp Asn Gly Ile Val Gln Ile Gly Val
770 775 780
Phe Asp Thr Ser Asp Gly Tyr Lys Tyr Phe Ala Pro Ala Asn Thr Val
785 790 795 800
Asn Asp Asn Ile Tyr Gly Gln Ala Val Glu Tyr Ser Gly Leu Val Arg
805 810 815
Val Gly Glu Asp Val Tyr Tyr Phe Gly Glu Thr Tyr Thr Ile Glu Thr
820 825 830
Gly Trp Ile Tyr Asp Met Glu Asn Glu Ser Asp Lys Tyr Tyr Phe Asn
835 840 845
Pro Glu Thr Lys Lys Ala Cys Lys Gly Ile Asn Leu Ile Asp Asp Ile
850 855 860
Lys Tyr Tyr Phe Asp Glu Lys Gly Ile Met Arg Thr Gly Leu Ile Ser
865 870 875 880
Phe Glu Asn Asn Asn Tyr Tyr Phe Asn Glu Asn Gly Glu Met Gln Phe
885 890 895
Gly Tyr Ile Asn Ile Glu Asp Lys Met Phe Tyr Phe Gly Glu Asp Gly
900 905 910
Val Met Gln Ile Gly Val Phe Asn Thr Pro Asp Gly Phe Lys Tyr Phe
915 920 925
Ala His Gln Asn Thr Leu Asp Glu Asn Phe Glu Gly Glu Ser Ile Asn
930 935 940
Tyr Thr Gly Trp Leu Asp Leu Asp Glu Lys Arg Tyr Tyr Phe Thr Asp
945 950 955 960
Glu Tyr Ile Ala Ala Thr Gly Ser Val Ile Ile Asp Gly Glu Glu Tyr
965 970 975
Tyr Phe Asp Pro Asp Thr Ala Gln Leu Val Ile Ser Glu His His His
980 985 990
His His His
995
<210> 28
<211> 593
<212> PRT
<213> 艰难梭菌
<400> 28
Met Ala Thr Gly Trp Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe Asn
1 5 10 15
Thr Asn Thr Ala Ile Ala Ser Thr Gly Tyr Thr Ile Ile Asn Gly Lys
20 25 30
His Phe Tyr Phe Asn Thr Asp Gly Ile Met Gln Ile Gly Val Phe Lys
35 40 45
Gly Pro Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr Asp Ala Asn
50 55 60
Asn Ile Glu Gly Gln Ala Ile Leu Tyr Gln Asn Glu Phe Leu Thr Leu
65 70 75 80
Asn Gly Lys Lys Tyr Tyr Phe Gly Ser Asp Ser Lys Ala Val Thr Gly
85 90 95
Trp Arg Ile Ile Asn Asn Lys Lys Tyr Tyr Phe Asn Pro Asn Asn Ala
100 105 110
Ile Ala Ala Ile His Leu Cys Thr Ile Asn Asn Asp Lys Tyr Tyr Phe
115 120 125
Ser Tyr Asp Gly Ile Leu Gln Asn Gly Tyr Ile Thr Ile Glu Arg Asn
130 135 140
Asn Phe Tyr Phe Asp Ala Asn Asn Glu Ser Lys Met Val Thr Gly Val
145 150 155 160
Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr His
165 170 175
Asn Asn Asn Ile Glu Gly Gln Ala Ile Val Tyr Gln Asn Lys Phe Leu
180 185 190
Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Asp Asn Asp Ser Lys Ala Val
195 200 205
Thr Gly Trp Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe Asn Leu Asn
210 215 220
Thr Ala Glu Ala Ala Thr Gly Trp Gln Thr Ile Asp Gly Lys Lys Tyr
225 230 235 240
Tyr Phe Asn Leu Asn Thr Ala Glu Ala Ala Thr Gly Trp Gln Thr Ile
245 250 255
Asp Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Phe Ile Ala Ser Thr
260 265 270
Gly Tyr Thr Ser Ile Asn Gly Lys His Phe Tyr Phe Asn Thr Asp Gly
275 280 285
Ile Met Gln Ile Gly Val Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe
290 295 300
Ala Pro Ala Asn Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile Leu
305 310 315 320
Tyr Gln Asn Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Gly
325 330 335
Ser Asp Ser Lys Ala Val Thr Gly Leu Arg Thr Ile Asp Gly Lys Lys
340 345 350
Tyr Tyr Phe Asn Thr Asn Thr Ala Val Ala Val Thr Gly Trp Gln Thr
355 360 365
Ile Asn Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Ser Ile Ala Ser
370 375 380
Thr Gly Tyr Thr Ile Ile Ser Gly Lys His Phe Tyr Phe Asn Thr Asp
385 390 395 400
Gly Ile Met Gln Ile Gly Val Phe Lys Gly Pro Asp Gly Phe Glu Tyr
405 410 415
Phe Ala Pro Ala Asn Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile
420 425 430
Arg Tyr Gln Asn Arg Phe Leu Tyr Leu His Asp Asn Ile Tyr Tyr Phe
435 440 445
Gly Asn Asn Ser Lys Ala Ala Thr Gly Trp Val Thr Ile Asp Gly Asn
450 455 460
Arg Tyr Tyr Phe Glu Pro Asn Thr Ala Met Gly Ala Asn Gly Tyr Lys
465 470 475 480
Thr Ile Asp Asn Lys Asn Phe Tyr Phe Arg Asn Gly Leu Pro Gln Ile
485 490 495
Gly Val Phe Lys Gly Ser Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn
500 505 510
Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile Arg Tyr Gln Asn Arg
515 520 525
Phe Leu His Leu Leu Gly Lys Ile Tyr Tyr Phe Gly Asn Asn Ser Lys
530 535 540
Ala Val Thr Gly Trp Gln Thr Ile Asn Gly Lys Val Tyr Tyr Phe Met
545 550 555 560
Pro Asp Thr Ala Met Ala Ala Ala Gly Gly Leu Phe Glu Ile Asp Gly
565 570 575
Val Ile Tyr Phe Phe Gly Val Asp Gly Val Lys Ala Pro Gly Ile Tyr
580 585 590
Gly
<210> 29
<211> 589
<212> PRT
<213> 艰难梭菌
<400> 29
Met Ala Thr Gly Trp Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe Asn
1 5 10 15
Thr Asn Thr Ala Ile Ala Ser Thr Gly Tyr Thr Ile Ile Asn Gly Lys
20 25 30
His Phe Tyr Phe Asn Thr Asp Gly Ile Met Gln Ile Gly Val Phe Lys
35 40 45
Gly Pro Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr Asp Ala Asn
50 55 60
Asn Ile Glu Gly Gln Ala Ile Leu Tyr Gln Asn Glu Phe Leu Thr Leu
65 70 75 80
Asn Gly Lys Lys Tyr Tyr Phe Gly Ser Asp Ser Lys Ala Val Thr Gly
85 90 95
Trp Arg Ile Ile Asn Asn Lys Lys Tyr Tyr Phe Asn Pro Asn Asn Ala
100 105 110
Ile Ala Ala Ile His Leu Cys Thr Ile Asn Asn Asp Lys Tyr Tyr Phe
115 120 125
Ser Tyr Asp Gly Ile Leu Gln Asn Gly Tyr Ile Thr Ile Glu Arg Asn
130 135 140
Asn Phe Tyr Phe Asp Ala Asn Asn Glu Ser Lys Met Val Thr Gly Val
145 150 155 160
Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr His
165 170 175
Asn Asn Asn Ile Glu Gly Gln Ala Ile Val Tyr Gln Asn Lys Phe Leu
180 185 190
Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Asp Asn Asp Ser Lys Ala Val
195 200 205
Thr Gly Trp Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe Asn Leu Asn
210 215 220
Thr Ala Glu Ala Ala Thr Gly Trp Gln Thr Ile Asp Gly Lys Lys Tyr
225 230 235 240
Tyr Phe Asn Leu Asn Thr Ala Glu Ala Ala Thr Gly Trp Gln Thr Ile
245 250 255
Asp Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Phe Ile Ala Ser Thr
260 265 270
Gly Tyr Thr Ser Ile Asn Gly Lys His Phe Tyr Phe Asn Thr Asp Gly
275 280 285
Ile Met Gln Ile Gly Val Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe
290 295 300
Ala Pro Ala Asn Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile Leu
305 310 315 320
Tyr Gln Asn Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Gly
325 330 335
Ser Asp Ser Lys Ala Val Thr Gly Leu Arg Thr Ile Asp Gly Lys Lys
340 345 350
Tyr Tyr Phe Asn Thr Asn Thr Ala Val Ala Val Thr Gly Trp Gln Thr
355 360 365
Ile Asn Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Ser Ile Ala Ser
370 375 380
Thr Gly Tyr Thr Ile Ile Ser Gly Lys His Phe Tyr Phe Asn Thr Asp
385 390 395 400
Gly Ile Met Gln Ile Gly Val Phe Lys Gly Pro Asp Gly Phe Glu Tyr
405 410 415
Phe Ala Pro Ala Asn Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile
420 425 430
Arg Tyr Gln Asn Arg Phe Leu Tyr Leu His Asp Asn Ile Tyr Tyr Phe
435 440 445
Gly Asn Asn Ser Lys Ala Ala Thr Gly Trp Val Thr Ile Asp Gly Asn
450 455 460
Arg Tyr Tyr Phe Glu Pro Asn Thr Ala Met Gly Ala Asn Gly Tyr Lys
465 470 475 480
Thr Ile Asp Asn Lys Asn Phe Tyr Phe Arg Asn Gly Leu Pro Gln Ile
485 490 495
Gly Val Phe Lys Gly Ser Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn
500 505 510
Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile Arg Tyr Gln Asn Arg
515 520 525
Phe Leu His Leu Leu Gly Lys Ile Tyr Tyr Phe Gly Asn Asn Ser Lys
530 535 540
Ala Val Thr Gly Trp Gln Thr Ile Asn Gly Lys Val Tyr Tyr Phe Met
545 550 555 560
Pro Asp Thr Ala Met Ala Ala Ala Gly Gly Leu Phe Glu Ile Asp Gly
565 570 575
Val Ile Tyr Phe Phe Gly Val Asp Gly Val Lys Ala Val
580 585
<210> 30
<211> 589
<212> PRT
<213> 艰难梭菌
<400> 30
Met Ala Thr Gly Trp Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe Asn
1 5 10 15
Thr Asn Thr Ala Ile Ala Ser Thr Gly Tyr Thr Ile Ile Asn Gly Lys
20 25 30
His Phe Tyr Phe Asn Thr Asp Gly Ile Met Gln Ile Gly Val Phe Lys
35 40 45
Gly Pro Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr Asp Ala Asn
50 55 60
Asn Ile Glu Gly Gln Ala Ile Leu Tyr Gln Asn Glu Phe Leu Thr Leu
65 70 75 80
Asn Gly Lys Lys Tyr Tyr Phe Gly Ser Asp Ser Lys Ala Val Thr Gly
85 90 95
Trp Arg Ile Ile Asn Asn Lys Lys Tyr Tyr Phe Asn Pro Asn Asn Ala
100 105 110
Ile Ala Ala Ile His Leu Cys Thr Ile Asn Asn Asp Lys Tyr Tyr Phe
115 120 125
Ser Tyr Asp Gly Ile Leu Gln Asn Gly Tyr Ile Thr Ile Glu Arg Asn
130 135 140
Asn Phe Tyr Phe Asp Ala Asn Asn Glu Ser Lys Met Val Thr Gly Val
145 150 155 160
Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr His
165 170 175
Asn Asn Asn Ile Glu Gly Gln Ala Ile Val Tyr Gln Asn Lys Phe Leu
180 185 190
Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Asp Asn Asp Ser Lys Ala Val
195 200 205
Thr Gly Trp Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe Asn Leu Asn
210 215 220
Thr Ala Glu Ala Ala Thr Gly Trp Gln Thr Ile Asp Gly Lys Lys Tyr
225 230 235 240
Tyr Phe Asn Leu Asn Thr Ala Glu Ala Ala Thr Gly Trp Gln Thr Ile
245 250 255
Asp Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Phe Ile Ala Ser Thr
260 265 270
Gly Tyr Thr Ser Ile Asn Gly Lys His Phe Tyr Phe Asn Thr Asp Gly
275 280 285
Ile Met Gln Ile Gly Val Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe
290 295 300
Ala Pro Ala Asn Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile Leu
305 310 315 320
Tyr Gln Asn Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Gly
325 330 335
Ser Asp Ser Lys Ala Val Thr Gly Leu Arg Thr Ile Asp Gly Lys Lys
340 345 350
Tyr Tyr Phe Asn Thr Asn Thr Ala Val Ala Val Thr Gly Trp Gln Thr
355 360 365
Ile Asn Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Ser Ile Ala Ser
370 375 380
Thr Gly Tyr Thr Ile Ile Ser Gly Lys His Phe Tyr Phe Asn Thr Asp
385 390 395 400
Gly Ile Met Gln Ile Gly Val Phe Lys Gly Pro Asp Gly Phe Glu Tyr
405 410 415
Phe Ala Pro Ala Asn Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile
420 425 430
Arg Tyr Gln Asn Arg Phe Leu Tyr Leu His Asp Asn Ile Tyr Tyr Phe
435 440 445
Gly Asn Asn Ser Lys Ala Ala Thr Gly Trp Val Thr Ile Asp Gly Asn
450 455 460
Arg Tyr Tyr Phe Glu Pro Asn Thr Ala Met Gly Ala Asn Gly Tyr Lys
465 470 475 480
Thr Ile Asp Asn Lys Asn Phe Tyr Phe Arg Asn Gly Leu Pro Gln Ile
485 490 495
Gly Val Phe Lys Gly Ser Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn
500 505 510
Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile Arg Tyr Gln Asn Arg
515 520 525
Phe Leu His Leu Leu Gly Lys Ile Tyr Tyr Phe Gly Asn Asn Ser Lys
530 535 540
Ala Val Thr Gly Trp Gln Thr Ile Asn Gly Lys Val Tyr Tyr Phe Met
545 550 555 560
Pro Asp Thr Ala Met Ala Ala Ala Gly Gly Leu Phe Glu Ile Asp Gly
565 570 575
Val Ile Tyr Phe Phe Gly Val Asp Gly Val Lys Ala Val
580 585
<210> 31
<211> 589
<212> PRT
<213> 艰难梭菌
<400> 31
Met Ala Thr Gly Trp Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe Asn
1 5 10 15
Thr Asn Thr Ala Ile Ala Ser Thr Gly Tyr Thr Ile Ile Asn Gly Lys
20 25 30
His Phe Tyr Phe Asn Thr Asp Gly Ile Met Gln Ile Gly Val Phe Lys
35 40 45
Gly Pro Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr Asp Ala Asn
50 55 60
Asn Ile Glu Gly Gln Ala Ile Leu Tyr Gln Asn Glu Phe Leu Thr Leu
65 70 75 80
Asn Gly Lys Lys Tyr Tyr Phe Gly Ser Asp Ser Lys Ala Val Thr Gly
85 90 95
Trp Arg Ile Ile Asn Asn Lys Lys Tyr Tyr Phe Asn Pro Asn Asn Ala
100 105 110
Ile Ala Ala Ile His Leu Cys Thr Ile Asn Asn Asp Lys Tyr Tyr Phe
115 120 125
Ser Tyr Asp Gly Ile Leu Gln Asn Gly Tyr Ile Thr Ile Glu Arg Asn
130 135 140
Asn Phe Tyr Phe Asp Ala Asn Asn Glu Ser Lys Met Val Thr Gly Val
145 150 155 160
Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn Thr His
165 170 175
Asn Asn Asn Ile Glu Gly Gln Ala Ile Val Tyr Gln Asn Lys Phe Leu
180 185 190
Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Asp Asn Asp Ser Lys Ala Val
195 200 205
Thr Gly Trp Gln Thr Ile Asp Gly Lys Lys Tyr Tyr Phe Asn Leu Asn
210 215 220
Thr Ala Glu Ala Ala Thr Gly Trp Gln Thr Ile Asp Gly Lys Lys Tyr
225 230 235 240
Tyr Phe Asn Leu Asn Thr Ala Glu Ala Ala Thr Gly Trp Gln Thr Ile
245 250 255
Asp Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Phe Ile Ala Ser Thr
260 265 270
Gly Tyr Thr Ser Ile Asn Gly Lys His Phe Tyr Phe Asn Thr Asp Gly
275 280 285
Ile Met Gln Ile Gly Val Phe Lys Gly Pro Asn Gly Phe Glu Tyr Phe
290 295 300
Ala Pro Ala Asn Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile Leu
305 310 315 320
Tyr Gln Asn Lys Phe Leu Thr Leu Asn Gly Lys Lys Tyr Tyr Phe Gly
325 330 335
Ser Asp Ser Lys Ala Val Thr Gly Leu Arg Thr Ile Asp Gly Lys Lys
340 345 350
Tyr Tyr Phe Asn Thr Asn Thr Ala Val Ala Val Thr Gly Trp Gln Thr
355 360 365
Ile Asn Gly Lys Lys Tyr Tyr Phe Asn Thr Asn Thr Ser Ile Ala Ser
370 375 380
Thr Gly Tyr Thr Ile Ile Ser Gly Lys His Phe Tyr Phe Asn Thr Asp
385 390 395 400
Gly Ile Met Gln Ile Gly Val Phe Lys Gly Pro Asp Gly Phe Glu Tyr
405 410 415
Phe Ala Pro Ala Asn Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile
420 425 430
Arg Tyr Gln Asn Arg Phe Leu Tyr Leu His Asp Asn Ile Tyr Tyr Phe
435 440 445
Gly Asn Asn Ser Lys Ala Ala Thr Gly Trp Val Thr Ile Asp Gly Asn
450 455 460
Arg Tyr Tyr Phe Glu Pro Asn Thr Ala Met Gly Ala Asn Gly Tyr Lys
465 470 475 480
Thr Ile Asp Asn Lys Asn Phe Tyr Phe Arg Asn Gly Leu Pro Gln Ile
485 490 495
Gly Val Phe Lys Gly Ser Asn Gly Phe Glu Tyr Phe Ala Pro Ala Asn
500 505 510
Thr Asp Ala Asn Asn Ile Glu Gly Gln Ala Ile Arg Tyr Gln Asn Arg
515 520 525
Phe Leu His Leu Leu Gly Lys Ile Tyr Tyr Phe Gly Asn Asn Ser Lys
530 535 540
Ala Val Thr Gly Trp Gln Thr Ile Asn Gly Lys Val Tyr Tyr Phe Met
545 550 555 560
Pro Asp Thr Ala Met Ala Ala Ala Gly Gly Leu Phe Glu Ile Asp Gly
565 570 575
Val Ile Tyr Phe Phe Gly Val Asp Gly Val Lys Ala Val
580 585
<210> 32
<211> 513
<212> PRT
<213> 艰难梭菌
<400> 32
Thr Gly Phe Val Thr Val Gly Asp Asp Lys Tyr Tyr Phe Asn Pro Ile
1 5 10 15
Asn Gly Gly Ala Ala Ser Ile Gly Glu Thr Ile Ile Asp Asp Lys Asn
20 25 30
Tyr Tyr Phe Asn Gln Ser Gly Val Leu Gln Thr Gly Val Phe Ser Thr
35 40 45
Glu Asp Gly Phe Lys Tyr Phe Ala Pro Ala Asn Thr Leu Asp Glu Asn
50 55 60
Leu Glu Gly Glu Ala Ile Asp Phe Thr Gly Lys Leu Ile Ile Asp Glu
65 70 75 80
Asn Ile Tyr Tyr Phe Asp Asp Asn Tyr Arg Gly Ala Val Glu Trp Lys
85 90 95
Glu Leu Asp Gly Glu Met His Tyr Phe Ser Pro Glu Thr Gly Lys Ala
100 105 110
Phe Lys Gly Leu Asn Gln Ile Gly Asp Tyr Lys Tyr Tyr Phe Asn Ser
115 120 125
Asp Gly Val Met Gln Lys Gly Phe Val Ser Ile Asn Asp Asn Lys His
130 135 140
Tyr Phe Asp Asp Ser Gly Val Met Lys Val Gly Tyr Thr Glu Ile Asp
145 150 155 160
Gly Lys His Phe Tyr Phe Ala Glu Asn Gly Glu Met Gln Ile Gly Val
165 170 175
Phe Asn Thr Glu Asp Gly Phe Lys Tyr Phe Ala His His Asn Glu Asp
180 185 190
Leu Gly Asn Glu Glu Gly Glu Glu Ile Ser Tyr Ser Gly Ile Leu Asn
195 200 205
Phe Asn Asn Lys Ile Tyr Tyr Phe Asp Asp Ser Phe Thr Ala Val Val
210 215 220
Gly Trp Lys Asp Leu Glu Asp Gly Ser Lys Tyr Tyr Phe Asp Glu Asp
225 230 235 240
Thr Ala Glu Ala Tyr Ile Gly Leu Ser Leu Ile Asn Asp Gly Gln Tyr
245 250 255
Tyr Phe Asn Asp Asp Gly Ile Met Gln Val Gly Phe Val Thr Ile Asn
260 265 270
Asp Lys Val Phe Tyr Phe Ser Asp Ser Gly Ile Ile Glu Ser Gly Val
275 280 285
Gln Asn Ile Asp Asp Asn Tyr Phe Tyr Ile Asp Asp Asn Gly Ile Val
290 295 300
Gln Ile Gly Val Phe Asp Thr Ser Asp Gly Tyr Lys Tyr Phe Ala Pro
305 310 315 320
Ala Asn Thr Val Asn Asp Asn Ile Tyr Gly Gln Ala Val Glu Tyr Ser
325 330 335
Gly Leu Val Arg Val Gly Glu Asp Val Tyr Tyr Phe Gly Glu Thr Tyr
340 345 350
Thr Ile Glu Thr Gly Trp Ile Tyr Asp Met Glu Asn Glu Ser Asp Lys
355 360 365
Tyr Tyr Phe Asn Pro Glu Thr Lys Lys Ala Cys Lys Gly Ile Asn Leu
370 375 380
Ile Asp Asp Ile Lys Tyr Tyr Phe Asp Glu Lys Gly Ile Met Arg Thr
385 390 395 400
Gly Leu Ile Ser Phe Glu Asn Asn Asn Tyr Tyr Phe Asn Glu Asn Gly
405 410 415
Glu Met Gln Phe Gly Tyr Ile Asn Ile Glu Asp Lys Met Phe Tyr Phe
420 425 430
Gly Glu Asp Gly Val Met Gln Ile Gly Val Phe Asn Thr Pro Asp Gly
435 440 445
Phe Lys Tyr Phe Ala His Gln Asn Thr Leu Asp Glu Asn Phe Glu Gly
450 455 460
Glu Ser Ile Asn Tyr Thr Gly Trp Leu Asp Leu Asp Glu Lys Arg Tyr
465 470 475 480
Tyr Phe Thr Asp Glu Tyr Ile Ala Ala Thr Gly Ser Val Ile Ile Asp
485 490 495
Gly Glu Glu Tyr Tyr Phe Asp Pro Asp Thr Ala Gln Leu Val Ile Ser
500 505 510
Glu
<210> 33
<211> 513
<212> PRT
<213> 艰难梭菌
<400> 33
Thr Gly Phe Val Thr Val Gly Asp Asp Lys Tyr Tyr Phe Asn Pro Ile
1 5 10 15
Asn Gly Gly Ala Ala Ser Ile Gly Glu Thr Ile Ile Asp Asp Lys Asn
20 25 30
Tyr Tyr Phe Asn Gln Ser Gly Val Leu Gln Thr Gly Val Phe Ser Thr
35 40 45
Glu Asp Gly Phe Lys Tyr Phe Ala Pro Ala Asn Thr Leu Asp Glu Asn
50 55 60
Leu Glu Gly Glu Ala Ile Asp Phe Thr Gly Lys Leu Ile Ile Asp Glu
65 70 75 80
Asn Ile Tyr Tyr Phe Asp Asp Asn Tyr Arg Gly Ala Val Glu Trp Lys
85 90 95
Glu Leu Asp Gly Glu Met His Tyr Phe Ser Pro Glu Thr Gly Lys Ala
100 105 110
Phe Lys Gly Leu Asn Gln Ile Gly Asp Tyr Lys Tyr Tyr Phe Asn Ser
115 120 125
Asp Gly Val Met Gln Lys Gly Phe Val Ser Ile Asn Asp Asn Lys His
130 135 140
Tyr Phe Asp Asp Ser Gly Val Met Lys Val Gly Tyr Thr Glu Ile Asp
145 150 155 160
Gly Lys His Phe Tyr Phe Ala Glu Asn Gly Glu Met Gln Ile Gly Val
165 170 175
Phe Asn Thr Glu Asp Gly Phe Lys Tyr Phe Ala His His Asn Glu Asp
180 185 190
Leu Gly Asn Glu Glu Gly Glu Glu Ile Ser Tyr Ser Gly Ile Leu Asn
195 200 205
Phe Asn Asn Lys Ile Tyr Tyr Phe Asp Asp Ser Phe Thr Ala Val Val
210 215 220
Gly Trp Lys Asp Leu Glu Asp Gly Ser Lys Tyr Tyr Phe Asp Glu Asp
225 230 235 240
Thr Ala Glu Ala Tyr Ile Gly Leu Ser Leu Ile Asn Asp Gly Gln Tyr
245 250 255
Tyr Phe Asn Asp Asp Gly Ile Met Gln Val Gly Phe Val Thr Ile Asn
260 265 270
Asp Lys Val Phe Tyr Phe Ser Asp Ser Gly Ile Ile Glu Ser Gly Val
275 280 285
Gln Asn Ile Asp Asp Asn Tyr Phe Tyr Ile Asp Asp Asn Gly Ile Val
290 295 300
Gln Ile Gly Val Phe Asp Thr Ser Asp Gly Tyr Lys Tyr Phe Ala Pro
305 310 315 320
Ala Asn Thr Val Asn Asp Asn Ile Tyr Gly Gln Ala Val Glu Tyr Ser
325 330 335
Gly Leu Val Arg Val Gly Glu Asp Val Tyr Tyr Phe Gly Glu Thr Tyr
340 345 350
Thr Ile Glu Thr Gly Trp Ile Tyr Asp Met Glu Asn Glu Ser Asp Lys
355 360 365
Tyr Tyr Phe Asn Pro Glu Thr Lys Lys Ala Cys Lys Gly Ile Asn Leu
370 375 380
Ile Asp Asp Ile Lys Tyr Tyr Phe Asp Glu Lys Gly Ile Met Arg Thr
385 390 395 400
Gly Leu Ile Ser Phe Glu Asn Asn Asn Tyr Tyr Phe Asn Glu Asn Gly
405 410 415
Glu Met Gln Phe Gly Tyr Ile Asn Ile Glu Asp Lys Met Phe Tyr Phe
420 425 430
Gly Glu Asp Gly Val Met Gln Ile Gly Val Phe Asn Thr Pro Asp Gly
435 440 445
Phe Lys Tyr Phe Ala His Gln Asn Thr Leu Asp Glu Asn Phe Glu Gly
450 455 460
Glu Ser Ile Asn Tyr Thr Gly Trp Leu Asp Leu Asp Glu Lys Arg Tyr
465 470 475 480
Tyr Phe Thr Asp Glu Tyr Ile Ala Ala Thr Gly Ser Val Ile Ile Asp
485 490 495
Gly Glu Glu Tyr Tyr Phe Asp Pro Asp Thr Ala Gln Leu Val Ile Ser
500 505 510
Glu
<210> 34
<211> 534
<212> PRT
<213> 艰难梭菌
<400> 34
Ser Gly Leu Ile Tyr Ile Asn Asp Ser Leu Tyr Tyr Phe Lys Pro Pro
1 5 10 15
Val Asn Asn Leu Ile Thr Gly Phe Val Thr Val Gly Asp Asp Lys Tyr
20 25 30
Tyr Phe Asn Pro Ile Asn Gly Gly Ala Ala Ser Ile Gly Glu Thr Ile
35 40 45
Ile Asp Asp Lys Asn Tyr Tyr Phe Asn Gln Ser Gly Val Leu Gln Thr
50 55 60
Gly Val Phe Ser Thr Glu Asp Gly Phe Lys Tyr Phe Ala Pro Ala Asn
65 70 75 80
Thr Leu Asp Glu Asn Leu Glu Gly Glu Ala Ile Asp Phe Thr Gly Lys
85 90 95
Leu Ile Ile Asp Glu Asn Ile Tyr Tyr Phe Asp Asp Asn Tyr Arg Gly
100 105 110
Ala Val Glu Trp Lys Glu Leu Asp Gly Glu Met His Tyr Phe Ser Pro
115 120 125
Glu Thr Gly Lys Ala Phe Lys Gly Leu Asn Gln Ile Gly Asp Tyr Lys
130 135 140
Tyr Tyr Phe Asn Ser Asp Gly Val Met Gln Lys Gly Phe Val Ser Ile
145 150 155 160
Asn Asp Asn Lys His Tyr Phe Asp Asp Ser Gly Val Met Lys Val Gly
165 170 175
Tyr Thr Glu Ile Asp Gly Lys His Phe Tyr Phe Ala Glu Asn Gly Glu
180 185 190
Met Gln Ile Gly Val Phe Asn Thr Glu Asp Gly Phe Lys Tyr Phe Ala
195 200 205
His His Asn Glu Asp Leu Gly Asn Glu Glu Gly Glu Glu Ile Ser Tyr
210 215 220
Ser Gly Ile Leu Asn Phe Asn Asn Lys Ile Tyr Tyr Phe Asp Asp Ser
225 230 235 240
Phe Thr Ala Val Val Gly Trp Lys Asp Leu Glu Asp Gly Ser Lys Tyr
245 250 255
Tyr Phe Asp Glu Asp Thr Ala Glu Ala Tyr Ile Gly Leu Ser Leu Ile
260 265 270
Asn Asp Gly Gln Tyr Tyr Phe Asn Asp Asp Gly Ile Met Gln Val Gly
275 280 285
Phe Val Thr Ile Asn Asp Lys Val Phe Tyr Phe Ser Asp Ser Gly Ile
290 295 300
Ile Glu Ser Gly Val Gln Asn Ile Asp Asp Asn Tyr Phe Tyr Ile Asp
305 310 315 320
Asp Asn Gly Ile Val Gln Ile Gly Val Phe Asp Thr Ser Asp Gly Tyr
325 330 335
Lys Tyr Phe Ala Pro Ala Asn Thr Val Asn Asp Asn Ile Tyr Gly Gln
340 345 350
Ala Val Glu Tyr Ser Gly Leu Val Arg Val Gly Glu Asp Val Tyr Tyr
355 360 365
Phe Gly Glu Thr Tyr Thr Ile Glu Thr Gly Trp Ile Tyr Asp Met Glu
370 375 380
Asn Glu Ser Asp Lys Tyr Tyr Phe Asn Pro Glu Thr Lys Lys Ala Cys
385 390 395 400
Lys Gly Ile Asn Leu Ile Asp Asp Ile Lys Tyr Tyr Phe Asp Glu Lys
405 410 415
Gly Ile Met Arg Thr Gly Leu Ile Ser Phe Glu Asn Asn Asn Tyr Tyr
420 425 430
Phe Asn Glu Asn Gly Glu Met Gln Phe Gly Tyr Ile Asn Ile Glu Asp
435 440 445
Lys Met Phe Tyr Phe Gly Glu Asp Gly Val Met Gln Ile Gly Val Phe
450 455 460
Asn Thr Pro Asp Gly Phe Lys Tyr Phe Ala His Gln Asn Thr Leu Asp
465 470 475 480
Glu Asn Phe Glu Gly Glu Ser Ile Asn Tyr Thr Gly Trp Leu Asp Leu
485 490 495
Asp Glu Lys Arg Tyr Tyr Phe Thr Asp Glu Tyr Ile Ala Ala Thr Gly
500 505 510
Ser Val Ile Ile Asp Gly Glu Glu Tyr Tyr Phe Asp Pro Asp Thr Ala
515 520 525
Gln Leu Val Ile Ser Glu
530
<210> 35
<211> 400
<212> PRT
<213> 艰难梭菌
<400> 35
Lys Gly Leu Asn Gln Ile Gly Asp Tyr Lys Tyr Tyr Phe Asn Ser Asp
1 5 10 15
Gly Val Met Gln Lys Gly Phe Val Ser Ile Asn Asp Asn Lys His Tyr
20 25 30
Phe Asp Asp Ser Gly Val Met Lys Val Gly Tyr Thr Glu Ile Asp Gly
35 40 45
Lys His Phe Tyr Phe Ala Glu Asn Gly Glu Met Gln Ile Gly Val Phe
50 55 60
Asn Thr Glu Asp Gly Phe Lys Tyr Phe Ala His His Asn Glu Asp Leu
65 70 75 80
Gly Asn Glu Glu Gly Glu Glu Ile Ser Tyr Ser Gly Ile Leu Asn Phe
85 90 95
Asn Asn Lys Ile Tyr Tyr Phe Asp Asp Ser Phe Thr Ala Val Val Gly
100 105 110
Trp Lys Asp Leu Glu Asp Gly Ser Lys Tyr Tyr Phe Asp Glu Asp Thr
115 120 125
Ala Glu Ala Tyr Ile Gly Leu Ser Leu Ile Asn Asp Gly Gln Tyr Tyr
130 135 140
Phe Asn Asp Asp Gly Ile Met Gln Val Gly Phe Val Thr Ile Asn Asp
145 150 155 160
Lys Val Phe Tyr Phe Ser Asp Ser Gly Ile Ile Glu Ser Gly Val Gln
165 170 175
Asn Ile Asp Asp Asn Tyr Phe Tyr Ile Asp Asp Asn Gly Ile Val Gln
180 185 190
Ile Gly Val Phe Asp Thr Ser Asp Gly Tyr Lys Tyr Phe Ala Pro Ala
195 200 205
Asn Thr Val Asn Asp Asn Ile Tyr Gly Gln Ala Val Glu Tyr Ser Gly
210 215 220
Leu Val Arg Val Gly Glu Asp Val Tyr Tyr Phe Gly Glu Thr Tyr Thr
225 230 235 240
Ile Glu Thr Gly Trp Ile Tyr Asp Met Glu Asn Glu Ser Asp Lys Tyr
245 250 255
Tyr Phe Asn Pro Glu Thr Lys Lys Ala Cys Lys Gly Ile Asn Leu Ile
260 265 270
Asp Asp Ile Lys Tyr Tyr Phe Asp Glu Lys Gly Ile Met Arg Thr Gly
275 280 285
Leu Ile Ser Phe Glu Asn Asn Asn Tyr Tyr Phe Asn Glu Asn Gly Glu
290 295 300
Met Gln Phe Gly Tyr Ile Asn Ile Glu Asp Lys Met Phe Tyr Phe Gly
305 310 315 320
Glu Asp Gly Val Met Gln Ile Gly Val Phe Asn Thr Pro Asp Gly Phe
325 330 335
Lys Tyr Phe Ala His Gln Asn Thr Leu Asp Glu Asn Phe Glu Gly Glu
340 345 350
Ser Ile Asn Tyr Thr Gly Trp Leu Asp Leu Asp Glu Lys Arg Tyr Tyr
355 360 365
Phe Thr Asp Glu Tyr Ile Ala Ala Thr Gly Ser Val Ile Ile Asp Gly
370 375 380
Glu Glu Tyr Tyr Phe Asp Pro Asp Thr Ala Gln Leu Val Ile Ser Glu
385 390 395 400

Claims (10)

1.一种多肽，其包含第一片段和第二片段，其中
(i) 所述第一片段是毒素A重复结构域片段；
(ii) 所述第二片段是毒素B重复结构域片段；
(iii) 所述第一片段具有第一近端；
(iv) 所述第二片段具有第二近端；并且
其中所述第一片段和所述第二片段彼此邻近，并且其中所述多肽诱导中和毒素A或毒素B或两者的抗体。
2.权利要求1的多肽，其中所述多肽在哺乳动物宿主中诱导针对艰难梭菌菌株的保护性免疫应答。
3.权利要求1-2中任一项的多肽，其中所述第一片段和/或所述第二片段包含少于25%、20%、18%或15%的α螺旋结构。
4.前述权利要求中任一项的多肽，其中所述第一片段和/或所述第二片段包含多于25%、30%、35%、38%或40%的β折叠结构。
5.前述权利要求中任一项的多肽，其中所述第一近端在短重复内。
6.前述权利要求中任一项的多肽，其中所述第二近端在短重复内。
7.前述权利要求中任一项的多肽，其中所述第一近端不破坏短重复-长重复-短重复部分。
8.前述权利要求中任一项的多肽，其中所述第二近端不破坏短重复-长重复-短重复部分。
9.前述权利要求中任一项的多肽，其中所述第一近端和所述第二近端不破坏短重复-长重复-短重复部分。
10.前述权利要求中任一项的多肽，其中所述第一近端不在毒素A的氨基酸1878-1940、2012-2074、2146-2208、2258-2322、2394-2456、2507-2569或2598-2660内。
CN201710239709.0A 2011-05-27 2012-05-25 免疫原性组合物 Pending CN107098977A (zh)

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