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。
本发明涉及来自艰难梭菌(clostridiumdifficile)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.11suppl: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,journalofinfection561:1-9(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,expertrev.antiinfect.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。
技术实现要素:
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,currentmicrobiology21:29-32(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(pnas102:18373-18378(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,pnas102:18373-18378(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,currentmicrobiology21:29-32(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
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srviii和toxb的第四个srii之间的连接的图示。
图4-融合体2中使用toxa的第二个srviii和toxb的第三个srii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lrii)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srviii和toxb的第三个sri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(aa2387-2706)、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(aa2387-2706)、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(aa2387-2706)、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(aa2387-2706)、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seqidno: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seqidno: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seqidno: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seqidno: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(toxa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(challengeassay)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(pnas102:18373-18378(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jascoj-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toxa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toxb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(seqidno:1)的氨基酸
为此原因,术语“短重复”可以指毒素a(seqidno:1)的氨基酸
类似地,术语“长重复”可以指毒素a(seqidno:1)的氨基酸1894-1924、2028-2058、2162-2192、2276-2306、2410-2440、2523-2553或2614-2644或毒素b(seqidno: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(seqidno:1)的氨基酸1874-1944、2008-2078、2142-2212、2254-2326、2390-2460、2503-2573或2595-2664,或毒素b(seqidno: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(seqidno:1)的氨基酸
在一个实施方案中,第一近端在毒素a的重复部分v(seqidno:1的氨基酸2307-2440或它们在不同菌株中的等同物)、vi(seqidno:1的氨基酸2441-2553或它们在不同菌株中的等同物)、vii(seqidno:1的氨基酸2554-2644或它们在不同菌株中的等同物)或viii(seqidno: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(seqidno: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(seqidno: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(seqidno:2的氨基酸1834-1926或它们在不同菌株中的等同物)、ii(seqidno:2的氨基酸1927-2057或它们在不同菌株中的等同物)、或iii(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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seqidno: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seqidno: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seqidno: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seqidno: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seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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(seqidno: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seqidno: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seqidno: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seqidno: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seqidno: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seqidno: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seqidno: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seqidno: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seqidno: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seqidno: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seqidno: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seqidno: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seqidno: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toxa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,gene40:39-46(1985)、murphy等人,proc.natl.acad.sci.usa83:8258-8262(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(pseudomonasaeruginosa)。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
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
用于多肽序列比较的参数包括以下内容:
算法:needleman和wunsch,j.molbiol.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(ricep.等人,trendsingenetics2000col.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seqidno: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seqidno:1中的序列全长的序列同一性。为了确定参考序列与seqidno: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seqidno: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
ii)与
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,(pnas102:18373-18378(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jascoj-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.acidsres.symp.ser.第215-223页(1980),horn等人,nucl.acidsres.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,science269:202-204(1995))进行,并且可以实现自动合成,例如使用asi431a肽合成仪(perkinelmer,paloalto,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,basicmethodsinmolecularbiology,(1986)和sambrook等人,molecularcloning:alaboratorymanual,第二版,coldspringharborlaboratorypress,coldspringharbor,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(scrapeloading)、弹道导入(ballisticintroduction)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,molecularcloning,alaboratorymanual(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vaccinedesign("thesubunitandadjuvantapproach"(powellm.f.和newmanm.j.编辑)(1995)plenumpressnewyork)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(consistingof)”、“组成(consistof)”和“组成(consistsof)”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(exempligratia),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(forexample)”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,genesv,由oxforduniversitypress出版,1994(isbn0-19-854287-9)、kendrew等人(编辑),theencyclopediaofmolecularbiology,由blackwellscienceltd.出版,1994(isbn0-632-02182-9)、和roberta.meyers(编辑),molecularbiologyandbiotechnology:acomprehensivedeskreference,由vchpublishers,inc.出版,1995(isbn1-56081-569-8)。
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(molecularweight)或分子量(molecularmass)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)(pnas2005vol102:18373-18378)。
toxa的c末端结构域的部分已知的3d结构(pnas2005greco等人,vol10218373-18378;naturestructural&molecularbiology2006vol13(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-thompsonjd等人,(1994)nucleicacidsres.,22,4673-4680)。更相似的序列为toxa的第三个srviii和toxb的第三个srii和toxb的第三个sriii。为了在toxb的这两个sr之间做出选择,使用部分的toxac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-arnoldk等人,(2006)bioinformatics,22,195-201)。使用toxa的第三个srviii,用toxb的第三个srii获得了最佳局部结构叠加(使用swisspdbviewer进行-guexn等人,(1997),electrophoresis18,2714-2723)。因此,设计了两个连接:第一个在toxa的第三个srviii和toxb的第四个srii之间(f1),并且第二个在toxa的第二个srviii和toxb的第三个srii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lrvii和toxb的lrii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(sri)和toxa的最后一个sr(第三个srviii)获得了最佳结构叠加。因此,在toxa的第二个srviii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srii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(seqidno: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(hanahand.«plasmidtransformationbysimanis.»inglover,d.m.(ed),dnacloning.irlpresslondon.(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λde3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200ml的lbt肉汤培养基±1%(w/v)葡萄糖+卡那霉素(50µg/ml),以获得0.1-0.2之间的o.d.600nm。在37℃,250rpm下过夜孵育培养物。
将该过夜培养物稀释在500ml的含有卡那霉素(50µg/ml)的lbt培养基至1:20,并且在37℃下以250rpm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1mm异丙基-β-d-1-硫代半乳糖苷(iptg;emdchemicalsinc.,目录号:5815)诱导重组蛋白的表达,并在23℃,250rpm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14000rpm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500mmnacl和蛋白酶抑制剂的混合物(完全的,roche)的20mmn-二(羟乙基)甘氨酸缓冲液(ph8.0)中重悬细菌沉淀。使用frenchpress系统20000psi裂解细菌。通过离心例如在4℃下以20000g30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(15ml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500mmnacl和不同浓度的咪唑(5-600mm)的20mmn-二(羟乙基)甘氨酸缓冲液(ph8.0)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(20mmn-二(羟乙基)甘氨酸、150mmnacl,含有或不含有5mmedtaph8.0)透析,使用biorad的dcproteinassay测定蛋白浓度。因此将蛋白混合,在0.22µm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2mmn-二(羟乙基)甘氨酸缓冲液(ph8.0)的deae纯化步骤,并且使用相同缓冲液但加入1mnacl的梯度洗脱。
实施例3:分离的艰难梭菌toxa和toxb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(seqidno:8和seqidno: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(hanahand.«plasmidtransformationbysimanis.»inglover,d.m.(ed),dnacloning.irlpresslondon.(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λde3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200ml的lbt肉汤培养基±1%(w/v)葡萄糖+卡那霉素(50µg/ml),以获得0.1-0.2之间的o.d.600nm。在37℃,250rpm下过夜孵育培养物。
在500ml的含有卡那霉素(50µg/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250rpm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1mm异丙基-β-d-1-硫代半乳糖苷(iptg;emdchemicalsinc.,目录号:5815)诱导重组蛋白的表达,并在23℃,250rpm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14000rpm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500mmnacl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20mmn-二(羟乙基)甘氨酸缓冲液(ph8.0)中重悬细菌沉淀。(roche目录1.01695.0001)。使用frenchpress系统2x20000psi裂解细菌。通过在4℃下以34000g或48000g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0.22µm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15ml)(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500mmnacl和不同浓度的咪唑(5-100mm)的20mmn-二(羟乙基)甘氨酸缓冲液(ph8.0)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500mmnacl和0.5%脱氧胆酸的20mmn-二(羟乙基)甘氨酸缓冲液(ph8.0)或含有150mmnacl的相同缓冲液进行第二次洗涤。使用含有500mmnacl和不同浓度的咪唑(10-500mm)的20mmn-二(羟乙基)甘氨酸缓冲液(ph8.0)进行洗脱。凝胶分析后,选择更纯的级分,添加5mmedta并装载到sec层析(superdextm200)上,用于在含有5mmedta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(20mmn-二(羟乙基)甘氨酸、150mmnacl,ph8.0)透析,使用biorad的rcdcproteinassay测定蛋白浓度。因此,将蛋白混合,在0.22µm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-coulterproteomelabtmxl-1分析超速离心机中以42000rpm旋转。
a.f1融合蛋白,500µg/ml,20mmn-二(羟乙基)甘氨酸,150mmnacl,ph8,0
b.f2融合蛋白,500µg/ml,20mmn-二(羟乙基)甘氨酸,150mmnacl,ph8,0
c.f3融合蛋白,500µg/ml,20mmn-二(羟乙基)甘氨酸,150mmnacl,ph8,0
d.f4融合蛋白,500µg/ml,20mmn-二(羟乙基)甘氨酸,150mmnacl,ph8,0
e.f5融合蛋白,500µg/ml,20mmn-二(羟乙基)甘氨酸,150mmnacl,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jascoj-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jascoj-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,analyticalbiochemistry,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(proteindatabank)。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toxa或toxb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10µg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toxbelisa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toxbelisa应答:方案
将toxa或toxb片段的样品在磷酸缓冲盐溶液(pbs)中以1µg/ml包被在高结合微量滴定板(nuncmaxisorptm)上,在4℃下过夜。在室温振荡下,用pbs-bsa1%将板封闭30分钟。将小鼠抗血清以1/500预稀释在pbs-bsa0.2%-tweentm0.05%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jacksonimmunolaboratoriesinc.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4mgo-苯二胺(opd)+5µlh2o2/10mlph4.50.1m柠檬酸盐缓冲液避光显色15分钟。用50µlhcl终止反应,并在490nm处读取相对于620nm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25µl的天然的毒素a(0.2µg/孔)并将板在室温下孵育30分钟。
孵育后,向每个孔中加入50µl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50µl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toxa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toxa评价。
血细胞凝集(haemagglutination)抑制效价描述于图12中。用抗toxa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toxb抗体,但使用f2、f3和f4融合体免疫后也诱导抗toxb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f54gly(seqidno:21)、f54new(seqidno:23)、f5toxb(seqidno:25)和f52new(seqidno: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:seqidno:21、seqidno:23、seqidno:25和seqidno: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seqidno:21、seqidno:23、seqidno:25和seqidno: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seqidno:21、seqidno:23、seqidno:25和seqidno: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:seqidno:21、seqidno:23、seqidno:25和seqidno: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seqidno:21、seqidno:23、seqidno:25和seqidno: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uvcd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。
seqidno:21、seqidno:23、seqidno:25和seqidno:27中描述的蛋白的近和远uvcd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toxa-toxb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f54gly(seqidno:21)、f54new(seqidno:23)、f5toxb(seqidno:25)和f52new(seqidno:27)免疫balb/c小鼠。
使用实施例6中描述的抗toxa和抗toxbelisa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2µg/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50µl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toxaelisa、抗toxbelisa、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>glaxosmithklinebiologicalss.a
<120>免疫原性组合物
<130>vb64650
<160>35
<170>fastseqforwindowsversion4.0
<210>1
<211>2710
<212>prt
<213>艰难梭菌
<400>1
metserleuileserlysglugluleuilelysleualatyrserile
151015
argproarggluasnglutyrlysthrileleuthrasnleuaspglu
202530
tyrasnlysleuthrthrasnasnasngluasnlystyrleuglnleu
354045
lyslysleuasngluserileaspvalphemetasnlystyrlysthr
505560
serserargasnargalaleuserasnleulyslysaspileleulys
65707580
gluvalileleuilelysasnserasnthrserprovalglulysasn
859095
leuhisphevaltrpileglyglygluvalseraspilealaleuglu
100105110
tyrilelysglntrpalaaspileasnalaglutyrasnilelysleu
115120125
trptyraspserglualapheleuvalasnthrleulyslysalaile
130135140
valgluserserthrthrglualaleuglnleuleugluglugluile
145150155160
glnasnproglnpheaspasnmetlysphetyrlyslysargmetglu
165170175
pheiletyraspargglnlysargpheileasntyrtyrlyssergln
180185190
ileasnlysprothrvalprothrileaspaspileilelysserhis
195200205
leuvalserglutyrasnargaspgluthrvalleuglusertyrarg
210215220
thrasnserleuarglysileasnserasnhisglyileaspilearg
225230235240
alaasnserleuphethrgluglngluleuleuasniletyrsergln
245250255
gluleuleuasnargglyasnleualaalaalaseraspilevalarg
260265270
leuleualaleulysasnpheglyglyvaltyrleuaspvalaspmet
275280285
leuproglyilehisseraspleuphelysthrileserargproser
290295300
serileglyleuaspargtrpglumetilelysleuglualailemet
305310315320
lystyrlyslystyrileasnasntyrthrsergluasnpheasplys
325330335
leuaspglnglnleulysaspasnphelysleuileilegluserlys
340345350
serglulyssergluilepheserlysleugluasnleuasnvalser
355360365
aspleugluilelysilealaphealaleuglyservalileasngln
370375380
alaleuileserlysglnglysertyrleuthrasnleuvalileglu
385390395400
glnvallysasnargtyrglnpheleuasnglnhisleuasnproala
405410415
ilegluseraspasnasnphethraspthrthrlysilephehisasp
420425430
serleupheasnseralathralagluasnsermetpheleuthrlys
435440445
ilealaprotyrleuglnvalglyphemetproglualaargserthr
450455460
ileserleuserglyproglyalatyralaseralatyrtyraspphe
465470475480
ileasnleuglngluasnthrileglulysthrleulysalaserasp
485490495
leuilegluphelyspheprogluasnasnleuserglnleuthrglu
500505510
glngluileasnserleutrpserpheaspglnalaseralalystyr
515520525
glnpheglulystyrvalargasptyrthrglyglyserleuserglu
530535540
aspasnglyvalasppheasnlysasnthralaleuasplysasntyr
545550555560
leuleuasnasnlysileproserasnasnvalgluglualaglyser
565570575
lysasntyrvalhistyrileileglnleuglnglyaspaspileser
580585590
tyrglualathrcysasnleupheserlysasnprolysasnserile
595600605
ileileglnargasnmetasngluseralalyssertyrpheleuser
610615620
aspaspglygluserileleugluleuasnlystyrargileproglu
625630635640
argleulysasnlysglulysvallysvalthrpheileglyhisgly
645650655
lysaspglupheasnthrsergluphealaargleuservalaspser
660665670
leuserasngluileserserpheleuaspthrilelysleuaspile
675680685
serprolysasnvalgluvalasnleuleuglycysasnmetpheser
690695700
tyrasppheasnvalglugluthrtyrproglylysleuleuleuser
705710715720
ilemetasplysilethrserthrleuproaspvalasnlysasnser
725730735
ilethrileglyalaasnglntyrgluvalargileasnserglugly
740745750
arglysgluleuleualahisserglylystrpileasnlysgluglu
755760765
alailemetseraspleuserserlysglutyrilephepheaspser
770775780
ileaspasnlysleulysalalysserlysasnileproglyleuala
785790795800
serilesergluaspilelysthrleuleuleuaspalaservalser
805810815
proaspthrlyspheileleuasnasnleulysleuasnilegluser
820825830
serileglyasptyriletyrtyrglulysleugluprovallysasn
835840845
ileilehisasnserileaspaspleuileaspglupheasnleuleu
850855860
gluasnvalseraspgluleutyrgluleulyslysleuasnasnleu
865870875880
aspglulystyrleuileserphegluaspileserlysasnasnser
885890895
thrtyrservalargpheileasnlysserasnglygluservaltyr
900905910
valgluthrglulysgluilepheserlystyrsergluhisilethr
915920925
lysgluileserthrilelysasnserileilethraspvalasngly
930935940
asnleuleuaspasnileglnleuasphisthrserglnvalasnthr
945950955960
leuasnalaalaphepheileglnserleuileasptyrserserasn
965970975
lysaspvalleuasnaspleuserthrservallysvalglnleutyr
980985990
alaglnleupheserthrglyleuasnthriletyraspserilegln
99510001005
leuvalasnleuileserasnalavalasnaspthrileasnvalleu
101010151020
prothrilethrgluglyileproilevalserthrileleuaspgly
1025103010351040
ileasnleuglyalaalailelysgluleuleuaspgluhisasppro
104510501055
leuleulyslysgluleuglualalysvalglyvalleualaileasn
106010651070
metserleuserilealaalathrvalalaserilevalglyilegly
107510801085
alagluvalthrilepheleuleuproilealaglyileseralagly
109010951100
ileproserleuvalasnasngluleuileleuhisasplysalathr
1105111011151120
servalvalasntyrpheasnhisleusergluserlyslystyrgly
112511301135
proleulysthrgluaspasplysileleuvalproileaspaspleu
114011451150
valilesergluileasppheasnasnasnserilelysleuglythr
115511601165
cysasnileleualametgluglyglyserglyhisthrvalthrgly
117011751180
asnileasphisphepheserserproserileserserhisilepro
1185119011951200
serleuseriletyrseralaileglyilegluthrgluasnleuasp
120512101215
pheserlyslysilemetmetleuproasnalaproserargvalphe
122012251230
trptrpgluthrglyalavalproglyleuargserleugluasnasp
123512401245
glythrargleuleuaspserileargaspleutyrproglylysphe
125012551260
tyrtrpargphetyralaphepheasptyralailethrthrleulys
1265127012751280
provaltyrgluaspthrasnilelysilelysleuasplysaspthr
128512901295
argasnpheilemetprothrilethrthrasngluileargasnlys
130013051310
leusertyrserpheaspglyalaglyglythrtyrserleuleuleu
131513201325
sersertyrproileserthrasnileasnleuserlysaspaspleu
133013351340
trpilepheasnileaspasngluvalarggluileserilegluasn
1345135013551360
glythrilelyslysglylysleuilelysaspvalleuserlysile
136513701375
aspileasnlysasnlysleuileileglyasnglnthrileaspphe
138013851390
serglyaspileaspasnlysaspargtyrilepheleuthrcysglu
139514001405
leuaspasplysileserleuileilegluileasnleuvalalalys
141014151420
sertyrserleuleuleuserglyasplysasntyrleuileserasn
1425143014351440
leuserasnthrileglulysileasnthrleuglyleuaspserlys
144514501455
asnilealatyrasntyrthraspgluserasnasnlystyrphegly
146014651470
alaileserlysthrserglnlysserileilehistyrlyslysasp
147514801485
serlysasnileleugluphetyrasnaspserthrleuglupheasn
149014951500
serlysasppheilealagluaspileasnvalphemetlysaspasp
1505151015151520
ileasnthrilethrglylystyrtyrvalaspasnasnthrasplys
152515301535
serileasppheserileserleuvalserlysasnglnvallysval
154015451550
asnglyleutyrleuasngluservaltyrsersertyrleuaspphe
155515601565
vallysasnseraspglyhishisasnthrserasnphemetasnleu
157015751580
pheleuaspasnileserphetrplysleupheglyphegluasnile
1585159015951600
asnphevalileasplystyrphethrleuvalglylysthrasnleu
160516101615
glytyrvalglupheilecysaspasnasnlysasnileaspiletyr
162016251630
pheglyglutrplysthrserserserlysserthrilephesergly
163516401645
asnglyargasnvalvalvalgluproiletyrasnproaspthrgly
165016551660
gluaspileserthrserleuaspphesertyrgluproleutyrgly
1665167016751680
ileaspargtyrileasnlysvalleuilealaproaspleutyrthr
168516901695
serleuileasnileasnthrasntyrtyrserasnglutyrtyrpro
170017051710
gluileilevalleuasnproasnthrphehislyslysvalasnile
171517201725
asnleuaspserserserpheglutyrlystrpserthrgluglyser
173017351740
asppheileleuvalargtyrleuglugluserasnlyslysileleu
1745175017551760
glnlysileargilelysglyileleuserasnthrglnserpheasn
176517701775
lysmetserileaspphelysaspilelyslysleuserleuglytyr
178017851790
ilemetserasnphelysserpheasnsergluasngluleuasparg
179518001805
asphisleuglyphelysileileaspasnlysthrtyrtyrtyrasp
181018151820
gluaspserlysleuvallysglyleuileasnileasnasnserleu
1825183018351840
phetyrpheaspproileglupheasnleuvalthrglytrpglnthr
184518501855
ileasnglylyslystyrtyrpheaspileasnthrglyalaalaleu
186018651870
thrsertyrlysileileasnglylyshisphetyrpheasnasnasp
187518801885
glyvalmetglnleuglyvalphelysglyproaspglypheglutyr
189018951900
phealaproalaasnthrglnasnasnasnilegluglyglnalaile
1905191019151920
valtyrglnserlyspheleuthrleuasnglylyslystyrtyrphe
192519301935
aspasnasnserlysalavalthrglytrpargileileasnasnglu
194019451950
lystyrtyrpheasnproasnasnalailealaalavalglyleugln
195519601965
valileaspasnasnlystyrtyrpheasnproaspthralaileile
197019751980
serlysglytrpglnthrvalasnglyserargtyrtyrpheaspthr
1985199019952000
aspthralailealapheasnglytyrlysthrileaspglylyshis
200520102015
phetyrpheaspseraspcysvalvallysileglyvalpheserthr
202020252030
serasnglypheglutyrphealaproalaasnthrtyrasnasnasn
203520402045
ilegluglyglnalailevaltyrglnserlyspheleuthrleuasn
205020552060
glylyslystyrtyrpheaspasnasnserlysalavalthrglyleu
2065207020752080
glnthrileaspserlyslystyrtyrpheasnthrasnthralaglu
208520902095
alaalathrglytrpglnthrileaspglylyslystyrtyrpheasn
210021052110
thrasnthralaglualaalathrglytrpglnthrileaspglylys
211521202125
lystyrtyrpheasnthrasnthralailealaserthrglytyrthr
213021352140
ileileasnglylyshisphetyrpheasnthraspglyilemetgln
2145215021552160
ileglyvalphelysglyproasnglypheglutyrphealaproala
216521702175
asnthraspalaasnasnilegluglyglnalaileleutyrglnasn
218021852190
glupheleuthrleuasnglylyslystyrtyrpheglyseraspser
219522002205
lysalavalthrglytrpargileileasnasnlyslystyrtyrphe
221022152220
asnproasnasnalailealaalailehisleucysthrileasnasn
2225223022352240
asplystyrtyrphesertyraspglyileleuglnasnglytyrile
224522502255
thrilegluargasnasnphetyrpheaspalaasnasngluserlys
226022652270
metvalthrglyvalphelysglyproasnglypheglutyrpheala
227522802285
proalaasnthrhisasnasnasnilegluglyglnalailevaltyr
229022952300
glnasnlyspheleuthrleuasnglylyslystyrtyrpheaspasn
2305231023152320
aspserlysalavalthrglytrpglnthrileaspglylyslystyr
232523302335
tyrpheasnleuasnthralaglualaalathrglytrpglnthrile
234023452350
aspglylyslystyrtyrpheasnleuasnthralaglualaalathr
235523602365
glytrpglnthrileaspglylyslystyrtyrpheasnthrasnthr
237023752380
pheilealaserthrglytyrthrserileasnglylyshisphetyr
2385239023952400
pheasnthraspglyilemetglnileglyvalphelysglyproasn
240524102415
glypheglutyrphealaproalaasnthraspalaasnasnileglu
242024252430
glyglnalaileleutyrglnasnlyspheleuthrleuasnglylys
243524402445
lystyrtyrpheglyseraspserlysalavalthrglyleuargthr
245024552460
ileaspglylyslystyrtyrpheasnthrasnthralavalalaval
2465247024752480
thrglytrpglnthrileasnglylyslystyrtyrpheasnthrasn
248524902495
thrserilealaserthrglytyrthrileileserglylyshisphe
250025052510
tyrpheasnthraspglyilemetglnileglyvalphelysglypro
251525202525
aspglypheglutyrphealaproalaasnthraspalaasnasnile
253025352540
gluglyglnalaileargtyrglnasnargpheleutyrleuhisasp
2545255025552560
asniletyrtyrpheglyasnasnserlysalaalathrglytrpval
256525702575
thrileaspglyasnargtyrtyrphegluproasnthralametgly
258025852590
alaasnglytyrlysthrileaspasnlysasnphetyrpheargasn
259526002605
glyleuproglnileglyvalphelysglyserasnglypheglutyr
261026152620
phealaproalaasnthraspalaasnasnilegluglyglnalaile
2625263026352640
argtyrglnasnargpheleuhisleuleuglylysiletyrtyrphe
264526502655
glyasnasnserlysalavalthrglytrpglnthrileasnglylys
266026652670
valtyrtyrphemetproaspthralametalaalaalaglyglyleu
267526802685
phegluileaspglyvaliletyrphepheglyvalaspglyvallys
269026952700
alaproglyiletyrgly
27052710
<210>2
<211>2366
<212>prt
<213>艰难梭菌
<400>2
metserleuvalasnarglysglnleuglulysmetalaasnvalarg
151015
pheargthrglngluaspglutyrvalalaileleuaspalaleuglu
202530
glutyrhisasnmetsergluasnthrvalvalglulystyrleulys
354045
leulysaspileasnserleuthraspiletyrileaspthrtyrlys
505560
lysserglyargasnlysalaleulyslysphelysglutyrleuval
65707580
thrgluvalleugluleulysasnasnasnleuthrprovalglulys
859095
asnleuhisphevaltrpileglyglyglnileasnaspthralaile
100105110
asntyrileasnglntrplysaspvalasnserasptyrasnvalasn
115120125
valphetyraspserasnalapheleuileasnthrleulyslysthr
130135140
valvalgluseralaileasnaspthrleugluserphearggluasn
145150155160
leuasnaspproargpheasptyrasnlysphephearglysargmet
165170175
gluileiletyrasplysglnlysasnpheileasntyrtyrlysala
180185190
glnargglugluasnprogluleuileileaspaspilevallysthr
195200205
tyrleuserasnglutyrserlysgluileaspgluleuasnthrtyr
210215220
ileglugluserleuasnlysilethrglnasnserglyasnaspval
225230235240
argasnpheglugluphelysasnglygluserpheasnleutyrglu
245250255
glngluleuvalgluargtrpasnleualaalaalaseraspileleu
260265270
argileseralaleulysgluileglyglymettyrleuaspvalasp
275280285
metleuproglyileglnproaspleuphegluserileglulyspro
290295300
serservalthrvalaspphetrpglumetthrlysleuglualaile
305310315320
metlystyrlysglutyrileproglutyrthrsergluhispheasp
325330335
metleuaspglugluvalglnserserphegluservalleualaser
340345350
lysserasplyssergluilepheserserleuglyaspmetgluala
355360365
serproleugluvallysilealapheasnserlysglyileileasn
370375380
glnglyleuileservallysaspsertyrcysserasnleuileval
385390395400
lysglnilegluasnargtyrlysileleuasnasnserleuasnpro
405410415
alailesergluaspasnasppheasnthrthrthrasnthrpheile
420425430
aspserilemetalaglualaasnalaaspasnglyargphemetmet
435440445
gluleuglylystyrleuargvalglyphepheproaspvallysthr
450455460
thrileasnleuserglyproglualatyralaalaalatyrglnasp
465470475480
leuleumetphelysgluglysermetasnilehisleuilegluala
485490495
aspleuargasnphegluileserlysthrasnileserglnserthr
500505510
gluglnglumetalaserleutrpserpheaspaspalaargalalys
515520525
alaglnphegluglutyrlysargasntyrphegluglyserleugly
530535540
gluaspaspasnleuasppheserglnasnilevalvalasplysglu
545550555560
tyrleuleuglulysileserserleualaargsersergluarggly
565570575
tyrilehistyrilevalglnleuglnglyasplysilesertyrglu
580585590
alaalacysasnleuphealalysthrprotyraspservalleuphe
595600605
glnlysasnilegluaspsergluilealatyrtyrtyrasnprogly
610615620
aspglygluileglngluileasplystyrlysileproserileile
625630635640
seraspargprolysilelysleuthrpheileglyhisglylysasp
645650655
glupheasnthraspilephealaglypheaspvalaspserleuser
660665670
thrgluileglualaalaileaspleualalysgluaspileserpro
675680685
lysserilegluileasnleuleuglycysasnmetphesertyrser
690695700
ileasnvalglugluthrtyrproglylysleuleuleulysvallys
705710715720
asplysilesergluleumetproserileserglnaspserileile
725730735
valseralaasnglntyrgluvalargileasnsergluglyargarg
740745750
gluleuleuasphisserglyglutrpileasnlysglugluserile
755760765
ilelysaspileserserlysglutyrileserpheasnprolysglu
770775780
asnlysilethrvallysserlysasnleuprogluleuserthrleu
785790795800
leuglngluileargasnasnserasnserseraspilegluleuglu
805810815
glulysvalmetleuthrglucysgluileasnvalileserasnile
820825830
aspthrglnilevalglugluargilegluglualalysasnleuthr
835840845
seraspserileasntyrilelysaspgluphelysleuilegluser
850855860
ileseraspalaleucysaspleulysglnglnasngluleugluasp
865870875880
serhispheileserphegluaspilesergluthraspgluglyphe
885890895
serileargpheileasnlysgluthrglygluserilephevalglu
900905910
thrglulysthrilepheserglutyralaasnhisilethrgluglu
915920925
ileserlysilelysglythrilepheaspthrvalasnglylysleu
930935940
vallyslysvalasnleuaspthrthrhisgluvalasnthrleuasn
945950955960
alaalaphepheileglnserleuileglutyrasnserserlysglu
965970975
serleuserasnleuservalalametlysvalglnvaltyralagln
980985990
leupheserthrglyleuasnthrilethraspalaalalysvalval
99510001005
gluleuvalserthralaleuaspgluthrileaspleuleuprothr
101010151020
leusergluglyleuproileilealathrileileaspglyvalser
1025103010351040
leuglyalaalailelysgluleusergluthrseraspproleuleu
104510501055
argglngluileglualalysileglyilemetalavalasnleuthr
106010651070
thralathrthralaileilethrserserleuglyilealasergly
107510801085
pheserileleuleuvalproleualaglyileseralaglyilepro
109010951100
serleuvalasnasngluleuvalleuargasplysalathrlysval
1105111011151120
valasptyrphelyshisvalserleuvalgluthrgluglyvalphe
112511301135
thrleuleuaspasplysilemetmetproglnaspaspleuvalile
114011451150
sergluileasppheasnasnasnserilevalleuglylyscysglu
115511601165
iletrpargmetgluglyglyserglyhisthrvalthraspaspile
117011751180
asphisphepheseralaproserilethrtyrarggluprohisleu
1185119011951200
seriletyraspvalleugluvalglnlysglugluleuaspleuser
120512101215
lysaspleumetvalleuproasnalaproasnargvalphealatrp
122012251230
gluthrglytrpthrproglyleuargserleugluasnaspglythr
123512401245
lysleuleuaspargileargaspasntyrgluglygluphetyrtrp
125012551260
argtyrphealapheilealaaspalaleuilethrthrleulyspro
1265127012751280
argtyrgluaspthrasnileargileasnleuaspserasnthrarg
128512901295
serpheilevalproileilethrthrglutyrileargglulysleu
130013051310
sertyrserphetyrglyserglyglythrtyralaleuserleuser
131513201325
glntyrasnmetglyileasnilegluleusergluseraspvaltrp
133013351340
ileileaspvalaspasnvalvalargaspvalthrilegluserasp
1345135013551360
lysilelyslysglyaspleuilegluglyileleuserthrleuser
136513701375
ileglugluasnlysileileleuasnserhisgluileasnpheser
138013851390
glygluvalasnglyserasnglyphevalserleuthrpheserile
139514001405
leugluglyileasnalaileilegluvalaspleuleuserlysser
141014151420
tyrlysleuleuileserglygluleulysileleumetleuasnser
1425143014351440
asnhisileglnglnlysileasptyrileglypheasnsergluleu
144514501455
glnlysasnileprotyrserphevalaspsergluglylysgluasn
146014651470
glypheileasnglyserthrlysgluglyleuphevalsergluleu
147514801485
proaspvalvalleuileserlysvaltyrmetaspaspserlyspro
149014951500
serpheglytyrtyrserasnasnleulysaspvallysvalilethr
1505151015151520
lysaspasnvalasnileleuthrglytyrtyrleulysaspaspile
152515301535
lysileserleuserleuthrleuglnaspglulysthrilelysleu
154015451550
asnservalhisleuaspgluserglyvalalagluileleulysphe
155515601565
metasnarglysglyasnthrasnthrseraspserleumetserphe
157015751580
leuglusermetasnilelysserilephevalasnpheleuglnser
1585159015951600
asnilelyspheileleuaspalaasnpheileileserglythrthr
160516101615
serileglyglnpheglupheilecysaspgluasnaspasnilegln
162016251630
protyrpheilelyspheasnthrleugluthrasntyrthrleutyr
163516401645
valglyasnargglnasnmetilevalgluproasntyraspleuasp
165016551660
aspserglyaspileserserthrvalileasnpheserglnlystyr
1665167016751680
leutyrglyileaspsercysvalasnlysvalvalileserproasn
168516901695
iletyrthraspgluileasnilethrprovaltyrgluthrasnasn
170017051710
thrtyrprogluvalilevalleuaspalaasntyrileasnglulys
171517201725
ileasnvalasnileasnaspleuserileargtyrvaltrpserasn
173017351740
aspglyasnasppheileleumetserthrserglugluasnlysval
1745175017551760
serglnvallysileargphevalasnvalphelysasplysthrleu
176517701775
alaasnlysleuserpheasnpheserasplysglnaspvalproval
178017851790
sergluileileleuserphethrprosertyrtyrgluaspglyleu
179518001805
ileglytyraspleuglyleuvalserleutyrasnglulysphetyr
181018151820
ileasnasnpheglymetmetvalserglyleuiletyrileasnasp
1825183018351840
serleutyrtyrphelysproprovalasnasnleuilethrglyphe
184518501855
valthrvalglyaspasplystyrtyrpheasnproileasnglygly
186018651870
alaalaserileglygluthrileileaspasplysasntyrtyrphe
187518801885
asnglnserglyvalleuglnthrglyvalpheserthrgluaspgly
189018951900
phelystyrphealaproalaasnthrleuaspgluasnleuglugly
1905191019151920
glualaileaspphethrglylysleuileileaspgluasniletyr
192519301935
tyrpheaspaspasntyrargglyalavalglutrplysgluleuasp
194019451950
glyglumethistyrpheserprogluthrglylysalaphelysgly
195519601965
leuasnglnileglyasptyrlystyrtyrpheasnseraspglyval
197019751980
metglnlysglyphevalserileasnaspasnlyshistyrpheasp
1985199019952000
aspserglyvalmetlysvalglytyrthrgluileaspglylyshis
200520102015
phetyrphealagluasnglyglumetglnileglyvalpheasnthr
202020252030
gluaspglyphelystyrphealahishisasngluaspleuglyasn
203520402045
glugluglyglugluilesertyrserglyileleuasnpheasnasn
205020552060
lysiletyrtyrpheaspaspserphethralavalvalglytrplys
2065207020752080
aspleugluaspglyserlystyrtyrpheaspgluaspthralaglu
208520902095
alatyrileglyleuserleuileasnaspglyglntyrtyrpheasn
210021052110
aspaspglyilemetglnvalglyphevalthrileasnasplysval
211521202125
phetyrpheseraspserglyileilegluserglyvalglnasnile
213021352140
aspaspasntyrphetyrileaspaspasnglyilevalglnilegly
2145215021552160
valpheaspthrseraspglytyrlystyrphealaproalaasnthr
216521702175
valasnaspasniletyrglyglnalavalglutyrserglyleuval
218021852190
argvalglygluaspvaltyrtyrpheglygluthrtyrthrileglu
219522002205
thrglytrpiletyraspmetgluasngluserasplystyrtyrphe
221022152220
asnprogluthrlyslysalacyslysglyileasnleuileaspasp
2225223022352240
ilelystyrtyrpheaspglulysglyilemetargthrglyleuile
224522502255
serphegluasnasnasntyrtyrpheasngluasnglyglumetgln
226022652270
pheglytyrileasnilegluasplysmetphetyrpheglygluasp
227522802285
glyvalmetglnileglyvalpheasnthrproaspglyphelystyr
229022952300
phealahisglnasnthrleuaspgluasnphegluglygluserile
2305231023152320
asntyrthrglytrpleuaspleuaspglulysargtyrtyrphethr
232523302335
aspglutyrilealaalathrglyservalileileaspglygluglu
234023452350
tyrtyrpheaspproaspthralaglnleuvalileserglu
235523602365
<210>3
<211>966
<212>prt
<213>艰难梭菌
<400>3
metglytrpglnthrileaspglylyslystyrtyrpheasnthrasn
151015
thralailealaserthrglytyrthrileileasnglylyshisphe
202530
tyrpheasnthraspglyilemetglnileglyvalphelysglypro
354045
asnglypheglutyrphealaproalaasnthraspalaasnasnile
505560
gluglyglnalaileleutyrglnasnglupheleuthrleuasngly
65707580
lyslystyrtyrpheglyseraspserlysalavalthrglytrparg
859095
ileileasnasnlyslystyrtyrpheasnproasnasnalaileala
100105110
alailehisleucysthrileasnasnasplystyrtyrphesertyr
115120125
aspglyileleuglnasnglytyrilethrilegluargasnasnphe
130135140
tyrpheaspalaasnasngluserlysmetvalthrglyvalphelys
145150155160
glyproasnglypheglutyrphealaproalaasnthrhisasnasn
165170175
asnilegluglyglnalailevaltyrglnasnlyspheleuthrleu
180185190
asnglylyslystyrtyrpheaspasnaspserlysalavalthrgly
195200205
trpglnthrileaspglylyslystyrtyrpheasnleuasnthrala
210215220
glualaalathrglytrpglnthrileaspglylyslystyrtyrphe
225230235240
asnleuasnthralaglualaalathrglytrpglnthrileaspgly
245250255
lyslystyrtyrpheasnthrasnthrpheilealaserthrglytyr
260265270
thrserileasnglylyshisphetyrpheasnthraspglyilemet
275280285
glnileglyvalphelysglyproasnglypheglutyrphealapro
290295300
alaasnthraspalaasnasnilegluglyglnalaileleutyrgln
305310315320
asnlyspheleuthrleuasnglylyslystyrtyrpheglyserasp
325330335
serlysalavalthrglyleuargthrileaspglylyslystyrtyr
340345350
pheasnthrasnthralavalalavalthrglytrpglnthrileasn
355360365
glylyslystyrtyrpheasnthrasnthrserilealaserthrgly
370375380
tyrthrileileserglylyshisphetyrpheasnthraspglyile
385390395400
metglnileglyvalphelysglyproaspglypheglutyrpheala
405410415
proalaasnthraspalaasnasnilegluglyglnalaileargtyr
420425430
glnasnargpheleutyrleuhisaspasniletyrtyrpheglyasn
435440445
asnserlysalaalathrglytrpvalthrileaspglyasnargtyr
450455460
tyrphegluproasnthralametglyalaasnglytyrlysthrile
465470475480
aspasnlysasnphetyrpheargasnglyleuproglnileglyval
485490495
phelysglyserasnglypheglutyrphealaproalaasnthrasp
500505510
alaasnasnilegluglyglnalaileargtyrglnasnargpheleu
515520525
hisleuleuglylysiletyrtyrpheglyasnasnserlysalaval
530535540
thrglytrpglnthrileasnglylysvaltyrtyrphemetproasp
545550555560
thralametalaalaalaglyglyleuphegluileaspglyvalile
565570575
tyrphepheglyvalaspglyvallysalaproglyphevalserile
580585590
asnaspasnlyshistyrpheaspaspserglyvalmetlysvalgly
595600605
tyrthrgluileaspglylyshisphetyrphealagluasnglyglu
610615620
metglnileglyvalpheasnthrgluaspglyphelystyrpheala
625630635640
hishisasngluaspleuglyasnglugluglyglugluilesertyr
645650655
serglyileleuasnpheasnasnlysiletyrtyrpheaspaspser
660665670
phethralavalvalglytrplysaspleugluaspglyserlystyr
675680685
tyrpheaspgluaspthralaglualatyrileglyleuserleuile
690695700
asnaspglyglntyrtyrpheasnaspaspglyilemetglnvalgly
705710715720
phevalthrileasnasplysvalphetyrpheseraspserglyile
725730735
ilegluserglyvalglnasnileaspaspasntyrphetyrileasp
740745750
aspasnglyilevalglnileglyvalpheaspthrseraspglytyr
755760765
lystyrphealaproalaasnthrvalasnaspasniletyrglygln
770775780
alavalglutyrserglyleuvalargvalglygluaspvaltyrtyr
785790795800
pheglygluthrtyrthrilegluthrglytrpiletyraspmetglu
805810815
asngluserasplystyrtyrpheasnprogluthrlyslysalacys
820825830
lysglyileasnleuileaspaspilelystyrtyrpheaspglulys
835840845
glyilemetargthrglyleuileserphegluasnasnasntyrtyr
850855860
pheasngluasnglyglumetglnpheglytyrileasnilegluasp
865870875880
lysmetphetyrpheglygluaspglyvalmetglnileglyvalphe
885890895
asnthrproaspglyphelystyrphealahisglnasnthrleuasp
900905910
gluasnphegluglygluserileasntyrthrglytrpleuaspleu
915920925
aspglulysargtyrtyrphethraspglutyrilealaalathrgly
930935940
servalileileaspglygluglutyrtyrpheaspproaspthrala
945950955960
glnleuvalileserglu
965
<210>4
<211>966
<212>prt
<213>艰难梭菌
<400>4
metglytrpglnthrileaspglylyslystyrtyrpheasnthrasn
151015
thralailealaserthrglytyrthrileileasnglylyshisphe
202530
tyrpheasnthraspglyilemetglnileglyvalphelysglypro
354045
asnglypheglutyrphealaproalaasnthraspalaasnasnile
505560
gluglyglnalaileleutyrglnasnglupheleuthrleuasngly
65707580
lyslystyrtyrpheglyseraspserlysalavalthrglytrparg
859095
ileileasnasnlyslystyrtyrpheasnproasnasnalaileala
100105110
alailehisleucysthrileasnasnasplystyrtyrphesertyr
115120125
aspglyileleuglnasnglytyrilethrilegluargasnasnphe
130135140
tyrpheaspalaasnasngluserlysmetvalthrglyvalphelys
145150155160
glyproasnglypheglutyrphealaproalaasnthrhisasnasn
165170175
asnilegluglyglnalailevaltyrglnasnlyspheleuthrleu
180185190
asnglylyslystyrtyrpheaspasnaspserlysalavalthrgly
195200205
trpglnthrileaspglylyslystyrtyrpheasnleuasnthrala
210215220
glualaalathrglytrpglnthrileaspglylyslystyrtyrphe
225230235240
asnleuasnthralaglualaalathrglytrpglnthrileaspgly
245250255
lyslystyrtyrpheasnthrasnthrpheilealaserthrglytyr
260265270
thrserileasnglylyshisphetyrpheasnthraspglyilemet
275280285
glnileglyvalphelysglyproasnglypheglutyrphealapro
290295300
alaasnthraspalaasnasnilegluglyglnalaileleutyrgln
305310315320
asnlyspheleuthrleuasnglylyslystyrtyrpheglyserasp
325330335
serlysalavalthrglyleuargthrileaspglylyslystyrtyr
340345350
pheasnthrasnthralavalalavalthrglytrpglnthrileasn
355360365
glylyslystyrtyrpheasnthrasnthrserilealaserthrgly
370375380
tyrthrileileserglylyshisphetyrpheasnthraspglyile
385390395400
metglnileglyvalphelysglyproaspglypheglutyrpheala
405410415
proalaasnthraspalaasnasnilegluglyglnalaileargtyr
420425430
glnasnargpheleutyrleuhisaspasniletyrtyrpheglyasn
435440445
asnserlysalaalathrglytrpvalthrileaspglyasnargtyr
450455460
tyrphegluproasnthralametglyalaasnglytyrlysthrile
465470475480
aspasnlysasnphetyrpheargasnglyleuproglnileglyval
485490495
phelysglyserasnglypheglutyrphealaproalaasnthrasp
500505510
alaasnasnilegluglyglnalaileargtyrglnasnargpheleu
515520525
hisleuleuglylysiletyrtyrpheglyasnasnserlysalaval
530535540
thrglytrpglnthrileasnglylysvaltyrtyrphemetproasp
545550555560
thralametalaalaalaglyglyleuasnglnileglyasptyrlys
565570575
tyrtyrpheasnseraspglyvalmetglnlysglyphevalserile
580585590
asnaspasnlyshistyrpheaspaspserglyvalmetlysvalgly
595600605
tyrthrgluileaspglylyshisphetyrphealagluasnglyglu
610615620
metglnileglyvalpheasnthrgluaspglyphelystyrpheala
625630635640
hishisasngluaspleuglyasnglugluglyglugluilesertyr
645650655
serglyileleuasnpheasnasnlysiletyrtyrpheaspaspser
660665670
phethralavalvalglytrplysaspleugluaspglyserlystyr
675680685
tyrpheaspgluaspthralaglualatyrileglyleuserleuile
690695700
asnaspglyglntyrtyrpheasnaspaspglyilemetglnvalgly
705710715720
phevalthrileasnasplysvalphetyrpheseraspserglyile
725730735
ilegluserglyvalglnasnileaspaspasntyrphetyrileasp
740745750
aspasnglyilevalglnileglyvalpheaspthrseraspglytyr
755760765
lystyrphealaproalaasnthrvalasnaspasniletyrglygln
770775780
alavalglutyrserglyleuvalargvalglygluaspvaltyrtyr
785790795800
pheglygluthrtyrthrilegluthrglytrpiletyraspmetglu
805810815
asngluserasplystyrtyrpheasnprogluthrlyslysalacys
820825830
lysglyileasnleuileaspaspilelystyrtyrpheaspglulys
835840845
glyilemetargthrglyleuileserphegluasnasnasntyrtyr
850855860
pheasngluasnglyglumetglnpheglytyrileasnilegluasp
865870875880
lysmetphetyrpheglygluaspglyvalmetglnileglyvalphe
885890895
asnthrproaspglyphelystyrphealahisglnasnthrleuasp
900905910
gluasnphegluglygluserileasntyrthrglytrpleuaspleu
915920925
aspglulysargtyrtyrphethraspglutyrilealaalathrgly
930935940
servalileileaspglygluglutyrtyrpheaspproaspthrala
945950955960
glnleuvalileserglu
965
<210>5
<211>833
<212>prt
<213>艰难梭菌
<400>5
metglytrpglnthrileaspglylyslystyrtyrpheasnthrasn
151015
thralailealaserthrglytyrthrileileasnglylyshisphe
202530
tyrpheasnthraspglyilemetglnileglyvalphelysglypro
354045
asnglypheglutyrphealaproalaasnthraspalaasnasnile
505560
gluglyglnalaileleutyrglnasnglupheleuthrleuasngly
65707580
lyslystyrtyrpheglyseraspserlysalavalthrglytrparg
859095
ileileasnasnlyslystyrtyrpheasnproasnasnalaileala
100105110
alailehisleucysthrileasnasnasplystyrtyrphesertyr
115120125
aspglyileleuglnasnglytyrilethrilegluargasnasnphe
130135140
tyrpheaspalaasnasngluserlysmetvalthrglyvalphelys
145150155160
glyproasnglypheglutyrphealaproalaasnthrhisasnasn
165170175
asnilegluglyglnalailevaltyrglnasnlyspheleuthrleu
180185190
asnglylyslystyrtyrpheaspasnaspserlysalavalthrgly
195200205
trpglnthrileaspglylyslystyrtyrpheasnleuasnthrala
210215220
glualaalathrglytrpglnthrileaspglylyslystyrtyrphe
225230235240
asnleuasnthralaglualaalathrglytrpglnthrileaspgly
245250255
lyslystyrtyrpheasnthrasnthrpheilealaserthrglytyr
260265270
thrserileasnglylyshisphetyrpheasnthraspglyilemet
275280285
glnileglyvalphelysglyproasnglypheglutyrphealapro
290295300
alaasnthraspalaasnasnilegluglyglnalaileleutyrgln
305310315320
asnlyspheleuthrleuasnglylyslystyrtyrpheglyserasp
325330335
serlysalavalthrglyleuargthrileaspglylyslystyrtyr
340345350
pheasnthrasnthralavalalavalthrglytrpglnthrileasn
355360365
glylyslystyrtyrpheasnthrasnthrserilealaserthrgly
370375380
tyrthrileileserglylyshisphetyrpheasnthraspglyile
385390395400
metglnileglyvalphelysglyproaspglypheglutyrpheala
405410415
proalaasnthraspalaasnasnilegluglyglnalaileargtyr
420425430
glnasnargpheleutyrleuhisaspasniletyrtyrpheglyasn
435440445
asnserlysalaalathrglytrpvalthrileaspglyasnargtyr
450455460
tyrphegluproasnthralametglyalaasnglytyrlysthrile
465470475480
aspasnlysasnphetyrpheargasnglyleuproglnileglyval
485490495
phelysglyserasnglypheglutyrphealahishisasngluasp
500505510
leuglyasnglugluglyglugluilesertyrserglyileleuasn
515520525
pheasnasnlysiletyrtyrpheaspaspserphethralavalval
530535540
glytrplysaspleugluaspglyserlystyrtyrpheaspgluasp
545550555560
thralaglualatyrileglyleuserleuileasnaspglyglntyr
565570575
tyrpheasnaspaspglyilemetglnvalglyphevalthrileasn
580585590
asplysvalphetyrpheseraspserglyileilegluserglyval
595600605
glnasnileaspaspasntyrphetyrileaspaspasnglyileval
610615620
glnileglyvalpheaspthrseraspglytyrlystyrphealapro
625630635640
alaasnthrvalasnaspasniletyrglyglnalavalglutyrser
645650655
glyleuvalargvalglygluaspvaltyrtyrpheglygluthrtyr
660665670
thrilegluthrglytrpiletyraspmetgluasngluserasplys
675680685
tyrtyrpheasnprogluthrlyslysalacyslysglyileasnleu
690695700
ileaspaspilelystyrtyrpheaspglulysglyilemetargthr
705710715720
glyleuileserphegluasnasnasntyrtyrpheasngluasngly
725730735
glumetglnpheglytyrileasnilegluasplysmetphetyrphe
740745750
glygluaspglyvalmetglnileglyvalpheasnthrproaspgly
755760765
phelystyrphealahisglnasnthrleuaspgluasnpheglugly
770775780
gluserileasntyrthrglytrpleuaspleuaspglulysargtyr
785790795800
tyrphethraspglutyrilealaalathrglyservalileileasp
805810815
glygluglutyrtyrpheaspproaspthralaglnleuvalileser
820825830
glu
<210>6
<211>1057
<212>prt
<213>艰难梭菌
<400>6
metglytrpglnthrileaspglylyslystyrtyrpheasnthrasn
151015
thralailealaserthrglytyrthrileileasnglylyshisphe
202530
tyrpheasnthraspglyilemetglnileglyvalphelysglypro
354045
asnglypheglutyrphealaproalaasnthraspalaasnasnile
505560
gluglyglnalaileleutyrglnasnglupheleuthrleuasngly
65707580
lyslystyrtyrpheglyseraspserlysalavalthrglytrparg
859095
ileileasnasnlyslystyrtyrpheasnproasnasnalaileala
100105110
alailehisleucysthrileasnasnasplystyrtyrphesertyr
115120125
aspglyileleuglnasnglytyrilethrilegluargasnasnphe
130135140
tyrpheaspalaasnasngluserlysmetvalthrglyvalphelys
145150155160
glyproasnglypheglutyrphealaproalaasnthrhisasnasn
165170175
asnilegluglyglnalailevaltyrglnasnlyspheleuthrleu
180185190
asnglylyslystyrtyrpheaspasnaspserlysalavalthrgly
195200205
trpglnthrileaspglylyslystyrtyrpheasnleuasnthrala
210215220
glualaalathrglytrpglnthrileaspglylyslystyrtyrphe
225230235240
asnleuasnthralaglualaalathrglytrpglnthrileaspgly
245250255
lyslystyrtyrpheasnthrasnthrpheilealaserthrglytyr
260265270
thrserileasnglylyshisphetyrpheasnthraspglyilemet
275280285
glnileglyvalphelysglyproasnglypheglutyrphealapro
290295300
alaasnthraspalaasnasnilegluglyglnalaileleutyrgln
305310315320
asnlyspheleuthrleuasnglylyslystyrtyrpheglyserasp
325330335
serlysalavalthrglyleuargthrileaspglylyslystyrtyr
340345350
pheasnthrasnthralavalalavalthrglytrpglnthrileasn
355360365
glylyslystyrtyrpheasnthrasnthrserilealaserthrgly
370375380
tyrthrileileserglylyshisphetyrpheasnthraspglyile
385390395400
metglnileglyvalphelysglyproaspglypheglutyrpheala
405410415
proalaasnthraspalaasnasnilegluglyglnalaileargtyr
420425430
glnasnargpheleutyrleuhisaspasniletyrtyrpheglyasn
435440445
asnserlysalaalathrglytrpvalthrileaspglyasnargtyr
450455460
tyrphegluproasnthralametglyalaasnglytyrlysthrile
465470475480
aspasnlysasnphetyrpheargasnglyleuproglnileglyval
485490495
phelysglyserasnglypheglutyrphealaproalaasnthrasp
500505510
alaasnasnilegluglyglnalaileargtyrglnasnargpheleu
515520525
hisleuleuglylysiletyrtyrpheglyasnasnserlysalaval
530535540
thrglytrpglnthrileasnglylysvaltyrtyrphemetproasp
545550555560
thralametalaalaalaglyglygluthrileileaspasplysasn
565570575
tyrtyrpheasnglnserglyvalleuglnthrglyvalpheserthr
580585590
gluaspglyphelystyrphealaproalaasnthrleuaspgluasn
595600605
leugluglyglualaileaspphethrglylysleuileileaspglu
610615620
asniletyrtyrpheaspaspasntyrargglyalavalglutrplys
625630635640
gluleuaspglyglumethistyrpheserprogluthrglylysala
645650655
phelysglyleuasnglnileglyasptyrlystyrtyrpheasnser
660665670
aspglyvalmetglnlysglyphevalserileasnaspasnlyshis
675680685
tyrpheaspaspserglyvalmetlysvalglytyrthrgluileasp
690695700
glylyshisphetyrphealagluasnglyglumetglnileglyval
705710715720
pheasnthrgluaspglyphelystyrphealahishisasngluasp
725730735
leuglyasnglugluglyglugluilesertyrserglyileleuasn
740745750
pheasnasnlysiletyrtyrpheaspaspserphethralavalval
755760765
glytrplysaspleugluaspglyserlystyrtyrpheaspgluasp
770775780
thralaglualatyrileglyleuserleuileasnaspglyglntyr
785790795800
tyrpheasnaspaspglyilemetglnvalglyphevalthrileasn
805810815
asplysvalphetyrpheseraspserglyileilegluserglyval
820825830
glnasnileaspaspasntyrphetyrileaspaspasnglyileval
835840845
glnileglyvalpheaspthrseraspglytyrlystyrphealapro
850855860
alaasnthrvalasnaspasniletyrglyglnalavalglutyrser
865870875880
glyleuvalargvalglygluaspvaltyrtyrpheglygluthrtyr
885890895
thrilegluthrglytrpiletyraspmetgluasngluserasplys
900905910
tyrtyrpheasnprogluthrlyslysalacyslysglyileasnleu
915920925
ileaspaspilelystyrtyrpheaspglulysglyilemetargthr
930935940
glyleuileserphegluasnasnasntyrtyrpheasngluasngly
945950955960
glumetglnpheglytyrileasnilegluasplysmetphetyrphe
965970975
glygluaspglyvalmetglnileglyvalpheasnthrproaspgly
980985990
phelystyrphealahisglnasnthrleuaspgluasnpheglugly
99510001005
gluserileasntyrthrglytrpleuaspleuaspglulysargtyr
101010151020
tyrphethraspglutyrilealaalathrglyservalileileasp
1025103010351040
glygluglutyrtyrpheaspproaspthralaglnleuvalileser
104510501055
glu
<210>7
<211>971
<212>prt
<213>艰难梭菌
<400>7
metglytrpglnthrileaspglylyslystyrtyrpheasnthrasn
151015
thralailealaserthrglytyrthrileileasnglylyshisphe
202530
tyrpheasnthraspglyilemetglnileglyvalphelysglypro
354045
asnglypheglutyrphealaproalaasnthraspalaasnasnile
505560
gluglyglnalaileleutyrglnasnglupheleuthrleuasngly
65707580
lyslystyrtyrpheglyseraspserlysalavalthrglytrparg
859095
ileileasnasnlyslystyrtyrpheasnproasnasnalaileala
100105110
alailehisleucysthrileasnasnasplystyrtyrphesertyr
115120125
aspglyileleuglnasnglytyrilethrilegluargasnasnphe
130135140
tyrpheaspalaasnasngluserlysmetvalthrglyvalphelys
145150155160
glyproasnglypheglutyrphealaproalaasnthrhisasnasn
165170175
asnilegluglyglnalailevaltyrglnasnlyspheleuthrleu
180185190
asnglylyslystyrtyrpheaspasnaspserlysalavalthrgly
195200205
trpglnthrileaspglylyslystyrtyrpheasnleuasnthrala
210215220
glualaalathrglytrpglnthrileaspglylyslystyrtyrphe
225230235240
asnleuasnthralaglualaalathrglytrpglnthrileaspgly
245250255
lyslystyrtyrpheasnthrasnthrpheilealaserthrglytyr
260265270
thrserileasnglylyshisphetyrpheasnthraspglyilemet
275280285
glnileglyvalphelysglyproasnglypheglutyrphealapro
290295300
alaasnthraspalaasnasnilegluglyglnalaileleutyrgln
305310315320
asnlyspheleuthrleuasnglylyslystyrtyrpheglyserasp
325330335
serlysalavalthrglyleuargthrileaspglylyslystyrtyr
340345350
pheasnthrasnthralavalalavalthrglytrpglnthrileasn
355360365
glylyslystyrtyrpheasnthrasnthrserilealaserthrgly
370375380
tyrthrileileserglylyshisphetyrpheasnthraspglyile
385390395400
metglnileglyvalphelysglyproaspglypheglutyrpheala
405410415
proalaasnthraspalaasnasnilegluglyglnalaileargtyr
420425430
glnasnargpheleutyrleuhisaspasniletyrtyrpheglyasn
435440445
asnserlysalaalathrglytrpvalthrileaspglyasnargtyr
450455460
tyrphegluproasnthralametglyalaasnglytyrlysthrile
465470475480
aspasnlysasnphetyrpheargasnglyleuproglnileglyval
485490495
phelysglyserasnglypheglutyrphealaproalaasnthrasp
500505510
alaasnasnilegluglyglnalaileargtyrglnasnargpheleu
515520525
hisleuleuglylysiletyrtyrpheglyasnasnserlysalaval
530535540
thrglytrpglnthrileasnglylysvaltyrtyrphemetproasp
545550555560
thralametalaalaalaglyglyleuphegluileaspglyvalile
565570575
tyrphepheglyvalaspglyvallysalaproglyiletyrglygly
580585590
glyphevalserileasnaspasnlyshistyrpheaspaspsergly
595600605
valmetlysvalglytyrthrgluileaspglylyshisphetyrphe
610615620
alagluasnglyglumetglnileglyvalpheasnthrgluaspgly
625630635640
phelystyrphealahishisasngluaspleuglyasngluglugly
645650655
glugluilesertyrserglyileleuasnpheasnasnlysiletyr
660665670
tyrpheaspaspserphethralavalvalglytrplysaspleuglu
675680685
aspglyserlystyrtyrpheaspgluaspthralaglualatyrile
690695700
glyleuserleuileasnaspglyglntyrtyrpheasnaspaspgly
705710715720
ilemetglnvalglyphevalthrileasnasplysvalphetyrphe
725730735
seraspserglyileilegluserglyvalglnasnileaspaspasn
740745750
tyrphetyrileaspaspasnglyilevalglnileglyvalpheasp
755760765
thrseraspglytyrlystyrphealaproalaasnthrvalasnasp
770775780
asniletyrglyglnalavalglutyrserglyleuvalargvalgly
785790795800
gluaspvaltyrtyrpheglygluthrtyrthrilegluthrglytrp
805810815
iletyraspmetgluasngluserasplystyrtyrpheasnproglu
820825830
thrlyslysalacyslysglyileasnleuileaspaspilelystyr
835840845
tyrpheaspglulysglyilemetargthrglyleuileserpheglu
850855860
asnasnasntyrtyrpheasngluasnglyglumetglnpheglytyr
865870875880
ileasnilegluasplysmetphetyrpheglygluaspglyvalmet
885890895
glnileglyvalpheasnthrproaspglyphelystyrphealahis
900905910
glnasnthrleuaspgluasnphegluglygluserileasntyrthr
915920925
glytrpleuaspleuaspglulysargtyrtyrphethraspglutyr
930935940
ilealaalathrglyservalileileaspglygluglutyrtyrphe
945950955960
aspproaspthralaglnleuvalileserglu
965970
<210>8
<211>321
<212>prt
<213>艰难梭菌
<400>8
metalaserthrglytyrthrserileasnglylyshisphetyrphe
151015
asnthraspglyilemetglnileglyvalphelysglyproasngly
202530
pheglutyrphealaproalaasnthraspalaasnasnileglugly
354045
glnalaileleutyrglnasnlyspheleuthrleuasnglylyslys
505560
tyrtyrpheglyseraspserlysalavalthrglyleuargthrile
65707580
aspglylyslystyrtyrpheasnthrasnthralavalalavalthr
859095
glytrpglnthrileasnglylyslystyrtyrpheasnthrasnthr
100105110
serilealaserthrglytyrthrileileserglylyshisphetyr
115120125
pheasnthraspglyilemetglnileglyvalphelysglyproasp
130135140
glypheglutyrphealaproalaasnthraspalaasnasnileglu
145150155160
glyglnalaileargtyrglnasnargpheleutyrleuhisaspasn
165170175
iletyrtyrpheglyasnasnserlysalaalathrglytrpvalthr
180185190
ileaspglyasnargtyrtyrphegluproasnthralametglyala
195200205
asnglytyrlysthrileaspasnlysasnphetyrpheargasngly
210215220
leuproglnileglyvalphelysglyserasnglypheglutyrphe
225230235240
alaproalaasnthraspalaasnasnilegluglyglnalailearg
245250255
tyrglnasnargpheleuhisleuleuglylysiletyrtyrphegly
260265270
asnasnserlysalavalthrglytrpglnthrileasnglylysval
275280285
tyrtyrphemetproaspthralametalaalaalaglyglyleuphe
290295300
gluileaspglyvaliletyrphepheglyvalaspglyvallysala
305310315320
pro
<210>9
<211>612
<212>prt
<213>艰难梭菌
<400>9
metileleumetserthrserglugluasnlysvalserglnvallys
151015
ileargphevalasnvalphelysasplysthrleualaasnlysleu
202530
serpheasnpheserasplysglnaspvalprovalsergluileile
354045
leuserphethrprosertyrtyrgluaspglyleuileglytyrasp
505560
leuglyleuvalserleutyrasnglulysphetyrileasnasnphe
65707580
glymetmetvalserglyleuiletyrileasnaspserleutyrtyr
859095
phelysproprovalasnasnleuilethrglyphevalthrvalgly
100105110
aspasplystyrtyrpheasnproileasnglyglyalaalaserile
115120125
glygluthrileileaspasplysasntyrtyrpheasnglnsergly
130135140
valleuglnthrglyvalpheserthrgluaspglyphelystyrphe
145150155160
alaproalaasnthrleuaspgluasnleugluglyglualaileasp
165170175
phethrglylysleuileileaspgluasniletyrtyrpheaspasp
180185190
asntyrargglyalavalglutrplysgluleuaspglyglumethis
195200205
tyrpheserprogluthrglylysalaphelysglyleuasnglnile
210215220
glyasptyrlystyrtyrpheasnseraspglyvalmetglnlysgly
225230235240
phevalserileasnaspasnlyshistyrpheaspaspserglyval
245250255
metlysvalglytyrthrgluileaspglylyshisphetyrpheala
260265270
gluasnglyglumetglnileglyvalpheasnthrgluaspglyphe
275280285
lystyrphealahishisasngluaspleuglyasnglugluglyglu
290295300
gluilesertyrserglyileleuasnpheasnasnlysiletyrtyr
305310315320
pheaspaspserphethralavalvalglytrplysaspleugluasp
325330335
glyserlystyrtyrpheaspgluaspthralaglualatyrilegly
340345350
leuserleuileasnaspglyglntyrtyrpheasnaspaspglyile
355360365
metglnvalglyphevalthrileasnasplysvalphetyrpheser
370375380
aspserglyileilegluserglyvalglnasnileaspaspasntyr
385390395400
phetyrileaspaspasnglyilevalglnileglyvalpheaspthr
405410415
seraspglytyrlystyrphealaproalaasnthrvalasnaspasn
420425430
iletyrglyglnalavalglutyrserglyleuvalargvalglyglu
435440445
aspvaltyrtyrpheglygluthrtyrthrilegluthrglytrpile
450455460
tyraspmetgluasngluserasplystyrtyrpheasnprogluthr
465470475480
lyslysalacyslysglyileasnleuileaspaspilelystyrtyr
485490495
pheaspglulysglyilemetargthrglyleuileserphegluasn
500505510
asnasntyrtyrpheasngluasnglyglumetglnpheglytyrile
515520525
asnilegluasplysmetphetyrpheglygluaspglyvalmetgln
530535540
ileglyvalpheasnthrproaspglyphelystyrphealahisgln
545550555560
asnthrleuaspgluasnphegluglygluserileasntyrthrgly
565570575
trpleuaspleuaspglulysargtyrtyrphethraspglutyrile
580585590
alaalathrglyservalileileaspglygluglutyrtyrpheasp
595600605
proaspthrala
610
<210>10
<211>587
<212>prt
<213>艰难梭菌
<400>10
metglytrpglnthrileaspglylyslystyrtyrpheasnthrasn
151015
thralailealaserthrglytyrthrileileasnglylyshisphe
202530
tyrpheasnthraspglyilemetglnileglyvalphelysglypro
354045
asnglypheglutyrphealaproalaasnthraspalaasnasnile
505560
gluglyglnalaileleutyrglnasnglupheleuthrleuasngly
65707580
lyslystyrtyrpheglyseraspserlysalavalthrglytrparg
859095
ileileasnasnlyslystyrtyrpheasnproasnasnalaileala
100105110
alailehisleucysthrileasnasnasplystyrtyrphesertyr
115120125
aspglyileleuglnasnglytyrilethrilegluargasnasnphe
130135140
tyrpheaspalaasnasngluserlysmetvalthrglyvalphelys
145150155160
glyproasnglypheglutyrphealaproalaasnthrhisasnasn
165170175
asnilegluglyglnalailevaltyrglnasnlyspheleuthrleu
180185190
asnglylyslystyrtyrpheaspasnaspserlysalavalthrgly
195200205
trpglnthrileaspglylyslystyrtyrpheasnleuasnthrala
210215220
glualaalathrglytrpglnthrileaspglylyslystyrtyrphe
225230235240
asnleuasnthralaglualaalathrglytrpglnthrileaspgly
245250255
lyslystyrtyrpheasnthrasnthrpheilealaserthrglytyr
260265270
thrserileasnglylyshisphetyrpheasnthraspglyilemet
275280285
glnileglyvalphelysglyproasnglypheglutyrphealapro
290295300
alaasnthraspalaasnasnilegluglyglnalaileleutyrgln
305310315320
asnlyspheleuthrleuasnglylyslystyrtyrpheglyserasp
325330335
serlysalavalthrglyleuargthrileaspglylyslystyrtyr
340345350
pheasnthrasnthralavalalavalthrglytrpglnthrileasn
355360365
glylyslystyrtyrpheasnthrasnthrserilealaserthrgly
370375380
tyrthrileileserglylyshisphetyrpheasnthraspglyile
385390395400
metglnileglyvalphelysglyproaspglypheglutyrpheala
405410415
proalaasnthraspalaasnasnilegluglyglnalaileargtyr
420425430
glnasnargpheleutyrleuhisaspasniletyrtyrpheglyasn
435440445
asnserlysalaalathrglytrpvalthrileaspglyasnargtyr
450455460
tyrphegluproasnthralametglyalaasnglytyrlysthrile
465470475480
aspasnlysasnphetyrpheargasnglyleuproglnileglyval
485490495
phelysglyserasnglypheglutyrphealaproalaasnthrasp
500505510
alaasnasnilegluglyglnalaileargtyrglnasnargpheleu
515520525
hisleuleuglylysiletyrtyrpheglyasnasnserlysalaval
530535540
thrglytrpglnthrileasnglylysvaltyrtyrphemetproasp
545550555560
thralametalaalaalaglyglyleuphegluileaspglyvalile
565570575
tyrphepheglyvalaspglyvallysalapro
580585
<210>11
<211>567
<212>prt
<213>艰难梭菌
<400>11
metglytrpglnthrileaspglylyslystyrtyrpheasnthrasn
151015
thralailealaserthrglytyrthrileileasnglylyshisphe
202530
tyrpheasnthraspglyilemetglnileglyvalphelysglypro
354045
asnglypheglutyrphealaproalaasnthraspalaasnasnile
505560
gluglyglnalaileleutyrglnasnglupheleuthrleuasngly
65707580
lyslystyrtyrpheglyseraspserlysalavalthrglytrparg
859095
ileileasnasnlyslystyrtyrpheasnproasnasnalaileala
100105110
alailehisleucysthrileasnasnasplystyrtyrphesertyr
115120125
aspglyileleuglnasnglytyrilethrilegluargasnasnphe
130135140
tyrpheaspalaasnasngluserlysmetvalthrglyvalphelys
145150155160
glyproasnglypheglutyrphealaproalaasnthrhisasnasn
165170175
asnilegluglyglnalailevaltyrglnasnlyspheleuthrleu
180185190
asnglylyslystyrtyrpheaspasnaspserlysalavalthrgly
195200205
trpglnthrileaspglylyslystyrtyrpheasnleuasnthrala
210215220
glualaalathrglytrpglnthrileaspglylyslystyrtyrphe
225230235240
asnleuasnthralaglualaalathrglytrpglnthrileaspgly
245250255
lyslystyrtyrpheasnthrasnthrpheilealaserthrglytyr
260265270
thrserileasnglylyshisphetyrpheasnthraspglyilemet
275280285
glnileglyvalphelysglyproasnglypheglutyrphealapro
290295300
alaasnthraspalaasnasnilegluglyglnalaileleutyrgln
305310315320
asnlyspheleuthrleuasnglylyslystyrtyrpheglyserasp
325330335
serlysalavalthrglyleuargthrileaspglylyslystyrtyr
340345350
pheasnthrasnthralavalalavalthrglytrpglnthrileasn
355360365
glylyslystyrtyrpheasnthrasnthrserilealaserthrgly
370375380
tyrthrileileserglylyshisphetyrpheasnthraspglyile
385390395400
metglnileglyvalphelysglyproaspglypheglutyrpheala
405410415
proalaasnthraspalaasnasnilegluglyglnalaileargtyr
420425430
glnasnargpheleutyrleuhisaspasniletyrtyrpheglyasn
435440445
asnserlysalaalathrglytrpvalthrileaspglyasnargtyr
450455460
tyrphegluproasnthralametglyalaasnglytyrlysthrile
465470475480
aspasnlysasnphetyrpheargasnglyleuproglnileglyval
485490495
phelysglyserasnglypheglutyrphealaproalaasnthrasp
500505510
alaasnasnilegluglyglnalaileargtyrglnasnargpheleu
515520525
hisleuleuglylysiletyrtyrpheglyasnasnserlysalaval
530535540
thrglytrpglnthrileasnglylysvaltyrtyrphemetproasp
545550555560
thralametalaalaalagly
565
<210>12
<211>505
<212>prt
<213>艰难梭菌
<400>12
metglytrpglnthrileaspglylyslystyrtyrpheasnthrasn
151015
thralailealaserthrglytyrthrileileasnglylyshisphe
202530
tyrpheasnthraspglyilemetglnileglyvalphelysglypro
354045
asnglypheglutyrphealaproalaasnthraspalaasnasnile
505560
gluglyglnalaileleutyrglnasnglupheleuthrleuasngly
65707580
lyslystyrtyrpheglyseraspserlysalavalthrglytrparg
859095
ileileasnasnlyslystyrtyrpheasnproasnasnalaileala
100105110
alailehisleucysthrileasnasnasplystyrtyrphesertyr
115120125
aspglyileleuglnasnglytyrilethrilegluargasnasnphe
130135140
tyrpheaspalaasnasngluserlysmetvalthrglyvalphelys
145150155160
glyproasnglypheglutyrphealaproalaasnthrhisasnasn
165170175
asnilegluglyglnalailevaltyrglnasnlyspheleuthrleu
180185190
asnglylyslystyrtyrpheaspasnaspserlysalavalthrgly
195200205
trpglnthrileaspglylyslystyrtyrpheasnleuasnthrala
210215220
glualaalathrglytrpglnthrileaspglylyslystyrtyrphe
225230235240
asnleuasnthralaglualaalathrglytrpglnthrileaspgly
245250255
lyslystyrtyrpheasnthrasnthrpheilealaserthrglytyr
260265270
thrserileasnglylyshisphetyrpheasnthraspglyilemet
275280285
glnileglyvalphelysglyproasnglypheglutyrphealapro
290295300
alaasnthraspalaasnasnilegluglyglnalaileleutyrgln
305310315320
asnlyspheleuthrleuasnglylyslystyrtyrpheglyserasp
325330335
serlysalavalthrglyleuargthrileaspglylyslystyrtyr
340345350
pheasnthrasnthralavalalavalthrglytrpglnthrileasn
355360365
glylyslystyrtyrpheasnthrasnthrserilealaserthrgly
370375380
tyrthrileileserglylyshisphetyrpheasnthraspglyile
385390395400
metglnileglyvalphelysglyproaspglypheglutyrpheala
405410415
proalaasnthraspalaasnasnilegluglyglnalaileargtyr
420425430
glnasnargpheleutyrleuhisaspasniletyrtyrpheglyasn
435440445
asnserlysalaalathrglytrpvalthrileaspglyasnargtyr
450455460
tyrphegluproasnthralametglyalaasnglytyrlysthrile
465470475480
aspasnlysasnphetyrpheargasnglyleuproglnileglyval
485490495
phelysglyserasnglypheglutyr
500505
<210>13
<211>567
<212>prt
<213>艰难梭菌
<400>13
metglytrpglnthrileaspglylyslystyrtyrpheasnthrasn
151015
thralailealaserthrglytyrthrileileasnglylyshisphe
202530
tyrpheasnthraspglyilemetglnileglyvalphelysglypro
354045
asnglypheglutyrphealaproalaasnthraspalaasnasnile
505560
gluglyglnalaileleutyrglnasnglupheleuthrleuasngly
65707580
lyslystyrtyrpheglyseraspserlysalavalthrglytrparg
859095
ileileasnasnlyslystyrtyrpheasnproasnasnalaileala
100105110
alailehisleucysthrileasnasnasplystyrtyrphesertyr
115120125
aspglyileleuglnasnglytyrilethrilegluargasnasnphe
130135140
tyrpheaspalaasnasngluserlysmetvalthrglyvalphelys
145150155160
glyproasnglypheglutyrphealaproalaasnthrhisasnasn
165170175
asnilegluglyglnalailevaltyrglnasnlyspheleuthrleu
180185190
asnglylyslystyrtyrpheaspasnaspserlysalavalthrgly
195200205
trpglnthrileaspglylyslystyrtyrpheasnleuasnthrala
210215220
glualaalathrglytrpglnthrileaspglylyslystyrtyrphe
225230235240
asnleuasnthralaglualaalathrglytrpglnthrileaspgly
245250255
lyslystyrtyrpheasnthrasnthrpheilealaserthrglytyr
260265270
thrserileasnglylyshisphetyrpheasnthraspglyilemet
275280285
glnileglyvalphelysglyproasnglypheglutyrphealapro
290295300
alaasnthraspalaasnasnilegluglyglnalaileleutyrgln
305310315320
asnlyspheleuthrleuasnglylyslystyrtyrpheglyserasp
325330335
serlysalavalthrglyleuargthrileaspglylyslystyrtyr
340345350
pheasnthrasnthralavalalavalthrglytrpglnthrileasn
355360365
glylyslystyrtyrpheasnthrasnthrserilealaserthrgly
370375380
tyrthrileileserglylyshisphetyrpheasnthraspglyile
385390395400
metglnileglyvalphelysglyproaspglypheglutyrpheala
405410415
proalaasnthraspalaasnasnilegluglyglnalaileargtyr
420425430
glnasnargpheleutyrleuhisaspasniletyrtyrpheglyasn
435440445
asnserlysalaalathrglytrpvalthrileaspglyasnargtyr
450455460
tyrphegluproasnthralametglyalaasnglytyrlysthrile
465470475480
aspasnlysasnphetyrpheargasnglyleuproglnileglyval
485490495
phelysglyserasnglypheglutyrphealaproalaasnthrasp
500505510
alaasnasnilegluglyglnalaileargtyrglnasnargpheleu
515520525
hisleuleuglylysiletyrtyrpheglyasnasnserlysalaval
530535540
thrglytrpglnthrileasnglylysvaltyrtyrphemetproasp
545550555560
thralametalaalaalagly
565
<210>14
<211>591
<212>prt
<213>艰难梭菌
<400>14
metglytrpglnthrileaspglylyslystyrtyrpheasnthrasn
151015
thralailealaserthrglytyrthrileileasnglylyshisphe
202530
tyrpheasnthraspglyilemetglnileglyvalphelysglypro
354045
asnglypheglutyrphealaproalaasnthraspalaasnasnile
505560
gluglyglnalaileleutyrglnasnglupheleuthrleuasngly
65707580
lyslystyrtyrpheglyseraspserlysalavalthrglytrparg
859095
ileileasnasnlyslystyrtyrpheasnproasnasnalaileala
100105110
alailehisleucysthrileasnasnasplystyrtyrphesertyr
115120125
aspglyileleuglnasnglytyrilethrilegluargasnasnphe
130135140
tyrpheaspalaasnasngluserlysmetvalthrglyvalphelys
145150155160
glyproasnglypheglutyrphealaproalaasnthrhisasnasn
165170175
asnilegluglyglnalailevaltyrglnasnlyspheleuthrleu
180185190
asnglylyslystyrtyrpheaspasnaspserlysalavalthrgly
195200205
trpglnthrileaspglylyslystyrtyrpheasnleuasnthrala
210215220
glualaalathrglytrpglnthrileaspglylyslystyrtyrphe
225230235240
asnleuasnthralaglualaalathrglytrpglnthrileaspgly
245250255
lyslystyrtyrpheasnthrasnthrpheilealaserthrglytyr
260265270
thrserileasnglylyshisphetyrpheasnthraspglyilemet
275280285
glnileglyvalphelysglyproasnglypheglutyrphealapro
290295300
alaasnthraspalaasnasnilegluglyglnalaileleutyrgln
305310315320
asnlyspheleuthrleuasnglylyslystyrtyrpheglyserasp
325330335
serlysalavalthrglyleuargthrileaspglylyslystyrtyr
340345350
pheasnthrasnthralavalalavalthrglytrpglnthrileasn
355360365
glylyslystyrtyrpheasnthrasnthrserilealaserthrgly
370375380
tyrthrileileserglylyshisphetyrpheasnthraspglyile
385390395400
metglnileglyvalphelysglyproaspglypheglutyrpheala
405410415
proalaasnthraspalaasnasnilegluglyglnalaileargtyr
420425430
glnasnargpheleutyrleuhisaspasniletyrtyrpheglyasn
435440445
asnserlysalaalathrglytrpvalthrileaspglyasnargtyr
450455460
tyrphegluproasnthralametglyalaasnglytyrlysthrile
465470475480
aspasnlysasnphetyrpheargasnglyleuproglnileglyval
485490495
phelysglyserasnglypheglutyrphealaproalaasnthrasp
500505510
alaasnasnilegluglyglnalaileargtyrglnasnargpheleu
515520525
hisleuleuglylysiletyrtyrpheglyasnasnserlysalaval
530535540
thrglytrpglnthrileasnglylysvaltyrtyrphemetproasp
545550555560
thralametalaalaalaglyglyleuphegluileaspglyvalile
565570575
tyrphepheglyvalaspglyvallysalaproglyiletyrgly
580585590
<210>15
<211>379
<212>prt
<213>艰难梭菌
<400>15
glyphevalserileasnaspasnlyshistyrpheaspaspsergly
151015
valmetlysvalglytyrthrgluileaspglylyshisphetyrphe
202530
alagluasnglyglumetglnileglyvalpheasnthrgluaspgly
354045
phelystyrphealahishisasngluaspleuglyasngluglugly
505560
glugluilesertyrserglyileleuasnpheasnasnlysiletyr
65707580
tyrpheaspaspserphethralavalvalglytrplysaspleuglu
859095
aspglyserlystyrtyrpheaspgluaspthralaglualatyrile
100105110
glyleuserleuileasnaspglyglntyrtyrpheasnaspaspgly
115120125
ilemetglnvalglyphevalthrileasnasplysvalphetyrphe
130135140
seraspserglyileilegluserglyvalglnasnileaspaspasn
145150155160
tyrphetyrileaspaspasnglyilevalglnileglyvalpheasp
165170175
thrseraspglytyrlystyrphealaproalaasnthrvalasnasp
180185190
asniletyrglyglnalavalglutyrserglyleuvalargvalgly
195200205
gluaspvaltyrtyrpheglygluthrtyrthrilegluthrglytrp
210215220
iletyraspmetgluasngluserasplystyrtyrpheasnproglu
225230235240
thrlyslysalacyslysglyileasnleuileaspaspilelystyr
245250255
tyrpheaspglulysglyilemetargthrglyleuileserpheglu
260265270
asnasnasntyrtyrpheasngluasnglyglumetglnpheglytyr
275280285
ileasnilegluasplysmetphetyrpheglygluaspglyvalmet
290295300
glnileglyvalpheasnthrproaspglyphelystyrphealahis
305310315320
glnasnthrleuaspgluasnphegluglygluserileasntyrthr
325330335
glytrpleuaspleuaspglulysargtyrtyrphethraspglutyr
340345350
ilealaalathrglyservalileileaspglygluglutyrtyrphe
355360365
aspproaspthralaglnleuvalileserglu
370375
<210>16
<211>399
<212>prt
<213>艰难梭菌
<400>16
glyleuasnglnileglyasptyrlystyrtyrpheasnseraspgly
151015
valmetglnlysglyphevalserileasnaspasnlyshistyrphe
202530
aspaspserglyvalmetlysvalglytyrthrgluileaspglylys
354045
hisphetyrphealagluasnglyglumetglnileglyvalpheasn
505560
thrgluaspglyphelystyrphealahishisasngluaspleugly
65707580
asnglugluglyglugluilesertyrserglyileleuasnpheasn
859095
asnlysiletyrtyrpheaspaspserphethralavalvalglytrp
100105110
lysaspleugluaspglyserlystyrtyrpheaspgluaspthrala
115120125
glualatyrileglyleuserleuileasnaspglyglntyrtyrphe
130135140
asnaspaspglyilemetglnvalglyphevalthrileasnasplys
145150155160
valphetyrpheseraspserglyileilegluserglyvalglnasn
165170175
ileaspaspasntyrphetyrileaspaspasnglyilevalglnile
180185190
glyvalpheaspthrseraspglytyrlystyrphealaproalaasn
195200205
thrvalasnaspasniletyrglyglnalavalglutyrserglyleu
210215220
valargvalglygluaspvaltyrtyrpheglygluthrtyrthrile
225230235240
gluthrglytrpiletyraspmetgluasngluserasplystyrtyr
245250255
pheasnprogluthrlyslysalacyslysglyileasnleuileasp
260265270
aspilelystyrtyrpheaspglulysglyilemetargthrglyleu
275280285
ileserphegluasnasnasntyrtyrpheasngluasnglyglumet
290295300
glnpheglytyrileasnilegluasplysmetphetyrpheglyglu
305310315320
aspglyvalmetglnileglyvalpheasnthrproaspglyphelys
325330335
tyrphealahisglnasnthrleuaspgluasnphegluglygluser
340345350
ileasntyrthrglytrpleuaspleuaspglulysargtyrtyrphe
355360365
thraspglutyrilealaalathrglyservalileileaspglyglu
370375380
glutyrtyrpheaspproaspthralaglnleuvalileserglu
385390395
<210>17
<211>328
<212>prt
<213>艰难梭菌
<400>17
phealahishisasngluaspleuglyasnglugluglyglugluile
151015
sertyrserglyileleuasnpheasnasnlysiletyrtyrpheasp
202530
aspserphethralavalvalglytrplysaspleugluaspglyser
354045
lystyrtyrpheaspgluaspthralaglualatyrileglyleuser
505560
leuileasnaspglyglntyrtyrpheasnaspaspglyilemetgln
65707580
valglyphevalthrileasnasplysvalphetyrpheseraspser
859095
glyileilegluserglyvalglnasnileaspaspasntyrphetyr
100105110
ileaspaspasnglyilevalglnileglyvalpheaspthrserasp
115120125
glytyrlystyrphealaproalaasnthrvalasnaspasniletyr
130135140
glyglnalavalglutyrserglyleuvalargvalglygluaspval
145150155160
tyrtyrpheglygluthrtyrthrilegluthrglytrpiletyrasp
165170175
metgluasngluserasplystyrtyrpheasnprogluthrlyslys
180185190
alacyslysglyileasnleuileaspaspilelystyrtyrpheasp
195200205
glulysglyilemetargthrglyleuileserphegluasnasnasn
210215220
tyrtyrpheasngluasnglyglumetglnpheglytyrileasnile
225230235240
gluasplysmetphetyrpheglygluaspglyvalmetglnilegly
245250255
valpheasnthrproaspglyphelystyrphealahisglnasnthr
260265270
leuaspgluasnphegluglygluserileasntyrthrglytrpleu
275280285
aspleuaspglulysargtyrtyrphethraspglutyrilealaala
290295300
thrglyservalileileaspglygluglutyrtyrpheaspproasp
305310315320
thralaglnleuvalileserglu
325
<210>18
<211>490
<212>prt
<213>艰难梭菌
<400>18
glygluthrileileaspasplysasntyrtyrpheasnglnsergly
151015
valleuglnthrglyvalpheserthrgluaspglyphelystyrphe
202530
alaproalaasnthrleuaspgluasnleugluglyglualaileasp
354045
phethrglylysleuileileaspgluasniletyrtyrpheaspasp
505560
asntyrargglyalavalglutrplysgluleuaspglyglumethis
65707580
tyrpheserprogluthrglylysalaphelysglyleuasnglnile
859095
glyasptyrlystyrtyrpheasnseraspglyvalmetglnlysgly
100105110
phevalserileasnaspasnlyshistyrpheaspaspserglyval
115120125
metlysvalglytyrthrgluileaspglylyshisphetyrpheala
130135140
gluasnglyglumetglnileglyvalpheasnthrgluaspglyphe
145150155160
lystyrphealahishisasngluaspleuglyasnglugluglyglu
165170175
gluilesertyrserglyileleuasnpheasnasnlysiletyrtyr
180185190
pheaspaspserphethralavalvalglytrplysaspleugluasp
195200205
glyserlystyrtyrpheaspgluaspthralaglualatyrilegly
210215220
leuserleuileasnaspglyglntyrtyrpheasnaspaspglyile
225230235240
metglnvalglyphevalthrileasnasplysvalphetyrpheser
245250255
aspserglyileilegluserglyvalglnasnileaspaspasntyr
260265270
phetyrileaspaspasnglyilevalglnileglyvalpheaspthr
275280285
seraspglytyrlystyrphealaproalaasnthrvalasnaspasn
290295300
iletyrglyglnalavalglutyrserglyleuvalargvalglyglu
305310315320
aspvaltyrtyrpheglygluthrtyrthrilegluthrglytrpile
325330335
tyraspmetgluasngluserasplystyrtyrpheasnprogluthr
340345350
lyslysalacyslysglyileasnleuileaspaspilelystyrtyr
355360365
pheaspglulysglyilemetargthrglyleuileserphegluasn
370375380
asnasntyrtyrpheasngluasnglyglumetglnpheglytyrile
385390395400
asnilegluasplysmetphetyrpheglygluaspglyvalmetgln
405410415
ileglyvalpheasnthrproaspglyphelystyrphealahisgln
420425430
asnthrleuaspgluasnphegluglygluserileasntyrthrgly
435440445
trpleuaspleuaspglulysargtyrtyrphethraspglutyrile
450455460
alaalathrglyservalileileaspglygluglutyrtyrpheasp
465470475480
proaspthralaglnleuvalileserglu
485490
<210>19
<211>379
<212>prt
<213>艰难梭菌
<400>19
glyphevalserileasnaspasnlyshistyrpheaspaspsergly
151015
valmetlysvalglytyrthrgluileaspglylyshisphetyrphe
202530
alagluasnglyglumetglnileglyvalpheasnthrgluaspgly
354045
phelystyrphealahishisasngluaspleuglyasngluglugly
505560
glugluilesertyrserglyileleuasnpheasnasnlysiletyr
65707580
tyrpheaspaspserphethralavalvalglytrplysaspleuglu
859095
aspglyserlystyrtyrpheaspgluaspthralaglualatyrile
100105110
glyleuserleuileasnaspglyglntyrtyrpheasnaspaspgly
115120125
ilemetglnvalglyphevalthrileasnasplysvalphetyrphe
130135140
seraspserglyileilegluserglyvalglnasnileaspaspasn
145150155160
tyrphetyrileaspaspasnglyilevalglnileglyvalpheasp
165170175
thrseraspglytyrlystyrphealaproalaasnthrvalasnasp
180185190
asniletyrglyglnalavalglutyrserglyleuvalargvalgly
195200205
gluaspvaltyrtyrpheglygluthrtyrthrilegluthrglytrp
210215220
iletyraspmetgluasngluserasplystyrtyrpheasnproglu
225230235240
thrlyslysalacyslysglyileasnleuileaspaspilelystyr
245250255
tyrpheaspglulysglyilemetargthrglyleuileserpheglu
260265270
asnasnasntyrtyrpheasngluasnglyglumetglnpheglytyr
275280285
ileasnilegluasplysmetphetyrpheglygluaspglyvalmet
290295300
glnileglyvalpheasnthrproaspglyphelystyrphealahis
305310315320
glnasnthrleuaspgluasnphegluglygluserileasntyrthr
325330335
glytrpleuaspleuaspglulysargtyrtyrphethraspglutyr
340345350
ilealaalathrglyservalileileaspglygluglutyrtyrphe
355360365
aspproaspthralaglnleuvalileserglu
370375
<210>20
<211>3339
<212>dna
<213>艰难梭菌
<400>20
atggcaaccggttggcagaccatcgatggcaaaaaatattattttaataccaacaccgca60
attgcaagcaccggctataccattatcaacggcaaacacttttattttaacaccgacggc120
attatgcagattggtgtgtttaaaggtccgaacggctttgaatactttgcaccggcaaat180
accgatgccaataatattgaaggccaggccattctgtatcagaatgaatttctgaccctg240
aacggcaaaaaatactactttggcagcgatagcaaagcagttaccggttggcgcatcatc300
aacaataagaaatattacttcaacccgaataatgcaattgcagcaattcatctgtgcacc360
attaacaacgacaaatattatttcagctatgacggtattctgcagaatggctacattacc420
atcgaacgcaacaacttttatttcgatgccaacaacgaaagcaaaatggtgaccggtgtt480
ttcaaaggccctaatggttttgagtatttcgctccggcaaacacccataataacaacatt540
gaaggtcaggcgatcgtttatcagaacaaattcctgacgctgaatggtaagaaatactat600
ttcgataatgacagcaaagccgtgaccggctggcagacaattgacgggaagaaatattac660
tttaatctgaataccgcagaagcagcaaccggttggcaaacgatcgacggtaaaaagtac720
tacttcaacctgaacacagccgaagcagccacaggatggcagactattgatggaaaaaaa780
tactatttcaacaccaacacctttattgcatctaccggttataccagcattaacggtaaa840
catttctacttcaacaccgatggtatcatgcagatcggcgttttcaaaggtccaaatggt900
ttcgaatactttgcccctgccaatacagatgcaaataacatcgagggtcaggcaatcctg960
taccaaaacaaatttctgaccctgaatgggaaaaaatattactttggtagcgattctaaa1020
gccgttaccggtctgcgtaccattgatggtaaaaaatactactttaatacgaatacagcc1080
gttgcggttacaggctggcagaccattaacgggaaaaaatactattttaacacaaatacc1140
agcattgcctcaacgggttataccattatttcgggtaaacacttctactttaataccgat1200
ggtattatgcaaatcggagtctttaaaggacctgatgggttcgaatattttgcgcctgcg1260
aacactgatgcgaacaatatcgaaggacaggcaatccgctatcagaatcgctttctgtat1320
ctgcacgacaacatctattattttggcaacaattcaaaagcagccaccggctgggttaca1380
attgatggcaaccgctactatttcgaaccgaataccgcaatgggtgcaaatggctacaaa1440
accatcgataataaaaatttctattttcgcaacggtctgccgcagatcggggtatttaaa1500
ggtagcaacggcttcgaatacttcgctccagcgaatacggacgcgaacaatattgagggt1560
caagcgattcgttatcaaaaccgttttctgcatctgctgggcaaaatctactactttggc1620
aataacagtaaagcagttactggatggcagacaatcaatggtaaagtgtactattttatg1680
ccggataccgccatggcagcagccggtggtctgtttgaaattgatggcgtgatctatttt1740
tttggtgtggatggtgttaaagcaccgggaatatacggtggtaccggctttgtgaccgtg1800
ggtgatgataaatactatttcaatccgattaacggtggtgcagcgagcattggcgaaacc1860
atcatcgatgacaaaaactattatttcaaccagagcggtgtgctgcagaccggtgtgttt1920
agcaccgaagatggctttaaatattttgcgccagcgaacaccctggatgaaaacctggaa1980
ggcgaagcgattgattttaccggcaaactgatcatcgatgaaaacatctattacttcgat2040
gataactatcgtggtgcggtggaatggaaagaactggatggcgaaatgcattatttttct2100
ccggaaaccggtaaagcgtttaaaggcctgaaccagatcggcgattacaaatactacttc2160
aacagcgatggcgtgatgcagaaaggctttgtgagcatcaacgataacaaacactatttc2220
gatgatagcggtgtgatgaaagtgggctataccgaaattgatggcaaacatttctacttc2280
gcggaaaacggcgaaatgcagattggcgtgttcaataccgaagatggtttcaaatacttc2340
gcgcaccataacgaagatctgggtaacgaagaaggcgaagaaattagctatagcggcatc2400
ctgaacttcaacaacaaaatctactactttgatgatagctttaccgcggtggtgggctgg2460
aaagatctggaagatggcagcaaatattatttcgatgaagataccgcggaagcgtatatt2520
ggcctgagcctgattaacgatggccagtactattttaacgatgatggcattatgcaggtg2580
ggtttcgtgaccattaatgataaagtgttctatttcagcgatagcggcattattgaaagc2640
ggcgtgcagaacattgatgataactacttctacatcgatgataacggcattgtgcagatc2700
ggcgtttttgataccagcgatggctacaaatatttcgcaccggccaataccgtgaacgat2760
aacatttatggccaggcggtggaatatagcggtctggtgcgtgtgggcgaagatgtgtat2820
tatttcggcgaaacctataccatcgaaaccggctggatttatgatatggaaaacgaaagc2880
gataaatattactttaatccggaaacgaaaaaagcgtgcaaaggcattaacctgatcgat2940
gatatcaaatactattttgatgaaaaaggcattatgcgtaccggtctgattagcttcgaa3000
aacaacaactattacttcaacgaaaacggtgaaatgcagttcggctacatcaacatcgaa3060
gataaaatgttctacttcggcgaagatggtgttatgcagattggtgtttttaacaccccg3120
gatggcttcaaatactttgcccatcagaataccctggatgaaaatttcgaaggtgaaagc3180
attaactataccggctggctggatctggatgaaaaacgctactacttcaccgatgaatac3240
attgcggcgaccggcagcgtgattattgatggcgaagaatactacttcgatccggatacc3300
gcgcagctggtgattagcgaacatcatcatcatcaccat3339
<210>21
<211>1113
<212>prt
<213>艰难梭菌
<400>21
metalathrglytrpglnthrileaspglylyslystyrtyrpheasn
151015
thrasnthralailealaserthrglytyrthrileileasnglylys
202530
hisphetyrpheasnthraspglyilemetglnileglyvalphelys
354045
glyproasnglypheglutyrphealaproalaasnthraspalaasn
505560
asnilegluglyglnalaileleutyrglnasnglupheleuthrleu
65707580
asnglylyslystyrtyrpheglyseraspserlysalavalthrgly
859095
trpargileileasnasnlyslystyrtyrpheasnproasnasnala
100105110
ilealaalailehisleucysthrileasnasnasplystyrtyrphe
115120125
sertyraspglyileleuglnasnglytyrilethrilegluargasn
130135140
asnphetyrpheaspalaasnasngluserlysmetvalthrglyval
145150155160
phelysglyproasnglypheglutyrphealaproalaasnthrhis
165170175
asnasnasnilegluglyglnalailevaltyrglnasnlyspheleu
180185190
thrleuasnglylyslystyrtyrpheaspasnaspserlysalaval
195200205
thrglytrpglnthrileaspglylyslystyrtyrpheasnleuasn
210215220
thralaglualaalathrglytrpglnthrileaspglylyslystyr
225230235240
tyrpheasnleuasnthralaglualaalathrglytrpglnthrile
245250255
aspglylyslystyrtyrpheasnthrasnthrpheilealaserthr
260265270
glytyrthrserileasnglylyshisphetyrpheasnthraspgly
275280285
ilemetglnileglyvalphelysglyproasnglypheglutyrphe
290295300
alaproalaasnthraspalaasnasnilegluglyglnalaileleu
305310315320
tyrglnasnlyspheleuthrleuasnglylyslystyrtyrphegly
325330335
seraspserlysalavalthrglyleuargthrileaspglylyslys
340345350
tyrtyrpheasnthrasnthralavalalavalthrglytrpglnthr
355360365
ileasnglylyslystyrtyrpheasnthrasnthrserilealaser
370375380
thrglytyrthrileileserglylyshisphetyrpheasnthrasp
385390395400
glyilemetglnileglyvalphelysglyproaspglypheglutyr
405410415
phealaproalaasnthraspalaasnasnilegluglyglnalaile
420425430
argtyrglnasnargpheleutyrleuhisaspasniletyrtyrphe
435440445
glyasnasnserlysalaalathrglytrpvalthrileaspglyasn
450455460
argtyrtyrphegluproasnthralametglyalaasnglytyrlys
465470475480
thrileaspasnlysasnphetyrpheargasnglyleuproglnile
485490495
glyvalphelysglyserasnglypheglutyrphealaproalaasn
500505510
thraspalaasnasnilegluglyglnalaileargtyrglnasnarg
515520525
pheleuhisleuleuglylysiletyrtyrpheglyasnasnserlys
530535540
alavalthrglytrpglnthrileasnglylysvaltyrtyrphemet
545550555560
proaspthralametalaalaalaglyglyleuphegluileaspgly
565570575
valiletyrphepheglyvalaspglyvallysalaproglyiletyr
580585590
glyglythrglyphevalthrvalglyaspasplystyrtyrpheasn
595600605
proileasnglyglyalaalaserileglygluthrileileaspasp
610615620
lysasntyrtyrpheasnglnserglyvalleuglnthrglyvalphe
625630635640
serthrgluaspglyphelystyrphealaproalaasnthrleuasp
645650655
gluasnleugluglyglualaileaspphethrglylysleuileile
660665670
aspgluasniletyrtyrpheaspaspasntyrargglyalavalglu
675680685
trplysgluleuaspglyglumethistyrpheserprogluthrgly
690695700
lysalaphelysglyleuasnglnileglyasptyrlystyrtyrphe
705710715720
asnseraspglyvalmetglnlysglyphevalserileasnaspasn
725730735
lyshistyrpheaspaspserglyvalmetlysvalglytyrthrglu
740745750
ileaspglylyshisphetyrphealagluasnglyglumetglnile
755760765
glyvalpheasnthrgluaspglyphelystyrphealahishisasn
770775780
gluaspleuglyasnglugluglyglugluilesertyrserglyile
785790795800
leuasnpheasnasnlysiletyrtyrpheaspaspserphethrala
805810815
valvalglytrplysaspleugluaspglyserlystyrtyrpheasp
820825830
gluaspthralaglualatyrileglyleuserleuileasnaspgly
835840845
glntyrtyrpheasnaspaspglyilemetglnvalglyphevalthr
850855860
ileasnasplysvalphetyrpheseraspserglyileilegluser
865870875880
glyvalglnasnileaspaspasntyrphetyrileaspaspasngly
885890895
ilevalglnileglyvalpheaspthrseraspglytyrlystyrphe
900905910
alaproalaasnthrvalasnaspasniletyrglyglnalavalglu
915920925
tyrserglyleuvalargvalglygluaspvaltyrtyrpheglyglu
930935940
thrtyrthrilegluthrglytrpiletyraspmetgluasngluser
945950955960
asplystyrtyrpheasnprogluthrlyslysalacyslysglyile
965970975
asnleuileaspaspilelystyrtyrpheaspglulysglyilemet
980985990
argthrglyleuileserphegluasnasnasntyrtyrpheasnglu
99510001005
asnglyglumetglnpheglytyrileasnilegluasplysmetphe
101010151020
tyrpheglygluaspglyvalmetglnileglyvalpheasnthrpro
1025103010351040
aspglyphelystyrphealahisglnasnthrleuaspgluasnphe
104510501055
gluglygluserileasntyrthrglytrpleuaspleuaspglulys
106010651070
argtyrtyrphethraspglutyrilealaalathrglyservalile
107510801085
ileaspglygluglutyrtyrpheaspproaspthralaglnleuval
109010951100
ilesergluhishishishishishis
11051110
<210>22
<211>3324
<212>dna
<213>艰难梭菌
<400>22
atggcaaccggttggcagaccatcgatggcaaaaaatattattttaataccaacaccgca60
attgcaagcaccggctataccattatcaacggcaaacacttttattttaacaccgacggc120
attatgcagattggtgtgtttaaaggtccgaacggctttgaatactttgcaccggcaaat180
accgatgccaataatattgaaggccaggccattctgtatcagaatgaatttctgaccctg240
aacggcaaaaaatactactttggcagcgatagcaaagcagttaccggttggcgcatcatc300
aacaataagaaatattacttcaacccgaataatgcaattgcagcaattcatctgtgcacc360
attaacaacgacaaatattatttcagctatgacggtattctgcagaatggctacattacc420
atcgaacgcaacaacttttatttcgatgccaacaacgaaagcaaaatggtgaccggtgtt480
ttcaaaggccctaatggttttgagtatttcgctccggcaaacacccataataacaacatt540
gaaggtcaggcgatcgtttatcagaacaaattcctgacgctgaatggtaagaaatactat600
ttcgataatgacagcaaagccgtgaccggctggcagacaattgacgggaagaaatattac660
tttaatctgaataccgcagaagcagcaaccggttggcaaacgatcgacggtaaaaagtac720
tacttcaacctgaacacagccgaagcagccacaggatggcagactattgatggaaaaaaa780
tactatttcaacaccaacacctttattgcatctaccggttataccagcattaacggtaaa840
catttctacttcaacaccgatggtatcatgcagatcggcgttttcaaaggtccaaatggt900
ttcgaatactttgcccctgccaatacagatgcaaataacatcgagggtcaggcaatcctg960
taccaaaacaaatttctgaccctgaatgggaaaaaatattactttggtagcgattctaaa1020
gccgttaccggtctgcgtaccattgatggtaaaaaatactactttaatacgaatacagcc1080
gttgcggttacaggctggcagaccattaacgggaaaaaatactattttaacacaaatacc1140
agcattgcctcaacgggttataccattatttcgggtaaacacttctactttaataccgat1200
ggtattatgcaaatcggagtctttaaaggacctgatgggttcgaatattttgcgcctgcg1260
aacactgatgcgaacaatatcgaaggacaggcaatccgctatcagaatcgctttctgtat1320
ctgcacgacaacatctattattttggcaacaattcaaaagcagccaccggctgggttaca1380
attgatggcaaccgctactatttcgaaccgaataccgcaatgggtgcaaatggctacaaa1440
accatcgataataaaaatttctattttcgcaacggtctgccgcagatcggggtatttaaa1500
ggtagcaacggcttcgaatacttcgctccagcgaatacggacgcgaacaatattgagggt1560
caagcgattcgttatcaaaaccgttttctgcatctgctgggcaaaatctactactttggc1620
aataacagtaaagcagttactggatggcagacaatcaatggtaaagtgtactattttatg1680
ccggataccgccatggcagcagccggtggtctgtttgaaattgatggcgtgatctatttt1740
tttggtgtggatggtgttaaagcagttaccggctttgtgaccgtgggtgatgataaatac1800
tatttcaatccgattaacggtggtgcagcgagcattggcgaaaccatcatcgatgacaaa1860
aactattatttcaaccagagcggtgtgctgcagaccggtgtgtttagcaccgaagatggc1920
tttaaatattttgcgccagcgaacaccctggatgaaaacctggaaggcgaagcgattgat1980
tttaccggcaaactgatcatcgatgaaaacatctattacttcgatgataactatcgtggt2040
gcggtggaatggaaagaactggatggcgaaatgcattatttttctccggaaaccggtaaa2100
gcgtttaaaggcctgaaccagatcggcgattacaaatactacttcaacagcgatggcgtg2160
atgcagaaaggctttgtgagcatcaacgataacaaacactatttcgatgatagcggtgtg2220
atgaaagtgggctataccgaaattgatggcaaacatttctacttcgcggaaaacggcgaa2280
atgcagattggcgtgttcaataccgaagatggtttcaaatacttcgcgcaccataacgaa2340
gatctgggtaacgaagaaggcgaagaaattagctatagcggcatcctgaacttcaacaac2400
aaaatctactactttgatgatagctttaccgcggtggtgggctggaaagatctggaagat2460
ggcagcaaatattatttcgatgaagataccgcggaagcgtatattggcctgagcctgatt2520
aacgatggccagtactattttaacgatgatggcattatgcaggtgggtttcgtgaccatt2580
aatgataaagtgttctatttcagcgatagcggcattattgaaagcggcgtgcagaacatt2640
gatgataactacttctacatcgatgataacggcattgtgcagatcggcgtttttgatacc2700
agcgatggctacaaatatttcgcaccggccaataccgtgaacgataacatttatggccag2760
gcggtggaatatagcggtctggtgcgtgtgggcgaagatgtgtattatttcggcgaaacc2820
tataccatcgaaaccggctggatttatgatatggaaaacgaaagcgataaatattacttt2880
aatccggaaacgaaaaaagcgtgcaaaggcattaacctgatcgatgatatcaaatactat2940
tttgatgaaaaaggcattatgcgtaccggtctgattagcttcgaaaacaacaactattac3000
ttcaacgaaaacggtgaaatgcagttcggctacatcaacatcgaagataaaatgttctac3060
ttcggcgaagatggtgttatgcagattggtgtttttaacaccccggatggcttcaaatac3120
tttgcccatcagaataccctggatgaaaatttcgaaggtgaaagcattaactataccggc3180
tggctggatctggatgaaaaacgctactacttcaccgatgaatacattgcggcgaccggc3240
agcgtgattattgatggcgaagaatactacttcgatccggataccgcgcagctggtgatt3300
agcgaacatcatcatcatcaccat3324
<210>23
<211>1108
<212>prt
<213>艰难梭菌
<400>23
metalathrglytrpglnthrileaspglylyslystyrtyrpheasn
151015
thrasnthralailealaserthrglytyrthrileileasnglylys
202530
hisphetyrpheasnthraspglyilemetglnileglyvalphelys
354045
glyproasnglypheglutyrphealaproalaasnthraspalaasn
505560
asnilegluglyglnalaileleutyrglnasnglupheleuthrleu
65707580
asnglylyslystyrtyrpheglyseraspserlysalavalthrgly
859095
trpargileileasnasnlyslystyrtyrpheasnproasnasnala
100105110
ilealaalailehisleucysthrileasnasnasplystyrtyrphe
115120125
sertyraspglyileleuglnasnglytyrilethrilegluargasn
130135140
asnphetyrpheaspalaasnasngluserlysmetvalthrglyval
145150155160
phelysglyproasnglypheglutyrphealaproalaasnthrhis
165170175
asnasnasnilegluglyglnalailevaltyrglnasnlyspheleu
180185190
thrleuasnglylyslystyrtyrpheaspasnaspserlysalaval
195200205
thrglytrpglnthrileaspglylyslystyrtyrpheasnleuasn
210215220
thralaglualaalathrglytrpglnthrileaspglylyslystyr
225230235240
tyrpheasnleuasnthralaglualaalathrglytrpglnthrile
245250255
aspglylyslystyrtyrpheasnthrasnthrpheilealaserthr
260265270
glytyrthrserileasnglylyshisphetyrpheasnthraspgly
275280285
ilemetglnileglyvalphelysglyproasnglypheglutyrphe
290295300
alaproalaasnthraspalaasnasnilegluglyglnalaileleu
305310315320
tyrglnasnlyspheleuthrleuasnglylyslystyrtyrphegly
325330335
seraspserlysalavalthrglyleuargthrileaspglylyslys
340345350
tyrtyrpheasnthrasnthralavalalavalthrglytrpglnthr
355360365
ileasnglylyslystyrtyrpheasnthrasnthrserilealaser
370375380
thrglytyrthrileileserglylyshisphetyrpheasnthrasp
385390395400
glyilemetglnileglyvalphelysglyproaspglypheglutyr
405410415
phealaproalaasnthraspalaasnasnilegluglyglnalaile
420425430
argtyrglnasnargpheleutyrleuhisaspasniletyrtyrphe
435440445
glyasnasnserlysalaalathrglytrpvalthrileaspglyasn
450455460
argtyrtyrphegluproasnthralametglyalaasnglytyrlys
465470475480
thrileaspasnlysasnphetyrpheargasnglyleuproglnile
485490495
glyvalphelysglyserasnglypheglutyrphealaproalaasn
500505510
thraspalaasnasnilegluglyglnalaileargtyrglnasnarg
515520525
pheleuhisleuleuglylysiletyrtyrpheglyasnasnserlys
530535540
alavalthrglytrpglnthrileasnglylysvaltyrtyrphemet
545550555560
proaspthralametalaalaalaglyglyleuphegluileaspgly
565570575
valiletyrphepheglyvalaspglyvallysalavalthrglyphe
580585590
valthrvalglyaspasplystyrtyrpheasnproileasnglygly
595600605
alaalaserileglygluthrileileaspasplysasntyrtyrphe
610615620
asnglnserglyvalleuglnthrglyvalpheserthrgluaspgly
625630635640
phelystyrphealaproalaasnthrleuaspgluasnleuglugly
645650655
glualaileaspphethrglylysleuileileaspgluasniletyr
660665670
tyrpheaspaspasntyrargglyalavalglutrplysgluleuasp
675680685
glyglumethistyrpheserprogluthrglylysalaphelysgly
690695700
leuasnglnileglyasptyrlystyrtyrpheasnseraspglyval
705710715720
metglnlysglyphevalserileasnaspasnlyshistyrpheasp
725730735
aspserglyvalmetlysvalglytyrthrgluileaspglylyshis
740745750
phetyrphealagluasnglyglumetglnileglyvalpheasnthr
755760765
gluaspglyphelystyrphealahishisasngluaspleuglyasn
770775780
glugluglyglugluilesertyrserglyileleuasnpheasnasn
785790795800
lysiletyrtyrpheaspaspserphethralavalvalglytrplys
805810815
aspleugluaspglyserlystyrtyrpheaspgluaspthralaglu
820825830
alatyrileglyleuserleuileasnaspglyglntyrtyrpheasn
835840845
aspaspglyilemetglnvalglyphevalthrileasnasplysval
850855860
phetyrpheseraspserglyileilegluserglyvalglnasnile
865870875880
aspaspasntyrphetyrileaspaspasnglyilevalglnilegly
885890895
valpheaspthrseraspglytyrlystyrphealaproalaasnthr
900905910
valasnaspasniletyrglyglnalavalglutyrserglyleuval
915920925
argvalglygluaspvaltyrtyrpheglygluthrtyrthrileglu
930935940
thrglytrpiletyraspmetgluasngluserasplystyrtyrphe
945950955960
asnprogluthrlyslysalacyslysglyileasnleuileaspasp
965970975
ilelystyrtyrpheaspglulysglyilemetargthrglyleuile
980985990
serphegluasnasnasntyrtyrpheasngluasnglyglumetgln
99510001005
pheglytyrileasnilegluasplysmetphetyrpheglygluasp
101010151020
glyvalmetglnileglyvalpheasnthrproaspglyphelystyr
1025103010351040
phealahisglnasnthrleuaspgluasnphegluglygluserile
104510501055
asntyrthrglytrpleuaspleuaspglulysargtyrtyrphethr
106010651070
aspglutyrilealaalathrglyservalileileaspglygluglu
107510801085
tyrtyrpheaspproaspthralaglnleuvalilesergluhishis
109010951100
hishishishis
1105
<210>24
<211>3387
<212>dna
<213>艰难梭菌
<400>24
atggcaaccggttggcagaccatcgatggcaaaaaatattattttaataccaacaccgca60
attgcaagcaccggctataccattatcaacggcaaacacttttattttaacaccgacggc120
attatgcagattggtgtgtttaaaggtccgaacggctttgaatactttgcaccggcaaat180
accgatgccaataatattgaaggccaggccattctgtatcagaatgaatttctgaccctg240
aacggcaaaaaatactactttggcagcgatagcaaagcagttaccggttggcgcatcatc300
aacaataagaaatattacttcaacccgaataatgcaattgcagcaattcatctgtgcacc360
attaacaacgacaaatattatttcagctatgacggtattctgcagaatggctacattacc420
atcgaacgcaacaacttttatttcgatgccaacaacgaaagcaaaatggtgaccggtgtt480
ttcaaaggccctaatggttttgagtatttcgctccggcaaacacccataataacaacatt540
gaaggtcaggcgatcgtttatcagaacaaattcctgacgctgaatggtaagaaatactat600
ttcgataatgacagcaaagccgtgaccggctggcagacaattgacgggaagaaatattac660
tttaatctgaataccgcagaagcagcaaccggttggcaaacgatcgacggtaaaaagtac720
tacttcaacctgaacacagccgaagcagccacaggatggcagactattgatggaaaaaaa780
tactatttcaacaccaacacctttattgcatctaccggttataccagcattaacggtaaa840
catttctacttcaacaccgatggtatcatgcagatcggcgttttcaaaggtccaaatggt900
ttcgaatactttgcccctgccaatacagatgcaaataacatcgagggtcaggcaatcctg960
taccaaaacaaatttctgaccctgaatgggaaaaaatattactttggtagcgattctaaa1020
gccgttaccggtctgcgtaccattgatggtaaaaaatactactttaatacgaatacagcc1080
gttgcggttacaggctggcagaccattaacgggaaaaaatactattttaacacaaatacc1140
agcattgcctcaacgggttataccattatttcgggtaaacacttctactttaataccgat1200
ggtattatgcaaatcggagtctttaaaggacctgatgggttcgaatattttgcgcctgcg1260
aacactgatgcgaacaatatcgaaggacaggcaatccgctatcagaatcgctttctgtat1320
ctgcacgacaacatctattattttggcaacaattcaaaagcagccaccggctgggttaca1380
attgatggcaaccgctactatttcgaaccgaataccgcaatgggtgcaaatggctacaaa1440
accatcgataataaaaatttctattttcgcaacggtctgccgcagatcggggtatttaaa1500
ggtagcaacggcttcgaatacttcgctccagcgaatacggacgcgaacaatattgagggt1560
caagcgattcgttatcaaaaccgttttctgcatctgctgggcaaaatctactactttggc1620
aataacagtaaagcagttactggatggcagacaatcaatggtaaagtgtactattttatg1680
ccggataccgccatggcagcagccggtggtctgtttgaaattgatggcgtgatctatttt1740
tttggtgtggatggtgttaaagcagtgagcggtctgatttatattaacgatagcctgtat1800
tactttaaaccaccggtgaataacctgattaccggctttgtgaccgtgggtgatgataaa1860
tactatttcaatccgattaacggtggtgcagcgagcattggcgaaaccatcatcgatgac1920
aaaaactattatttcaaccagagcggtgtgctgcagaccggtgtgtttagcaccgaagat1980
ggctttaaatattttgcgccagcgaacaccctggatgaaaacctggaaggcgaagcgatt2040
gattttaccggcaaactgatcatcgatgaaaacatctattacttcgatgataactatcgt2100
ggtgcggtggaatggaaagaactggatggcgaaatgcattatttttctccggaaaccggt2160
aaagcgtttaaaggcctgaaccagatcggcgattacaaatactacttcaacagcgatggc2220
gtgatgcagaaaggctttgtgagcatcaacgataacaaacactatttcgatgatagcggt2280
gtgatgaaagtgggctataccgaaattgatggcaaacatttctacttcgcggaaaacggc2340
gaaatgcagattggcgtgttcaataccgaagatggtttcaaatacttcgcgcaccataac2400
gaagatctgggtaacgaagaaggcgaagaaattagctatagcggcatcctgaacttcaac2460
aacaaaatctactactttgatgatagctttaccgcggtggtgggctggaaagatctggaa2520
gatggcagcaaatattatttcgatgaagataccgcggaagcgtatattggcctgagcctg2580
attaacgatggccagtactattttaacgatgatggcattatgcaggtgggtttcgtgacc2640
attaatgataaagtgttctatttcagcgatagcggcattattgaaagcggcgtgcagaac2700
attgatgataactacttctacatcgatgataacggcattgtgcagatcggcgtttttgat2760
accagcgatggctacaaatatttcgcaccggccaataccgtgaacgataacatttatggc2820
caggcggtggaatatagcggtctggtgcgtgtgggcgaagatgtgtattatttcggcgaa2880
acctataccatcgaaaccggctggatttatgatatggaaaacgaaagcgataaatattac2940
tttaatccggaaacgaaaaaagcgtgcaaaggcattaacctgatcgatgatatcaaatac3000
tattttgatgaaaaaggcattatgcgtaccggtctgattagcttcgaaaacaacaactat3060
tacttcaacgaaaacggtgaaatgcagttcggctacatcaacatcgaagataaaatgttc3120
tacttcggcgaagatggtgttatgcagattggtgtttttaacaccccggatggcttcaaa3180
tactttgcccatcagaataccctggatgaaaatttcgaaggtgaaagcattaactatacc3240
ggctggctggatctggatgaaaaacgctactacttcaccgatgaatacattgcggcgacc3300
ggcagcgtgattattgatggcgaagaatactacttcgatccggataccgcgcagctggtg3360
attagcgaacatcatcatcatcaccat3387
<210>25
<211>1129
<212>prt
<213>艰难梭菌
<400>25
metalathrglytrpglnthrileaspglylyslystyrtyrpheasn
151015
thrasnthralailealaserthrglytyrthrileileasnglylys
202530
hisphetyrpheasnthraspglyilemetglnileglyvalphelys
354045
glyproasnglypheglutyrphealaproalaasnthraspalaasn
505560
asnilegluglyglnalaileleutyrglnasnglupheleuthrleu
65707580
asnglylyslystyrtyrpheglyseraspserlysalavalthrgly
859095
trpargileileasnasnlyslystyrtyrpheasnproasnasnala
100105110
ilealaalailehisleucysthrileasnasnasplystyrtyrphe
115120125
sertyraspglyileleuglnasnglytyrilethrilegluargasn
130135140
asnphetyrpheaspalaasnasngluserlysmetvalthrglyval
145150155160
phelysglyproasnglypheglutyrphealaproalaasnthrhis
165170175
asnasnasnilegluglyglnalailevaltyrglnasnlyspheleu
180185190
thrleuasnglylyslystyrtyrpheaspasnaspserlysalaval
195200205
thrglytrpglnthrileaspglylyslystyrtyrpheasnleuasn
210215220
thralaglualaalathrglytrpglnthrileaspglylyslystyr
225230235240
tyrpheasnleuasnthralaglualaalathrglytrpglnthrile
245250255
aspglylyslystyrtyrpheasnthrasnthrpheilealaserthr
260265270
glytyrthrserileasnglylyshisphetyrpheasnthraspgly
275280285
ilemetglnileglyvalphelysglyproasnglypheglutyrphe
290295300
alaproalaasnthraspalaasnasnilegluglyglnalaileleu
305310315320
tyrglnasnlyspheleuthrleuasnglylyslystyrtyrphegly
325330335
seraspserlysalavalthrglyleuargthrileaspglylyslys
340345350
tyrtyrpheasnthrasnthralavalalavalthrglytrpglnthr
355360365
ileasnglylyslystyrtyrpheasnthrasnthrserilealaser
370375380
thrglytyrthrileileserglylyshisphetyrpheasnthrasp
385390395400
glyilemetglnileglyvalphelysglyproaspglypheglutyr
405410415
phealaproalaasnthraspalaasnasnilegluglyglnalaile
420425430
argtyrglnasnargpheleutyrleuhisaspasniletyrtyrphe
435440445
glyasnasnserlysalaalathrglytrpvalthrileaspglyasn
450455460
argtyrtyrphegluproasnthralametglyalaasnglytyrlys
465470475480
thrileaspasnlysasnphetyrpheargasnglyleuproglnile
485490495
glyvalphelysglyserasnglypheglutyrphealaproalaasn
500505510
thraspalaasnasnilegluglyglnalaileargtyrglnasnarg
515520525
pheleuhisleuleuglylysiletyrtyrpheglyasnasnserlys
530535540
alavalthrglytrpglnthrileasnglylysvaltyrtyrphemet
545550555560
proaspthralametalaalaalaglyglyleuphegluileaspgly
565570575
valiletyrphepheglyvalaspglyvallysalavalserglyleu
580585590
iletyrileasnaspserleutyrtyrphelysproprovalasnasn
595600605
leuilethrglyphevalthrvalglyaspasplystyrtyrpheasn
610615620
proileasnglyglyalaalaserileglygluthrileileaspasp
625630635640
lysasntyrtyrpheasnglnserglyvalleuglnthrglyvalphe
645650655
serthrgluaspglyphelystyrphealaproalaasnthrleuasp
660665670
gluasnleugluglyglualaileaspphethrglylysleuileile
675680685
aspgluasniletyrtyrpheaspaspasntyrargglyalavalglu
690695700
trplysgluleuaspglyglumethistyrpheserprogluthrgly
705710715720
lysalaphelysglyleuasnglnileglyasptyrlystyrtyrphe
725730735
asnseraspglyvalmetglnlysglyphevalserileasnaspasn
740745750
lyshistyrpheaspaspserglyvalmetlysvalglytyrthrglu
755760765
ileaspglylyshisphetyrphealagluasnglyglumetglnile
770775780
glyvalpheasnthrgluaspglyphelystyrphealahishisasn
785790795800
gluaspleuglyasnglugluglyglugluilesertyrserglyile
805810815
leuasnpheasnasnlysiletyrtyrpheaspaspserphethrala
820825830
valvalglytrplysaspleugluaspglyserlystyrtyrpheasp
835840845
gluaspthralaglualatyrileglyleuserleuileasnaspgly
850855860
glntyrtyrpheasnaspaspglyilemetglnvalglyphevalthr
865870875880
ileasnasplysvalphetyrpheseraspserglyileilegluser
885890895
glyvalglnasnileaspaspasntyrphetyrileaspaspasngly
900905910
ilevalglnileglyvalpheaspthrseraspglytyrlystyrphe
915920925
alaproalaasnthrvalasnaspasniletyrglyglnalavalglu
930935940
tyrserglyleuvalargvalglygluaspvaltyrtyrpheglyglu
945950955960
thrtyrthrilegluthrglytrpiletyraspmetgluasngluser
965970975
asplystyrtyrpheasnprogluthrlyslysalacyslysglyile
980985990
asnleuileaspaspilelystyrtyrpheaspglulysglyilemet
99510001005
argthrglyleuileserphegluasnasnasntyrtyrpheasnglu
101010151020
asnglyglumetglnpheglytyrileasnilegluasplysmetphe
1025103010351040
tyrpheglygluaspglyvalmetglnileglyvalpheasnthrpro
104510501055
aspglyphelystyrphealahisglnasnthrleuaspgluasnphe
106010651070
gluglygluserileasntyrthrglytrpleuaspleuaspglulys
107510801085
argtyrtyrphethraspglutyrilealaalathrglyservalile
109010951100
ileaspglygluglutyrtyrpheaspproaspthralaglnleuval
1105111011151120
ilesergluhishishishishishis
1125
<210>26
<211>2985
<212>dna
<213>艰难梭菌
<400>26
atggcaaccggttggcagaccatcgatggcaaaaaatattattttaataccaacaccgca60
attgcaagcaccggctataccattatcaacggcaaacacttttattttaacaccgacggc120
attatgcagattggtgtgtttaaaggtccgaacggctttgaatactttgcaccggcaaat180
accgatgccaataatattgaaggccaggccattctgtatcagaatgaatttctgaccctg240
aacggcaaaaaatactactttggcagcgatagcaaagcagttaccggttggcgcatcatc300
aacaataagaaatattacttcaacccgaataatgcaattgcagcaattcatctgtgcacc360
attaacaacgacaaatattatttcagctatgacggtattctgcagaatggctacattacc420
atcgaacgcaacaacttttatttcgatgccaacaacgaaagcaaaatggtgaccggtgtt480
ttcaaaggccctaatggttttgagtatttcgctccggcaaacacccataataacaacatt540
gaaggtcaggcgatcgtttatcagaacaaattcctgacgctgaatggtaagaaatactat600
ttcgataatgacagcaaagccgtgaccggctggcagacaattgacgggaagaaatattac660
tttaatctgaataccgcagaagcagcaaccggttggcaaacgatcgacggtaaaaagtac720
tacttcaacctgaacacagccgaagcagccacaggatggcagactattgatggaaaaaaa780
tactatttcaacaccaacacctttattgcatctaccggttataccagcattaacggtaaa840
catttctacttcaacaccgatggtatcatgcagatcggcgttttcaaaggtccaaatggt900
ttcgaatactttgcccctgccaatacagatgcaaataacatcgagggtcaggcaatcctg960
taccaaaacaaatttctgaccctgaatgggaaaaaatattactttggtagcgattctaaa1020
gccgttaccggtctgcgtaccattgatggtaaaaaatactactttaatacgaatacagcc1080
gttgcggttacaggctggcagaccattaacgggaaaaaatactattttaacacaaatacc1140
agcattgcctcaacgggttataccattatttcgggtaaacacttctactttaataccgat1200
ggtattatgcaaatcggagtctttaaaggacctgatgggttcgaatattttgcgcctgcg1260
aacactgatgcgaacaatatcgaaggacaggcaatccgctatcagaatcgctttctgtat1320
ctgcacgacaacatctattattttggcaacaattcaaaagcagccaccggctgggttaca1380
attgatggcaaccgctactatttcgaaccgaataccgcaatgggtgcaaatggctacaaa1440
accatcgataataaaaatttctattttcgcaacggtctgccgcagatcggggtatttaaa1500
ggtagcaacggcttcgaatacttcgctccagcgaatacggacgcgaacaatattgagggt1560
caagcgattcgttatcaaaaccgttttctgcatctgctgggcaaaatctactactttggc1620
aataacagtaaagcagttactggatggcagacaatcaatggtaaagtgtactattttatg1680
ccggataccgccatggcagcagccggtggtctgtttgaaattgatggcgtgatctatttt1740
tttggtgtggatggtgttaaagcagtgaaaggcctgaaccagatcggcgattacaaatac1800
tacttcaacagcgatggcgtgatgcagaaaggctttgtgagcatcaacgataacaaacac1860
tatttcgatgatagcggtgtgatgaaagtgggctataccgaaattgatggcaaacatttc1920
tacttcgcggaaaacggcgaaatgcagattggcgtgttcaataccgaagatggtttcaaa1980
tacttcgcgcaccataacgaagatctgggtaacgaagaaggcgaagaaattagctatagc2040
ggcatcctgaacttcaacaacaaaatctactactttgatgatagctttaccgcggtggtg2100
ggctggaaagatctggaagatggcagcaaatattatttcgatgaagataccgcggaagcg2160
tatattggcctgagcctgattaacgatggccagtactattttaacgatgatggcattatg2220
caggtgggtttcgtgaccattaatgataaagtgttctatttcagcgatagcggcattatt2280
gaaagcggcgtgcagaacattgatgataactacttctacatcgatgataacggcattgtg2340
cagatcggcgtttttgataccagcgatggctacaaatatttcgcaccggccaataccgtg2400
aacgataacatttatggccaggcggtggaatatagcggtctggtgcgtgtgggcgaagat2460
gtgtattatttcggcgaaacctataccatcgaaaccggctggatttatgatatggaaaac2520
gaaagcgataaatattactttaatccggaaacgaaaaaagcgtgcaaaggcattaacctg2580
atcgatgatatcaaatactattttgatgaaaaaggcattatgcgtaccggtctgattagc2640
ttcgaaaacaacaactattacttcaacgaaaacggtgaaatgcagttcggctacatcaac2700
atcgaagataaaatgttctacttcggcgaagatggtgttatgcagattggtgtttttaac2760
accccggatggcttcaaatactttgcccatcagaataccctggatgaaaatttcgaaggt2820
gaaagcattaactataccggctggctggatctggatgaaaaacgctactacttcaccgat2880
gaatacattgcggcgaccggcagcgtgattattgatggcgaagaatactacttcgatccg2940
gataccgcgcagctggtgattagcgaacatcatcatcatcaccat2985
<210>27
<211>995
<212>prt
<213>艰难梭菌
<400>27
metalathrglytrpglnthrileaspglylyslystyrtyrpheasn
151015
thrasnthralailealaserthrglytyrthrileileasnglylys
202530
hisphetyrpheasnthraspglyilemetglnileglyvalphelys
354045
glyproasnglypheglutyrphealaproalaasnthraspalaasn
505560
asnilegluglyglnalaileleutyrglnasnglupheleuthrleu
65707580
asnglylyslystyrtyrpheglyseraspserlysalavalthrgly
859095
trpargileileasnasnlyslystyrtyrpheasnproasnasnala
100105110
ilealaalailehisleucysthrileasnasnasplystyrtyrphe
115120125
sertyraspglyileleuglnasnglytyrilethrilegluargasn
130135140
asnphetyrpheaspalaasnasngluserlysmetvalthrglyval
145150155160
phelysglyproasnglypheglutyrphealaproalaasnthrhis
165170175
asnasnasnilegluglyglnalailevaltyrglnasnlyspheleu
180185190
thrleuasnglylyslystyrtyrpheaspasnaspserlysalaval
195200205
thrglytrpglnthrileaspglylyslystyrtyrpheasnleuasn
210215220
thralaglualaalathrglytrpglnthrileaspglylyslystyr
225230235240
tyrpheasnleuasnthralaglualaalathrglytrpglnthrile
245250255
aspglylyslystyrtyrpheasnthrasnthrpheilealaserthr
260265270
glytyrthrserileasnglylyshisphetyrpheasnthraspgly
275280285
ilemetglnileglyvalphelysglyproasnglypheglutyrphe
290295300
alaproalaasnthraspalaasnasnilegluglyglnalaileleu
305310315320
tyrglnasnlyspheleuthrleuasnglylyslystyrtyrphegly
325330335
seraspserlysalavalthrglyleuargthrileaspglylyslys
340345350
tyrtyrpheasnthrasnthralavalalavalthrglytrpglnthr
355360365
ileasnglylyslystyrtyrpheasnthrasnthrserilealaser
370375380
thrglytyrthrileileserglylyshisphetyrpheasnthrasp
385390395400
glyilemetglnileglyvalphelysglyproaspglypheglutyr
405410415
phealaproalaasnthraspalaasnasnilegluglyglnalaile
420425430
argtyrglnasnargpheleutyrleuhisaspasniletyrtyrphe
435440445
glyasnasnserlysalaalathrglytrpvalthrileaspglyasn
450455460
argtyrtyrphegluproasnthralametglyalaasnglytyrlys
465470475480
thrileaspasnlysasnphetyrpheargasnglyleuproglnile
485490495
glyvalphelysglyserasnglypheglutyrphealaproalaasn
500505510
thraspalaasnasnilegluglyglnalaileargtyrglnasnarg
515520525
pheleuhisleuleuglylysiletyrtyrpheglyasnasnserlys
530535540
alavalthrglytrpglnthrileasnglylysvaltyrtyrphemet
545550555560
proaspthralametalaalaalaglyglyleuphegluileaspgly
565570575
valiletyrphepheglyvalaspglyvallysalavallysglyleu
580585590
asnglnileglyasptyrlystyrtyrpheasnseraspglyvalmet
595600605
glnlysglyphevalserileasnaspasnlyshistyrpheaspasp
610615620
serglyvalmetlysvalglytyrthrgluileaspglylyshisphe
625630635640
tyrphealagluasnglyglumetglnileglyvalpheasnthrglu
645650655
aspglyphelystyrphealahishisasngluaspleuglyasnglu
660665670
gluglyglugluilesertyrserglyileleuasnpheasnasnlys
675680685
iletyrtyrpheaspaspserphethralavalvalglytrplysasp
690695700
leugluaspglyserlystyrtyrpheaspgluaspthralagluala
705710715720
tyrileglyleuserleuileasnaspglyglntyrtyrpheasnasp
725730735
aspglyilemetglnvalglyphevalthrileasnasplysvalphe
740745750
tyrpheseraspserglyileilegluserglyvalglnasnileasp
755760765
aspasntyrphetyrileaspaspasnglyilevalglnileglyval
770775780
pheaspthrseraspglytyrlystyrphealaproalaasnthrval
785790795800
asnaspasniletyrglyglnalavalglutyrserglyleuvalarg
805810815
valglygluaspvaltyrtyrpheglygluthrtyrthrilegluthr
820825830
glytrpiletyraspmetgluasngluserasplystyrtyrpheasn
835840845
progluthrlyslysalacyslysglyileasnleuileaspaspile
850855860
lystyrtyrpheaspglulysglyilemetargthrglyleuileser
865870875880
phegluasnasnasntyrtyrpheasngluasnglyglumetglnphe
885890895
glytyrileasnilegluasplysmetphetyrpheglygluaspgly
900905910
valmetglnileglyvalpheasnthrproaspglyphelystyrphe
915920925
alahisglnasnthrleuaspgluasnphegluglygluserileasn
930935940
tyrthrglytrpleuaspleuaspglulysargtyrtyrphethrasp
945950955960
glutyrilealaalathrglyservalileileaspglygluglutyr
965970975
tyrpheaspproaspthralaglnleuvalilesergluhishishis
980985990
hishishis
995
<210>28
<211>593
<212>prt
<213>艰难梭菌
<400>28
metalathrglytrpglnthrileaspglylyslystyrtyrpheasn
151015
thrasnthralailealaserthrglytyrthrileileasnglylys
202530
hisphetyrpheasnthraspglyilemetglnileglyvalphelys
354045
glyproasnglypheglutyrphealaproalaasnthraspalaasn
505560
asnilegluglyglnalaileleutyrglnasnglupheleuthrleu
65707580
asnglylyslystyrtyrpheglyseraspserlysalavalthrgly
859095
trpargileileasnasnlyslystyrtyrpheasnproasnasnala
100105110
ilealaalailehisleucysthrileasnasnasplystyrtyrphe
115120125
sertyraspglyileleuglnasnglytyrilethrilegluargasn
130135140
asnphetyrpheaspalaasnasngluserlysmetvalthrglyval
145150155160
phelysglyproasnglypheglutyrphealaproalaasnthrhis
165170175
asnasnasnilegluglyglnalailevaltyrglnasnlyspheleu
180185190
thrleuasnglylyslystyrtyrpheaspasnaspserlysalaval
195200205
thrglytrpglnthrileaspglylyslystyrtyrpheasnleuasn
210215220
thralaglualaalathrglytrpglnthrileaspglylyslystyr
225230235240
tyrpheasnleuasnthralaglualaalathrglytrpglnthrile
245250255
aspglylyslystyrtyrpheasnthrasnthrpheilealaserthr
260265270
glytyrthrserileasnglylyshisphetyrpheasnthraspgly
275280285
ilemetglnileglyvalphelysglyproasnglypheglutyrphe
290295300
alaproalaasnthraspalaasnasnilegluglyglnalaileleu
305310315320
tyrglnasnlyspheleuthrleuasnglylyslystyrtyrphegly
325330335
seraspserlysalavalthrglyleuargthrileaspglylyslys
340345350
tyrtyrpheasnthrasnthralavalalavalthrglytrpglnthr
355360365
ileasnglylyslystyrtyrpheasnthrasnthrserilealaser
370375380
thrglytyrthrileileserglylyshisphetyrpheasnthrasp
385390395400
glyilemetglnileglyvalphelysglyproaspglypheglutyr
405410415
phealaproalaasnthraspalaasnasnilegluglyglnalaile
420425430
argtyrglnasnargpheleutyrleuhisaspasniletyrtyrphe
435440445
glyasnasnserlysalaalathrglytrpvalthrileaspglyasn
450455460
argtyrtyrphegluproasnthralametglyalaasnglytyrlys
465470475480
thrileaspasnlysasnphetyrpheargasnglyleuproglnile
485490495
glyvalphelysglyserasnglypheglutyrphealaproalaasn
500505510
thraspalaasnasnilegluglyglnalaileargtyrglnasnarg
515520525
pheleuhisleuleuglylysiletyrtyrpheglyasnasnserlys
530535540
alavalthrglytrpglnthrileasnglylysvaltyrtyrphemet
545550555560
proaspthralametalaalaalaglyglyleuphegluileaspgly
565570575
valiletyrphepheglyvalaspglyvallysalaproglyiletyr
580585590
gly
<210>29
<211>589
<212>prt
<213>艰难梭菌
<400>29
metalathrglytrpglnthrileaspglylyslystyrtyrpheasn
151015
thrasnthralailealaserthrglytyrthrileileasnglylys
202530
hisphetyrpheasnthraspglyilemetglnileglyvalphelys
354045
glyproasnglypheglutyrphealaproalaasnthraspalaasn
505560
asnilegluglyglnalaileleutyrglnasnglupheleuthrleu
65707580
asnglylyslystyrtyrpheglyseraspserlysalavalthrgly
859095
trpargileileasnasnlyslystyrtyrpheasnproasnasnala
100105110
ilealaalailehisleucysthrileasnasnasplystyrtyrphe
115120125
sertyraspglyileleuglnasnglytyrilethrilegluargasn
130135140
asnphetyrpheaspalaasnasngluserlysmetvalthrglyval
145150155160
phelysglyproasnglypheglutyrphealaproalaasnthrhis
165170175
asnasnasnilegluglyglnalailevaltyrglnasnlyspheleu
180185190
thrleuasnglylyslystyrtyrpheaspasnaspserlysalaval
195200205
thrglytrpglnthrileaspglylyslystyrtyrpheasnleuasn
210215220
thralaglualaalathrglytrpglnthrileaspglylyslystyr
225230235240
tyrpheasnleuasnthralaglualaalathrglytrpglnthrile
245250255
aspglylyslystyrtyrpheasnthrasnthrpheilealaserthr
260265270
glytyrthrserileasnglylyshisphetyrpheasnthraspgly
275280285
ilemetglnileglyvalphelysglyproasnglypheglutyrphe
290295300
alaproalaasnthraspalaasnasnilegluglyglnalaileleu
305310315320
tyrglnasnlyspheleuthrleuasnglylyslystyrtyrphegly
325330335
seraspserlysalavalthrglyleuargthrileaspglylyslys
340345350
tyrtyrpheasnthrasnthralavalalavalthrglytrpglnthr
355360365
ileasnglylyslystyrtyrpheasnthrasnthrserilealaser
370375380
thrglytyrthrileileserglylyshisphetyrpheasnthrasp
385390395400
glyilemetglnileglyvalphelysglyproaspglypheglutyr
405410415
phealaproalaasnthraspalaasnasnilegluglyglnalaile
420425430
argtyrglnasnargpheleutyrleuhisaspasniletyrtyrphe
435440445
glyasnasnserlysalaalathrglytrpvalthrileaspglyasn
450455460
argtyrtyrphegluproasnthralametglyalaasnglytyrlys
465470475480
thrileaspasnlysasnphetyrpheargasnglyleuproglnile
485490495
glyvalphelysglyserasnglypheglutyrphealaproalaasn
500505510
thraspalaasnasnilegluglyglnalaileargtyrglnasnarg
515520525
pheleuhisleuleuglylysiletyrtyrpheglyasnasnserlys
530535540
alavalthrglytrpglnthrileasnglylysvaltyrtyrphemet
545550555560
proaspthralametalaalaalaglyglyleuphegluileaspgly
565570575
valiletyrphepheglyvalaspglyvallysalaval
580585
<210>30
<211>589
<212>prt
<213>艰难梭菌
<400>30
metalathrglytrpglnthrileaspglylyslystyrtyrpheasn
151015
thrasnthralailealaserthrglytyrthrileileasnglylys
202530
hisphetyrpheasnthraspglyilemetglnileglyvalphelys
354045
glyproasnglypheglutyrphealaproalaasnthraspalaasn
505560
asnilegluglyglnalaileleutyrglnasnglupheleuthrleu
65707580
asnglylyslystyrtyrpheglyseraspserlysalavalthrgly
859095
trpargileileasnasnlyslystyrtyrpheasnproasnasnala
100105110
ilealaalailehisleucysthrileasnasnasplystyrtyrphe
115120125
sertyraspglyileleuglnasnglytyrilethrilegluargasn
130135140
asnphetyrpheaspalaasnasngluserlysmetvalthrglyval
145150155160
phelysglyproasnglypheglutyrphealaproalaasnthrhis
165170175
asnasnasnilegluglyglnalailevaltyrglnasnlyspheleu
180185190
thrleuasnglylyslystyrtyrpheaspasnaspserlysalaval
195200205
thrglytrpglnthrileaspglylyslystyrtyrpheasnleuasn
210215220
thralaglualaalathrglytrpglnthrileaspglylyslystyr
225230235240
tyrpheasnleuasnthralaglualaalathrglytrpglnthrile
245250255
aspglylyslystyrtyrpheasnthrasnthrpheilealaserthr
260265270
glytyrthrserileasnglylyshisphetyrpheasnthraspgly
275280285
ilemetglnileglyvalphelysglyproasnglypheglutyrphe
290295300
alaproalaasnthraspalaasnasnilegluglyglnalaileleu
305310315320
tyrglnasnlyspheleuthrleuasnglylyslystyrtyrphegly
325330335
seraspserlysalavalthrglyleuargthrileaspglylyslys
340345350
tyrtyrpheasnthrasnthralavalalavalthrglytrpglnthr
355360365
ileasnglylyslystyrtyrpheasnthrasnthrserilealaser
370375380
thrglytyrthrileileserglylyshisphetyrpheasnthrasp
385390395400
glyilemetglnileglyvalphelysglyproaspglypheglutyr
405410415
phealaproalaasnthraspalaasnasnilegluglyglnalaile
420425430
argtyrglnasnargpheleutyrleuhisaspasniletyrtyrphe
435440445
glyasnasnserlysalaalathrglytrpvalthrileaspglyasn
450455460
argtyrtyrphegluproasnthralametglyalaasnglytyrlys
465470475480
thrileaspasnlysasnphetyrpheargasnglyleuproglnile
485490495
glyvalphelysglyserasnglypheglutyrphealaproalaasn
500505510
thraspalaasnasnilegluglyglnalaileargtyrglnasnarg
515520525
pheleuhisleuleuglylysiletyrtyrpheglyasnasnserlys
530535540
alavalthrglytrpglnthrileasnglylysvaltyrtyrphemet
545550555560
proaspthralametalaalaalaglyglyleuphegluileaspgly
565570575
valiletyrphepheglyvalaspglyvallysalaval
580585
<210>31
<211>589
<212>prt
<213>艰难梭菌
<400>31
metalathrglytrpglnthrileaspglylyslystyrtyrpheasn
151015
thrasnthralailealaserthrglytyrthrileileasnglylys
202530
hisphetyrpheasnthraspglyilemetglnileglyvalphelys
354045
glyproasnglypheglutyrphealaproalaasnthraspalaasn
505560
asnilegluglyglnalaileleutyrglnasnglupheleuthrleu
65707580
asnglylyslystyrtyrpheglyseraspserlysalavalthrgly
859095
trpargileileasnasnlyslystyrtyrpheasnproasnasnala
100105110
ilealaalailehisleucysthrileasnasnasplystyrtyrphe
115120125
sertyraspglyileleuglnasnglytyrilethrilegluargasn
130135140
asnphetyrpheaspalaasnasngluserlysmetvalthrglyval
145150155160
phelysglyproasnglypheglutyrphealaproalaasnthrhis
165170175
asnasnasnilegluglyglnalailevaltyrglnasnlyspheleu
180185190
thrleuasnglylyslystyrtyrpheaspasnaspserlysalaval
195200205
thrglytrpglnthrileaspglylyslystyrtyrpheasnleuasn
210215220
thralaglualaalathrglytrpglnthrileaspglylyslystyr
225230235240
tyrpheasnleuasnthralaglualaalathrglytrpglnthrile
245250255
aspglylyslystyrtyrpheasnthrasnthrpheilealaserthr
260265270
glytyrthrserileasnglylyshisphetyrpheasnthraspgly
275280285
ilemetglnileglyvalphelysglyproasnglypheglutyrphe
290295300
alaproalaasnthraspalaasnasnilegluglyglnalaileleu
305310315320
tyrglnasnlyspheleuthrleuasnglylyslystyrtyrphegly
325330335
seraspserlysalavalthrglyleuargthrileaspglylyslys
340345350
tyrtyrpheasnthrasnthralavalalavalthrglytrpglnthr
355360365
ileasnglylyslystyrtyrpheasnthrasnthrserilealaser
370375380
thrglytyrthrileileserglylyshisphetyrpheasnthrasp
385390395400
glyilemetglnileglyvalphelysglyproaspglypheglutyr
405410415
phealaproalaasnthraspalaasnasnilegluglyglnalaile
420425430
argtyrglnasnargpheleutyrleuhisaspasniletyrtyrphe
435440445
glyasnasnserlysalaalathrglytrpvalthrileaspglyasn
450455460
argtyrtyrphegluproasnthralametglyalaasnglytyrlys
465470475480
thrileaspasnlysasnphetyrpheargasnglyleuproglnile
485490495
glyvalphelysglyserasnglypheglutyrphealaproalaasn
500505510
thraspalaasnasnilegluglyglnalaileargtyrglnasnarg
515520525
pheleuhisleuleuglylysiletyrtyrpheglyasnasnserlys
530535540
alavalthrglytrpglnthrileasnglylysvaltyrtyrphemet
545550555560
proaspthralametalaalaalaglyglyleuphegluileaspgly
565570575
valiletyrphepheglyvalaspglyvallysalaval
580585
<210>32
<211>513
<212>prt
<213>艰难梭菌
<400>32
thrglyphevalthrvalglyaspasplystyrtyrpheasnproile
151015
asnglyglyalaalaserileglygluthrileileaspasplysasn
202530
tyrtyrpheasnglnserglyvalleuglnthrglyvalpheserthr
354045
gluaspglyphelystyrphealaproalaasnthrleuaspgluasn
505560
leugluglyglualaileaspphethrglylysleuileileaspglu
65707580
asniletyrtyrpheaspaspasntyrargglyalavalglutrplys
859095
gluleuaspglyglumethistyrpheserprogluthrglylysala
100105110
phelysglyleuasnglnileglyasptyrlystyrtyrpheasnser
115120125
aspglyvalmetglnlysglyphevalserileasnaspasnlyshis
130135140
tyrpheaspaspserglyvalmetlysvalglytyrthrgluileasp
145150155160
glylyshisphetyrphealagluasnglyglumetglnileglyval
165170175
pheasnthrgluaspglyphelystyrphealahishisasngluasp
180185190
leuglyasnglugluglyglugluilesertyrserglyileleuasn
195200205
pheasnasnlysiletyrtyrpheaspaspserphethralavalval
210215220
glytrplysaspleugluaspglyserlystyrtyrpheaspgluasp
225230235240
thralaglualatyrileglyleuserleuileasnaspglyglntyr
245250255
tyrpheasnaspaspglyilemetglnvalglyphevalthrileasn
260265270
asplysvalphetyrpheseraspserglyileilegluserglyval
275280285
glnasnileaspaspasntyrphetyrileaspaspasnglyileval
290295300
glnileglyvalpheaspthrseraspglytyrlystyrphealapro
305310315320
alaasnthrvalasnaspasniletyrglyglnalavalglutyrser
325330335
glyleuvalargvalglygluaspvaltyrtyrpheglygluthrtyr
340345350
thrilegluthrglytrpiletyraspmetgluasngluserasplys
355360365
tyrtyrpheasnprogluthrlyslysalacyslysglyileasnleu
370375380
ileaspaspilelystyrtyrpheaspglulysglyilemetargthr
385390395400
glyleuileserphegluasnasnasntyrtyrpheasngluasngly
405410415
glumetglnpheglytyrileasnilegluasplysmetphetyrphe
420425430
glygluaspglyvalmetglnileglyvalpheasnthrproaspgly
435440445
phelystyrphealahisglnasnthrleuaspgluasnpheglugly
450455460
gluserileasntyrthrglytrpleuaspleuaspglulysargtyr
465470475480
tyrphethraspglutyrilealaalathrglyservalileileasp
485490495
glygluglutyrtyrpheaspproaspthralaglnleuvalileser
500505510
glu
<210>33
<211>513
<212>prt
<213>艰难梭菌
<400>33
thrglyphevalthrvalglyaspasplystyrtyrpheasnproile
151015
asnglyglyalaalaserileglygluthrileileaspasplysasn
202530
tyrtyrpheasnglnserglyvalleuglnthrglyvalpheserthr
354045
gluaspglyphelystyrphealaproalaasnthrleuaspgluasn
505560
leugluglyglualaileaspphethrglylysleuileileaspglu
65707580
asniletyrtyrpheaspaspasntyrargglyalavalglutrplys
859095
gluleuaspglyglumethistyrpheserprogluthrglylysala
100105110
phelysglyleuasnglnileglyasptyrlystyrtyrpheasnser
115120125
aspglyvalmetglnlysglyphevalserileasnaspasnlyshis
130135140
tyrpheaspaspserglyvalmetlysvalglytyrthrgluileasp
145150155160
glylyshisphetyrphealagluasnglyglumetglnileglyval
165170175
pheasnthrgluaspglyphelystyrphealahishisasngluasp
180185190
leuglyasnglugluglyglugluilesertyrserglyileleuasn
195200205
pheasnasnlysiletyrtyrpheaspaspserphethralavalval
210215220
glytrplysaspleugluaspglyserlystyrtyrpheaspgluasp
225230235240
thralaglualatyrileglyleuserleuileasnaspglyglntyr
245250255
tyrpheasnaspaspglyilemetglnvalglyphevalthrileasn
260265270
asplysvalphetyrpheseraspserglyileilegluserglyval
275280285
glnasnileaspaspasntyrphetyrileaspaspasnglyileval
290295300
glnileglyvalpheaspthrseraspglytyrlystyrphealapro
305310315320
alaasnthrvalasnaspasniletyrglyglnalavalglutyrser
325330335
glyleuvalargvalglygluaspvaltyrtyrpheglygluthrtyr
340345350
thrilegluthrglytrpiletyraspmetgluasngluserasplys
355360365
tyrtyrpheasnprogluthrlyslysalacyslysglyileasnleu
370375380
ileaspaspilelystyrtyrpheaspglulysglyilemetargthr
385390395400
glyleuileserphegluasnasnasntyrtyrpheasngluasngly
405410415
glumetglnpheglytyrileasnilegluasplysmetphetyrphe
420425430
glygluaspglyvalmetglnileglyvalpheasnthrproaspgly
435440445
phelystyrphealahisglnasnthrleuaspgluasnpheglugly
450455460
gluserileasntyrthrglytrpleuaspleuaspglulysargtyr
465470475480
tyrphethraspglutyrilealaalathrglyservalileileasp
485490495
glygluglutyrtyrpheaspproaspthralaglnleuvalileser
500505510
glu
<210>34
<211>534
<212>prt
<213>艰难梭菌
<400>34
serglyleuiletyrileasnaspserleutyrtyrphelyspropro
151015
valasnasnleuilethrglyphevalthrvalglyaspasplystyr
202530
tyrpheasnproileasnglyglyalaalaserileglygluthrile
354045
ileaspasplysasntyrtyrpheasnglnserglyvalleuglnthr
505560
glyvalpheserthrgluaspglyphelystyrphealaproalaasn
65707580
thrleuaspgluasnleugluglyglualaileaspphethrglylys
859095
leuileileaspgluasniletyrtyrpheaspaspasntyrarggly
100105110
alavalglutrplysgluleuaspglyglumethistyrpheserpro
115120125
gluthrglylysalaphelysglyleuasnglnileglyasptyrlys
130135140
tyrtyrpheasnseraspglyvalmetglnlysglyphevalserile
145150155160
asnaspasnlyshistyrpheaspaspserglyvalmetlysvalgly
165170175
tyrthrgluileaspglylyshisphetyrphealagluasnglyglu
180185190
metglnileglyvalpheasnthrgluaspglyphelystyrpheala
195200205
hishisasngluaspleuglyasnglugluglyglugluilesertyr
210215220
serglyileleuasnpheasnasnlysiletyrtyrpheaspaspser
225230235240
phethralavalvalglytrplysaspleugluaspglyserlystyr
245250255
tyrpheaspgluaspthralaglualatyrileglyleuserleuile
260265270
asnaspglyglntyrtyrpheasnaspaspglyilemetglnvalgly
275280285
phevalthrileasnasplysvalphetyrpheseraspserglyile
290295300
ilegluserglyvalglnasnileaspaspasntyrphetyrileasp
305310315320
aspasnglyilevalglnileglyvalpheaspthrseraspglytyr
325330335
lystyrphealaproalaasnthrvalasnaspasniletyrglygln
340345350
alavalglutyrserglyleuvalargvalglygluaspvaltyrtyr
355360365
pheglygluthrtyrthrilegluthrglytrpiletyraspmetglu
370375380
asngluserasplystyrtyrpheasnprogluthrlyslysalacys
385390395400
lysglyileasnleuileaspaspilelystyrtyrpheaspglulys
405410415
glyilemetargthrglyleuileserphegluasnasnasntyrtyr
420425430
pheasngluasnglyglumetglnpheglytyrileasnilegluasp
435440445
lysmetphetyrpheglygluaspglyvalmetglnileglyvalphe
450455460
asnthrproaspglyphelystyrphealahisglnasnthrleuasp
465470475480
gluasnphegluglygluserileasntyrthrglytrpleuaspleu
485490495
aspglulysargtyrtyrphethraspglutyrilealaalathrgly
500505510
servalileileaspglygluglutyrtyrpheaspproaspthrala
515520525
glnleuvalileserglu
530
<210>35
<211>400
<212>prt
<213>艰难梭菌
<400>35
lysglyleuasnglnileglyasptyrlystyrtyrpheasnserasp
151015
glyvalmetglnlysglyphevalserileasnaspasnlyshistyr
202530
pheaspaspserglyvalmetlysvalglytyrthrgluileaspgly
354045
lyshisphetyrphealagluasnglyglumetglnileglyvalphe
505560
asnthrgluaspglyphelystyrphealahishisasngluaspleu
65707580
glyasnglugluglyglugluilesertyrserglyileleuasnphe
859095
asnasnlysiletyrtyrpheaspaspserphethralavalvalgly
100105110
trplysaspleugluaspglyserlystyrtyrpheaspgluaspthr
115120125
alaglualatyrileglyleuserleuileasnaspglyglntyrtyr
130135140
pheasnaspaspglyilemetglnvalglyphevalthrileasnasp
145150155160
lysvalphetyrpheseraspserglyileilegluserglyvalgln
165170175
asnileaspaspasntyrphetyrileaspaspasnglyilevalgln
180185190
ileglyvalpheaspthrseraspglytyrlystyrphealaproala
195200205
asnthrvalasnaspasniletyrglyglnalavalglutyrsergly
210215220
leuvalargvalglygluaspvaltyrtyrpheglygluthrtyrthr
225230235240
ilegluthrglytrpiletyraspmetgluasngluserasplystyr
245250255
tyrpheasnprogluthrlyslysalacyslysglyileasnleuile
260265270
aspaspilelystyrtyrpheaspglulysglyilemetargthrgly
275280285
leuileserphegluasnasnasntyrtyrpheasngluasnglyglu
290295300
metglnpheglytyrileasnilegluasplysmetphetyrphegly
305310315320
gluaspglyvalmetglnileglyvalpheasnthrproaspglyphe
325330335
lystyrphealahisglnasnthrleuaspgluasnphegluglyglu
340345350
serileasntyrthrglytrpleuaspleuaspglulysargtyrtyr
355360365
phethraspglutyrilealaalathrglyservalileileaspgly
370375380
gluglutyrtyrpheaspproaspthralaglnleuvalileserglu
385390395400