CN110036105A - 经修饰的肽 - Google Patents
经修饰的肽 Download PDFInfo
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- CN110036105A CN110036105A CN201780074202.2A CN201780074202A CN110036105A CN 110036105 A CN110036105 A CN 110036105A CN 201780074202 A CN201780074202 A CN 201780074202A CN 110036105 A CN110036105 A CN 110036105A
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Abstract
本发明涉及抗微生物剂的领域。特别地,本发明涉及包含肽聚糖水解酶的序列和与肽聚糖水解酶异源的肽序列的多肽,其中所述异源肽序列包含长度为16、17、18、19或20个氨基酸的特定序列基序。本发明还涉及相应的核酸、载体、噬菌体、宿主细胞、组合物和试剂盒。本发明还涉及所述多肽、核酸、载体、噬菌体、宿主细胞、组合物和试剂盒在用于通过手术或疗法治疗人或动物体的方法中或在人或动物体上实施的诊断方法中的用途。根据本发明的多肽、核酸、载体、噬菌体、宿主细胞、组合物和试剂盒也可以在例如食物或饲料中、在化妆品中用作抗微生物剂或者用作消毒剂。
Description
发明背景
I.发明领域
本发明涉及抗微生物剂的领域。特别地,本发明涉及包含肽聚糖水解酶的序列和与肽聚糖水解酶异源的肽序列的多肽,其中所述异源肽序列包含长度为16、17、18、19或20个氨基酸的特定序列基序。本发明还涉及相应的核酸、载体、噬菌体、宿主细胞、组合物和试剂盒。本发明还涉及所述多肽、核酸、载体、噬菌体、宿主细胞、组合物和试剂盒在用于通过手术或疗法治疗人或动物体的方法中或在人或动物体上实施的诊断方法中的用途。根据本发明的多肽、核酸、载体、噬菌体、宿主细胞、组合物和试剂盒也可以在例如食物或饲料中、在化妆品中用作抗微生物剂或者用作消毒剂。
II.相关技术的描述
对常规抗生素的抗性正在成为人类日益增加的健康风险。新的抗生素抗性机制正在出现并迅速在全球蔓延。因此,在不久的将来,治疗常见传染病的能力可能变得越来越困难。在本领域中已经容易理解这种危险,并且探索了对抗感染原的新方法。
这些新方法之一是肽聚糖水解酶与抗微生物肽的融合物。在WO 2010/149792中,已经显示此类融合物在治疗许多细菌中是有效的。WO 2010/149792公开了肽聚糖水解酶和肽的各种组合。有趣的是,并且由于未知原因,并非所有肽聚糖水解酶和肽的组合都同样有效。虽然与29聚体抗微生物肽SMAP-29肽(SEQ ID NO:1)的组合显示出非常高的抗微生物活性,但是与相同的肽聚糖水解酶组合的其他肽仅在较小的程度上增加抗微生物活性。
因此,本领域仍需要进一步改善此类抗细菌剂的设计。
因此,本发明人的目的是提供新的抗微生物剂,与肽聚糖水解酶与抗微生物肽的随机组合相比,其提供改善的结果。
通过如下和所附权利要求中阐述的主题解决了该问题。
发明概述
令人惊讶地,本发明的发明人发现,如果使用展现出某些通用氨基酸序列基序的肽,则可以有目的地产生肽聚糖水解酶和肽的有效抗微生物组合。通过应用该模式,发明人使先前存在的抗微生物肽变得更有效。此外,通过引入相应的突变,本发明人甚至成功地将完全不相关的肽(即先前未知任何抗微生物活性)从头转化为这方面的有用化合物。
在第一方面,本发明涉及包含序列基序的多肽,所述序列基序:
i)长度为16、17、18、19或20个氨基酸;
ii)包含至少40%并且至多60%选自由赖氨酸、精氨酸和组氨酸组成的第一组氨基酸的氨基酸,
其中每个氨基酸独立地选自所述第一组,
其中选自该第一组的每个氨基酸在所述序列基序中单独地排列、与选自所述第一组的另一个氨基酸一起成对地排列,或者与选自所述第一组的另外2个氨基酸成区块(block)排列,但不与选自所述第一组的3个或更多个氨基酸成区块出现,其中至少2对的选自所述第一组的氨基酸存在于所述序列基序中,并且其中至多一个具有成排的选自所述第一组的3个氨基酸的区块存在于所述序列基序中,附加条件是如果此类具有所述第一组的3个氨基酸的区块存在于所述序列基序中,则在相对于所述3个氨基酸的区块的第一个氨基酸的位置12、-11、-8、-5、-4、+6、+7、+10、+13和+14处的氨基酸不选自所述第一组,条件是相应的位置可以在所述序列基序中找到,
iii)包含至少40%并且至多60%选自由丙氨酸、甘氨酸、异亮氨酸、亮氨酸、苯丙氨酸、丝氨酸、苏氨酸、色氨酸、酪氨酸和缬氨酸组成的第二组氨基酸的氨基酸,
其中每个氨基酸独立地选自所述第二组,
其中如果选自所述第一组的和选自所述第二组的氨基酸的总和产生100%的所述序列基序,则至少三个不同的氨基酸选自该第二组;
其中如果所述序列基序含有至少两个单一非相邻的苯丙氨酸残基,并且这些苯丙氨酸残基中的至少一个直接在赖氨酸残基之后,则所述序列基序不包含序列AFV,并且
其中如果所述序列基序含有至少三个非相邻的组氨酸残基,则所述序列基序不包含序列AALTH(SEQ ID NO:2),
iv)其中若存在于所述基序中的话,所述序列基序的剩余氨基酸选自由天冬酰胺、天冬氨酸、谷氨酰胺、谷氨酸、甲硫氨酸或半胱氨酸组成的第三组,其中所述氨基酸的每一个独立地选自所述第三组,并且其中谷氨酰胺可以仅选择一次并且此外其中所述选择可以不包含谷氨酰胺和谷氨酸的组合,并且
其中所述多肽不包含SMAP-29肽(SEQ ID NO:1)的序列。
本发明多肽的特别优选的实施方案是本发明的融合蛋白,其中本发明的多肽另外包含肽聚糖水解酶的序列。
在其他方面,本发明涉及编码本发明多肽的核酸,包含本发明核酸的载体或噬菌体以及包含本发明多肽、核酸、载体和/或噬菌体的宿主细胞。
在另一方面,本发明还涉及包含根据本发明的多肽、核酸、载体、噬菌体和/或宿主细胞的组合物。此类组合物优选是包含药学上可接受的载体、稀释剂或赋形剂的药物组合物。
在另一方面,本发明考虑了试剂盒,其包含本发明多肽、核酸、载体、噬菌体和/或宿主细胞,并且进一步包含肽聚糖水解酶,或分别编码或包含所述肽聚糖水解酶的核酸、载体、噬菌体和/或宿主细胞。
最后,本发明涉及本发明的多肽、核酸、载体、噬菌体、宿主细胞、组合物和/或试剂盒,其用于治疗,特别是用于治疗或预防细菌感染的方法。
发明详述
I.定义
如本文所用,术语“多肽”特别是指通过肽键以特定序列连接的氨基酸残基的聚合物。多肽的氨基酸残基可以通过例如各种基团诸如碳水化合物和磷酸盐的共价附接修饰。其他物质可以与多肽更松散地缔合,例如血红素或脂质,产生缀合的多肽,其也包含在如本文所用的术语“多肽”中。如本文所用,该术语还旨在涵盖蛋白质。因此,术语“多肽”还涵盖例如两个或更多个氨基酸聚合物链的复合物。术语“多肽”确实涵盖多肽的实施方案,所述多肽任选地展现出本领域中通常使用的修饰,例如,生物素化,乙酰化,聚乙二醇化,氨基、SH-或羧基基团(例如保护基团)的化学变化等。如从下面的描述中可变得明显,根据本发明的多肽可以是人工工程化改造的多肽,其在自然界中不以此种形式存在。此类多肽可以例如展现出相对于天然存在的多肽的人工突变,或者可以包含异源序列,或者可以是天然存在的多肽的片段,该片段在自然界中不以此种形式存在。此外,根据本发明的多肽可以是融合蛋白,即表示在自然界中不以此种组合存在的至少两个氨基酸序列的连接。如本文所用,术语“多肽”不限于氨基酸聚合物链的特定长度。但是,最小长度为16个氨基酸。通常但非必要地,本发明的典型多肽的长度将不超过约1000个氨基酸。本发明的多肽可以是例如至多约750个氨基酸长、至多约500个氨基酸长或至多约300个氨基酸长。因此,本发明多肽的可能长度范围可以例如为16至1000个氨基酸、16至约50个氨基酸、约200至约750个氨基酸,或约225至约600个氨基酸,或约250至约350个氨基酸,但不限于此。
如本文所用,术语“肽聚糖水解酶”在本领域中通常是理解的。它是指能够水解细菌(例如革兰氏阴性细菌)的肽聚糖的任何多肽。该术语不限于特定的酶切割机制。就切割机制而言,肽聚糖水解酶可以是例如内肽酶、几丁质酶、T4样胞壁质酶(muraminidase)、lambda样胞壁质酶、N-乙酰基-胞壁酰-L-丙氨酸-酰胺酶(酰胺酶)、胞壁酰-L-丙氨酸-酰胺酶、胞壁质酶(muramidase)、裂解性转糖基酶(C)、裂解性转糖基酶(M)、N-乙酰基-胞壁质酶(溶菌酶)、N-乙酰基-氨基葡萄糖苷酶或转糖基酶。此外,该术语涵盖天然存在的肽聚糖水解酶,例如真核、原核或病毒(特别是噬菌体)来源的肽聚糖水解酶。该术语涵盖例如脊椎动物溶菌酶(例如鸡蛋清溶菌酶和人溶菌酶)、内溶素(例如KZ144内溶素或Lys394内溶素)、病毒体相关肽聚糖水解酶(VAPGH)、细菌素(例如溶葡萄球菌素)和自溶素。“肽聚糖水解酶”也可以是能够水解细菌的肽聚糖的合成或人工修饰的多肽。例如,其中已调换/交换两种或更多种内溶素的结构域的酶促活性改组内溶素确实有资格作为“肽聚糖水解酶”,就像截短的内溶素一样,其中仅保留酶活性结构域。活性可以通过本领域技术人员熟知的测定法测量,例如抗细菌测定法,其例如在Briers等人(J.Biochem.Biophys Methods;2007;70:531-533)或Donovan等人(J.FEMS Microbiol Lett.2006Dec;265(1)和相似的出版物中描述。
如果在本文中提及“氨基酸残基”,则通常意指L-氨基酸残基。
如本文所用,术语“内溶素”是指适于水解细菌细胞壁的源自噬菌体的酶。内溶素包含至少一个具有以下活性中的至少一种的“酶促活性结构域”(EAD):内肽酶、几丁质酶、T4样胞壁质酶、lambda样胞壁质酶、N-乙酰基-胞壁酰-L-丙氨酸-酰胺酶(酰胺酶)、胞壁酰-L-丙氨酸-酰胺酶、胞壁质酶、裂解性转糖基酶(C)、裂解性转糖基酶(M)、N-乙酰基-胞壁质酶(溶菌酶)、N-乙酰基-氨基葡萄糖苷酶或转糖基酶。此外,内溶素还可以含有无酶促活性,但是与宿主细菌的细胞壁结合的区域,即所谓的CBD(细胞壁结合结构域)。
如本文所用,术语“包含”不应解释为限于含义“由......组成”(即排除存在另外的其他物质)。相反,“包含”意味着可以任选地存在另外的物质。术语“包含”涵盖特别设想的实施方案,其落入其范围“由......组成”(即排除存在另外的其他物质)和“包含但不由......组成”(即需要存在另外的其他物质),其中前者更优选。
当在本文中使用时,词语“一”或“一个”的使用可以意指“一个”,但是它也与“一个或多个”、“至少一个”和“一个或超出一个”的含义一致。
II.多肽
如已经提到的,本发明首先涉及包含序列基序的多肽,所述序列基序:
i)长度为16、17、18、19或20个氨基酸;
ii)包含至少40%并且至多60%选自由赖氨酸、精氨酸和组氨酸组成的第一组氨基酸的氨基酸,
其中每个氨基酸独立地选自所述第一组,
其中选自该第一组的每个氨基酸在所述序列基序中单独地排列、与选自所述第一组的另一个氨基酸一起成对地排列,或者在与选自所述第一组的另外两个氨基酸成区块排列,但不与选自所述第一组的3个或更多个氨基酸成区块中出现,其中至少2对的选自所述第一组的氨基酸存在于所述序列基序中,并且其中至多一个成排的具有选自所述第一组的3个氨基酸的区块存在于所述序列基序中,附加条件是如果此类具有所述第一组的3个氨基酸的区块存在于所述序列基序中,则在相对于所述3个氨基酸的区块的第一个氨基酸的位置12、-11、-8、-5、-4、+6、+7、+10、+13和+14处的氨基酸不选自所述第一组,条件是相应的位置可以在所述序列基序中找到,
iii)包含至少40%并且至多60%选自由丙氨酸、甘氨酸、异亮氨酸、亮氨酸、苯丙氨酸、丝氨酸、苏氨酸、色氨酸、酪氨酸和缬氨酸组成的第二组氨基酸的氨基酸,
其中每个氨基酸独立地选自所述第二组,
其中如果选自所述第一组的和选自所述第二组的氨基酸的总和产生100%的所述序列基序,则至少三个不同的氨基酸选自该第二组;
其中如果所述序列基序含有至少两个单一非相邻的苯丙氨酸残基,并且这些苯丙氨酸残基中的至少一个直接在赖氨酸残基之后,则所述序列基序不包含序列AFV,并且
其中如果所述序列基序含有至少三个非相邻的组氨酸残基,则所述序列基序不包含序列AALTH(SEQ ID NO:2),
iv)其中若存在于所述基序中的话,所述序列基序的剩余氨基酸选自由天冬酰胺、天冬氨酸、谷氨酰胺、谷氨酸、甲硫氨酸或半胱氨酸组成的第三组,其中所述氨基酸的每一个独立地选自所述第三组,并且其中谷氨酰胺可以仅选择一次并且此外其中所述选择可以不包含谷氨酰胺和谷氨酸的组合,并且
其中所述融合蛋白不包含SMAP-29肽(SEQ ID NO:1)的序列。
以上在i)至iii)中定义的序列基序可以仅代表本发明多肽的序列的一部分,即本发明的多肽比序列基序长。或者,序列基序可以是本发明多肽的序列,即本发明多肽的序列与序列基序的序列相同。此外,如从图1中提供的实例中明显的是,本发明的多肽可能包含一个或多个此类序列基序。例如,20聚体基序可以固有地包含也遵从上述标准的16聚体基序。因此,本发明的多肽包含如上定义的“一个”序列基序的事实可以不理解为本发明的多肽仅可以包含“一个”序列基序,而没有也遵从上述限制的另外的(例如重叠的)序列基序。
本发明多肽的序列基序长度可以是16、17、18、19或20个氨基酸。优选地,序列基序的长度为17、18或19个氨基酸,甚至更优选地长度为17或18个氨基酸。
本发明多肽的序列基序包含至少40%和至多60%选自第一组氨基酸的氨基酸。所述第一组由赖氨酸、精氨酸和组氨酸组成。如果序列基序长16个氨基酸,则它将展现出选自该第一组的至少7个和至多9个氨基酸。如果序列基序长17个氨基酸,则它将展现出选自该第一组的至少7个和至多10个氨基酸。如果序列基序长18个氨基酸,则它将展现出选自该第一组的至少8个和至多10个氨基酸。如果序列基序长19个氨基酸,则它将展现出选自该第一组的至少8个和至多11个氨基酸。如果序列基序长20个氨基酸,则它将展现出选自该第一组的至少8个和至多12个氨基酸。
用于在该第一组内选择的优选氨基酸是赖氨酸和精氨酸。优选地,序列基序不包含超过50%的组氨酸残基。甚至更优选地,序列基序不包含超过25%的组氨酸残基。在本发明的一些实施方案中,序列基序仅包含一个组氨酸残基或甚至不包含组氨酸残基。
选自第一组的氨基酸是独立选择的。例如,这意味着如果给定的序列基序包含例如选自第一组的八个氨基酸,则这八个氨基酸残基的每一个可以与先前的或后续的选择独立地选自第一组。因此,所选择的氨基酸可以包含所有三种类型的氨基酸(赖氨酸、精氨酸和组氨酸),可以是相同的(例如分别为8个赖氨酸或8个精氨酸残基),或者可以仅包含三种类型的氨基酸的两种(例如赖氨酸和精氨酸)。同样,独立选择不规定个别选择的氨基酸之间的任何特定比例。例如,选自该第一组的8个氨基酸可以是8个赖氨酸残基,7个精氨酸残基和1个组氨酸残基或3个精氨酸、4个赖氨酸和1个组氨酸残基,但不限于此。
序列基序内的选自第一组的氨基酸残基的定位受到某些限制。选自该第一组的每个氨基酸可以仅在所述序列基序中单独地排列、与选自第一组的另一个氨基酸一起成对地排列,或者与选自第一组的另外两个氨基酸成区块排列。
“单独地”意指选自所述第一组的氨基酸,例如赖氨酸(K)在N端和C端侧翼都没有来自所述第一组的另一个氨基酸。相邻的氨基酸残基可以选自第二组,或视情况而定,选自第三组(例如LKE、N-KE(在基序的N端处)、LK-C(在基序的C端处))。值得注意的是,不考虑在本发明多肽内但在序列基序之外的潜在的其他氨基酸用于该位置确定。因此认为在序列基序的两个末端之一处的来自第一组的氨基酸是单独定位的,即使序列基序外的前面的(N端)或后续的(C端)氨基酸残基偶然也是精氨酸、组氨酸或赖氨酸残基。
“与选自第一组的另外的氨基酸一起成对地”意指在序列基序内,选自第一组的氨基酸与选自第一组的另外的氨基酸直接相邻。由此,这两个氨基酸形成选自第一组的一对氨基酸。继而,所述对在C端和N端侧翼有来自第二组,或视情况而定,来自第三组的氨基酸(例如LKRE(SEQ ID NO:3)、N-KRE(在基序的N端处)、LKR-C(在基序的C端处))。再次不考虑在本发明多肽内但在序列基序之外的潜在的其他氨基酸用于该位置确定。
“与选自第一组的另外2个氨基酸成区块地”意指选自第一组的三个氨基酸彼此直接相邻。所述区块(或三联体)在C端和N端侧翼有来自第二组,或视情况而定,来自第三组的氨基酸(例如LKRKE(SEQ ID NO:4)、N-KRKE(在基序的N端处;SEQ ID NO:5)、LKRK-C(在基序的C端处;SEQ ID NO:6))。再次不考虑在本发明多肽内但在序列基序之外的潜在的其他氨基酸用于该位置确定。对于以此类方式(三联体;具有第一组的3个氨基酸的区块)排列的氨基酸,必须满足另外的位置要求,即在相对于3个氨基酸的区块的第一个氨基酸的位置-12、-11、-8、-5、-4、+6、+7、+10、+13和+14处无一氨基酸选自所述第一组的氨基酸,条件是相应的位置可以在所述序列基序中找到。负值表示三联体的第一个氨基酸的N端的位置;正值是指三联体的第一个氨基酸的C端的位置。用于位置计算的基础是三联体的第一个(N端)氨基酸(例如三联体的直接N端的氨基酸为-1,三联体的直接C端的氨基酸为+3)。因此,该限制排除了像RRRGLR(SEQ ID NO:7)的序列,因为位置+6(H)是第一组的氨基酸。相应的位置(-12、-11、-8、-5、-4、+6、+7、+10、+13和+14)是否存在于序列基序中将取决于序列基序内三联体的位置和序列基序的长度。例如,如果三联体位于序列基序的N端处,则所有负值都是废弃的(即不需要考虑)。如果三联体位于序列基序的C端处,则同样适用于正值。然而,在优选的实施方案中,序列基序根本不包含第一组的氨基酸的此类三联体区块,即不包含由选自第一组的3个氨基酸组成的区块。
应当理解,单独地、与选自第一组的另外氨基酸一起成对地以及在与选自第一组的另外2个氨基酸成区块的位置要求是不重叠的,并且这些术语是互斥的(例如,三联体不是“单独地”和/或“一起成对地”的情况等)。
对选自第一组的氨基酸的进一步的位置要求是,序列基序必须包含至少2对选自第一组的氨基酸。然而,优选的是,并非所有选自第一组的氨基酸都在序列基序中成对地排列。
本发明多肽的序列基序不包含选自第一组的4个(四联体)或更多个氨基酸(五联体、六联体等)的区块(即第一组的氨基酸不与选自第一组的3个或更多个氨基酸成区块中出现)。因此,序列基序可以例如不包含诸如“KRKK”(SEQ ID NO:8)或“RRRR”(SEQ ID NO:9)的序列。
由于第一组的氨基酸仅占序列基序的40%至60%,因此剩余的氨基酸需要选自其他氨基酸残基。如上所述,序列基序还包含至少40%并且至多60%的选自第二组氨基酸的氨基酸。所述第二组由氨基酸残基丙氨酸、甘氨酸、异亮氨酸、亮氨酸、苯丙氨酸、丝氨酸、苏氨酸、色氨酸、酪氨酸和缬氨酸组成。如前文对于第一组氨基酸,第二组的氨基酸的每一个同样原则上独立地选择,即每个氨基酸与任何先前的选择或后续的选择独立地选自所述第二组。
然而,对于第二组,对独立选择的此种一般原则存在一些限制。如果选自第一组和选自第二组的氨基酸的总和产生100%的序列基序的氨基酸(即序列基序中没有来自第三组的氨基酸),则适用第一个限制。在此类情境下,至少三个不同的氨基酸必须选自第二组。在此类情境下,第二组的氨基酸可以例如不仅限于缬氨酸和色氨酸残基。
进一步的(位置的)限制是如果序列基序含有至少两个单一非相邻苯丙氨酸残基并且这些苯丙氨酸残基中的至少一个(在N端)直接在赖氨酸残基之后(即KF),则序列基序可以不包含三联体序列AFV(丙氨酸、苯丙氨酸、缬氨酸)。不相邻的苯丙氨酸残基是在序列中不成排出现,而是被一个或多个其他氨基酸分开的苯丙氨酸残基。单一苯丙氨酸残基意味着它们不是一对苯丙氨酸残基的一部分或几个苯丙氨酸残基的区块的一部分,而是单独定位在序列基序中。
下一个限制是如果序列基序含有至少三个单一非相邻的组氨酸残基,则序列基序不包含序列AALTH(即丙氨酸、丙氨酸、赖氨酸、苏氨酸、组氨酸)。不相邻的组氨酸残基是不成排出现,而是被一个或多个其他氨基酸分开的组氨酸残基。单一组氨酸残基意味着它们不是一对组氨酸残基的一部分或几个组氨酸残基的区块的一部分,而是单独定位在序列基序中。
在优选的实施方案中,少于5个异亮氨酸残基(例如4、3、2、1或0)选自所述第二组,特别是如果多肽不包含肽聚糖水解酶的序列和/或具有短的长度,例如具有在16至50个氨基酸的范围中的长度。
有可能的是,本发明多肽的序列基序不排他地由选自第一和第二组的氨基酸构成(即它们一起占小于100%)。在此类情境下,所述序列基序的剩余氨基酸选自第三组氨基酸,所述组由天冬酰胺、天冬氨酸、谷氨酰胺、谷氨酸、甲硫氨酸和半胱氨酸组成。如前文对于第一和第二组氨基酸,第三组的氨基酸的每一个同样原则上独立地选择,即每个氨基酸与任何先前或后续的选择独立地选自所述第二组。然而,如前文对于第二组,从所述第三组选择氨基酸存在一些限制:谷氨酰胺可以仅选择一次并且也不允许平行选择谷氨酰胺和谷氨酸,即如果谷氨酰胺存在于序列基序中,则不可以存在谷氨酸,并且反之亦然)。优选地,选自第三组的氨基酸限于天冬酰胺、天冬氨酸、谷氨酰胺和谷氨酸,即所选择的第三组氨基酸不包含甲硫氨酸或半胱氨酸残基。
在优选的实施方案中,序列基序仅包含来自第三组的单一氨基酸残基或者甚至更优选根本不包含来自第三组的氨基酸残基。
在本发明的优选实施方案中,序列基序中所选择的氨基酸的排列遵从图1(和分别地图2a、2b、2c、2d和2e)中描绘的可能的序列基序备选之一中所述的要求。图1指出对于给定的16聚体、17聚体、18聚体、19聚体或20聚体的特定位置处,不可以存在选自第一组的氨基酸。在这些位置处,仅可以存在选自第二和/或第三组(如果有的话)的氨基酸。优选地,第二组的氨基酸存在于所述位置处。第一组的氨基酸仅可以存在于序列基序的任何剩余位置处。这并不意味着在这些剩余位置处仅可以找到第一组的氨基酸。如果仍然满足第一和第二组的总体百分比要求,也可以在这些剩余位置处找到第二和任选地第三组的氨基酸。
优选地,本发明多肽的序列基序是螺旋状结构的。
本发明多肽的序列基序不包含除第一、第二或第三组中定义的那些之外的任何其他氨基酸残基。特别地,本发明多肽的序列基序不包含任何脯氨酸残基,并且如果第三组限于天冬酰胺、天冬氨酸、谷氨酰胺和谷氨酸,则也不包含甲硫氨酸和半胱氨酸。
然而,正如在SMAP-29序列中一样,脯氨酸残基可以非常好地存在于本发明的多肽中。例如,优选脯氨酸残基定位于序列基序的N端或C端的1至10个,优选地1至5个氨基酸残基内,其中优选后者。在本发明多肽还包含肽聚糖水解酶的序列的情况下(参见下文),优选在肽聚糖水解酶的序列和序列基序之间找到此类脯氨酸残基。优选地,序列基序在肽聚糖水解酶的序列的N端,并且脯氨酸残基位于其间的某处,通常接近序列基序。
根据本发明的多肽不包含SEQ ID NO:1的序列。在一些实施方案中,根据本发明的多肽可以包含SEQ ID NO:10。然而,在优选的实施方案中,根据本发明的多肽也不包含SEQID NO:10的序列。
根据本发明的多肽优选是自然界中不存在的人工多肽。此类人工构建的序列的实例为SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ IDNO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21、SEQ IDNO:22和SEQ ID NO:23。其他实例为SEQ ID NO:71、SEQ ID NO:72、SEQ ID NO:73和SEQ IDNO:74。因此根据本发明的多肽的特别优选的实例为包含任何以下各项的多肽:SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ IDNO:17、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:22、SEQ IDNO:23、SEQ ID NO:71、SEQ ID NO:72、SEQ ID NO:73或SEQ ID NO:74。
在优选的实施方案中,根据本发明的多肽另外包含肽聚糖水解酶的序列。此类多肽(代表本发明的融合蛋白)包含:
a)肽聚糖水解酶的序列,和
b)肽序列,所述肽序列优选与肽聚糖水解酶是异源的,并且其中所述(异源)肽序列包含序列基序,所述序列基序:
i)长度为16、17、18、19或20个氨基酸;
ii)包含至少40%并且至多60%选自由赖氨酸、精氨酸和组氨酸组成的第一组氨基酸的氨基酸;
其中每个氨基酸独立地选自所述第一组;
其中选自该第一组的每个氨基酸在所述序列基序中单独地排列、与选自第一组的另一个氨基酸一起成对地排列,或者在与选自第一组的另外两个氨基酸成区块排列,但不与选自第一组的3个或更多个氨基酸成区块出现,其中至少2对的选自第一组的氨基酸存在于所述序列基序中,并且其中至多一个具有成排的选自第一组的3个氨基酸的区块存在于所述序列基序中,附加条件是如果此类具有第一组的3个氨基酸的区块存在于所述序列基序中,则在相对于3个氨基酸区块的第一个氨基酸的位置-12、-11、-8、-5、-4、+6、+7、+10、+13和+14处的氨基酸不选自所述第一组,,条件是相应的位置可以在所述序列基序中找到
iii)包含至少40%并且至多60%选自由丙氨酸、甘氨酸、异亮氨酸、亮氨酸、苯丙氨酸、丝氨酸、苏氨酸、色氨酸、酪氨酸和缬氨酸组成的第二组氨基酸的氨基酸,
其中每个氨基酸独立地选自所述第二组,
其中如果选自第一组的和选自第二组的氨基酸的总和产生100%的序列基序,则至少三个不同的氨基酸选自该第二组;
其中如果序列基序含有至少两个单一非相邻的苯丙氨酸残基,并且这些苯丙氨酸残基中的至少一个直接在赖氨酸残基之后,则序列基序不包含序列AFV,并且
其中如果序列基序含有至少三个单一非相邻的组氨酸残基,则序列基序不包含序列AALTH(SEQ ID NO:2),
iv)其中如果存在于基序中的话,所述序列基序的剩余氨基酸选自由天冬酰胺、天冬氨酸、谷氨酰胺、谷氨酸、甲硫氨酸或半胱氨酸组成的第三组,其中所述氨基酸的每一个独立地选自所述第三组,并且其中谷氨酰胺可以仅选择一次并且此外其中选择可以不包含谷氨酰胺和谷氨酸的组合,并且
c)其中所述融合蛋白不包含SEQ ID NO:1的序列。
应当理解,已经在上面详细解释的本发明多肽的序列基序的特点和特征确实同样适用于(异源)肽序列的序列基序。
本发明的融合蛋白的肽聚糖水解酶可以是能够降解细菌肽聚糖的任何肽聚糖水解酶。就酶活性而言,此类肽聚糖水解酶可以是例如内肽酶、N-乙酰基-胞壁酰-L-丙氨酸-酰胺酶(酰胺酶)、N-乙酰基-胞壁质酶、N-乙酰基-氨基葡萄糖苷酶或裂解性转糖基酶,并因此适用于降解细菌细胞壁的肽聚糖。优选地,肽聚糖水解酶降解革兰氏阴性细菌的肽聚糖,例如肺炎克雷伯氏菌、大肠杆菌或铜绿假单胞菌。
细菌细胞壁的肽聚糖结构总体上在很大程度上保守,具有微小的修饰(Schleifer&Kandler 1972)。细菌物种具有由不同氨基酸组成的肽间桥或者甚至可以缺乏肽间桥。在缺乏肽间桥的肽聚糖结构中,二氨基庚二酸(DAP)或内消旋-二氨基庚二酸(mDAP;氨基酸,代表赖氨酸的epsilon-羧基衍生物,其是肽聚糖的典型组分)(二氨基庚二酸是残基,替换氨基酸L-Lys并直接交联至相对肽链的末端氨基酸D-Ala。因此,存在有限类型的可以通过肽聚糖水解酶水解的可用的化学键。肽聚糖水解酶展现出至少一个具有如上所列的酶活性的酶结构域。此外,肽聚糖水解酶在一些情况下含有至少一个适于结合肽聚糖并支持肽聚糖水解酶的酶活性的结构域。结合结构域通常称为细胞壁结合结构域(CBD)。
肽聚糖水解酶的实例是脊椎动物溶菌酶(例如鸡蛋清溶菌酶和人溶菌酶)、内溶素(例如KZ144内溶素或Lys394内溶素)、病毒体相关肽聚糖水解酶(VAPGH)、细菌素(例如溶葡萄球菌素)和自溶素。最优选地,本发明的融合蛋白的肽聚糖水解酶是内溶素。最优选地,肽聚糖水解酶是内溶素。特别优选的肽聚糖水解酶序列列为SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26和SEQ ID NO:27。
对革兰氏阴性和革兰氏阳性细菌的肽聚糖降解活性可以通过本领域熟知的测定法测量,例如,通过溶胞壁(muralytic)测定法,其中革兰氏阴性细菌的外膜被透化或除去(例如用氯仿),以允许推定的酶进入肽聚糖层。如果酶是有活性的,则肽聚糖层的降解将导致浊度的下降,这可以通过光度测定测量(参见例如Briers et al.,J.Biochem.BiophysMethods 70:531-533,(2007)或Schmelcher et al.,Bacteriophage endolysins asnovel antimicrobials.Schmelcher M,Donovan DM,Loessner MJ.FutureMicrobiol.2012Oct;7(10):1147-7)。
根据本发明的融合蛋白优选同样展现出肽聚糖降解酶的活性,即能够降解细菌肽聚糖。优选地,本发明的融合蛋白将能够降解革兰氏阴性细菌的细菌的肽聚糖,例如肺炎克雷伯氏菌、大肠杆菌或铜绿假单胞菌。
包含本发明的序列基序的肽序列优选与肽聚糖水解酶序列是异源的。因此,包含本发明的序列基序的肽序列和肽聚糖水解酶序列优选不在天然存在的多肽链中一起存在。甚至更优选地,序列基序和肽聚糖水解酶序列不在天然存在的多肽链中一起存在。
在本发明的融合蛋白中,包含本发明的序列基序的肽序列优选是不在自然界中存在的人工肽序列。包含根据本发明的序列基序的异源肽的特别优选的实例是SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ IDNO:17、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21、SEQ ID NO:22、SEQ IDNO:23、SEQ ID NO:71、SEQ ID NO:72、SEQ ID NO:73和SEQ ID NO:74。
在本发明的融合蛋白的优选实施方案中,(异源)肽序列(或序列基序)通过另外的居间氨基酸残基(接头)与肽聚糖水解酶序列连接,例如氨基酸残基甘氨酸、丝氨酸和丝氨酸(Gly-Ser-Ser),甘氨酸、丙氨酸、甘氨酸和丙氨酸(Gly-Ala-Gly-Ala;SEQ ID NO:28),甘氨酸、丙氨酸、甘氨酸、丙氨酸、甘氨酸、丙氨酸、甘氨酸和丙氨酸(Gly-Ala-Gly-Ala-Gly-Ala-Gly-Ala;SEQ ID NO:29)或甘氨酸、丙氨酸、甘氨酸、丙氨酸、甘氨酸、丙氨酸、甘氨酸、丙氨酸、甘氨酸、丙氨酸、甘氨酸和丙氨酸(Gly-Ala-Gly-Ala-Gly-Ala-Gly-Ala-Gly-Ala-Gly-Ala;SEQ ID NO:30)。
本发明的多肽,以及特别是本发明的融合蛋白,当然可以包含其他氨基酸序列元件,例如,一个或多个标签,例如His标签、Strep标签、Avi标签、Myc标签、Gst标签、JS标签、半胱氨酸标签、FLAG标签或本领域已知的其他标签,硫氧还蛋白,麦芽糖结合蛋白(MBP)等。
在该上下文中,本发明的多肽可以另外包含标签,例如用于纯化。优选的是His6标签(SEQ ID NO:31),优选在根据本发明的多肽的C端和/或N端处。所述标签可以通过另外的氨基酸残基与多肽连接,例如由于克隆原因。优选地,所述标签可以通过至少1、2、3、4、5、6、7、8、9或10个另外的氨基酸残基与蛋白质连接。在一些实施方案中,所述另外的氨基酸残基可以不被蛋白酶识别和/或切割。在其他实施方案中,所述另外的氨基酸残基被蛋白酶识别和/或切割。在优选的实施方案中,本发明的多肽包含在其C端处通过另外的氨基酸残基赖氨酸和甘氨酸(Lys-Gly)或亮氨酸和谷氨酸(Leu-Glu)与多肽连接的His6标签。在另一个优选的实施方案中,本发明的多肽包含在其N端处通过另外的氨基酸残基赖氨酸和甘氨酸(Lys-Gly)或亮氨酸和谷氨酸(Leu-Glu)与多肽连接的His6标签。在另一个优选的实施方案中,多肽包含在其N和C端处通过另外的氨基酸残基赖氨酸和甘氨酸(Lys-Gly)或亮氨酸和谷氨酸(Leu-Glu)与多肽连接的His6标签。
本发明的特别优选的融合蛋白可以包含SEQ ID NO:32、SEQ ID NO:33、SEQ IDNO:34、SEQ ID NO:35、SEQ ID NO:36、SEQ ID NO:37、SEQ ID NO:38、SEQ ID NO:39、SEQ IDNO:40、SEQ ID NO:41、SEQ ID NO:42和SEQ ID NO:43的序列。根据本发明的融合蛋白的另一组包含SEQ ID NO:75、SEQ ID NO:76、SEQ ID NO:77或SEQ ID NO:78。
根据本发明的多肽可以通过本领域已知的标准方法产生,例如,通过在适当的宿主细胞中重组表达编码相应多肽的核酸。如果本发明的多肽另外包含例如氨基酸序列段(stretch)或标签等,则可以通过使用如例如Sambrook et al.2001,Molecular Cloning:ALaboratory Manual所述的标准克隆技术连接所需的个别核酸序列来产生此类融合蛋白。此类多肽可以用本领域已知的方法(例如在重组DNA表达系统中)同样产生。根据本发明的相对短的多肽,例如,长度多达50个氨基酸,也可以例如通过合成方法产生。
III.核酸、载体、噬菌体和宿主细胞
本发明还涉及编码本发明的一种或多种本发明多肽的核酸。本发明的核酸可以采用核酸可以想到的所有形式。特别地,根据本发明的核酸可以是RNA、DNA或其杂合体。它们可以是单链或双链的。它们可以具有小转录物或整个基因组(例如噬菌体基因组)的大小。如本文所用,编码本发明的一种或多种本发明多肽的核酸可以是反映有义链的核酸。同样,也涵盖反义链。核酸可以涵盖用于表达本发明多肽的异源启动子。特别优选的核酸编码根据本发明的融合蛋白。
在进一步的方面,本发明涉及包含根据本发明的核酸的载体。此类载体可以是例如允许表达本发明多肽的表达载体。所述表达可以是组成型或诱导型。载体也可以是包含本发明核酸序列的克隆载体,用于克隆目的。
本发明还涉及包含本发明核酸,特别是包含编码根据本发明的融合蛋白的本发明核酸的噬菌体。
本发明还涉及(分离的)宿主细胞,其包含根据本发明的多肽、核酸、载体或噬菌体。宿主细胞特别地可以选自由细菌细胞和酵母细胞组成的组。适当时,其他合适的宿主细胞可以是永生化细胞系,例如哺乳动物(特别是人)源的。特别优选的宿主细胞包含根据本发明的融合蛋白。
IV.组合物
在进一步的方面,本发明涉及包含根据本发明的多肽、根据本发明的核酸、根据本发明的载体、根据本发明的噬菌体和/或根据本发明的宿主细胞的组合物。
本发明的特别优选的组合物包含根据本发明的融合蛋白。其他优选的组合物包含根据本发明的多肽和肽聚糖水解酶。
根据本发明的组合物可以是包含药学上可接受的稀释剂、赋形剂或载体的药物组合物。
在更进一步的方面,根据本发明的组合物是化妆品组合物。几种细菌物种可以对患者身体的环境暴露表面(例如皮肤)造成刺激。为了防止此类刺激或者为了消除所述细菌病原体的微小表现,可以采用特殊的化妆品制剂,其包含足量的本发明的多肽、核酸、载体、宿主细胞和/或组合物以实现粉刺溶解效应。
V.试剂盒
在进一步的方面,本发明涉及试剂盒,其包含根据本发明的多肽、根据本发明的核酸、根据本发明的载体、根据本发明的噬菌体和/或根据本发明的宿主细胞,并且进一步包含肽聚糖水解酶或分别编码或包含此类肽聚糖水解酶的核酸、载体、噬菌体和/或宿主细胞。优选地,试剂盒包含根据本发明的多肽和/或肽聚糖水解酶。
本发明特别优选的试剂盒包含根据本发明的多肽,但不包含本发明的融合蛋白,即试剂盒中的多肽不包含肽聚糖水解酶的序列。
在进一步的实施方案中,本发明的试剂盒包含至少一种其他抗微生物剂,例如抗生素或抗微生物肽。
VI.用途
在进一步的方面,本发明涉及根据本发明的多肽、根据本发明的核酸、根据本发明的载体、根据本发明的噬菌体、根据本发明的宿主细胞和/或根据本发明的组合物,其用于通过手术或疗法治疗人或动物体的方法中或在人或动物体上实施的诊断方法中。在此类情境下,可以利用本发明多肽的抗细菌活性,特别是如果使用本发明的融合蛋白。
此类方法通常包括向受试者施用有效量的本发明多肽(例如本发明的融合蛋白)、核酸、载体、噬菌体、宿主细胞或组合物。受试者可以是例如人或动物,更优选人受试者。特别地,本发明的多肽、本发明的核酸、本发明的载体、本发明的噬菌体、本发明的宿主细胞和/或本发明的组合物可以用于治疗或预防细菌感染的方法,例如革兰氏阴性细菌感染。治疗的方法可以包括治疗和/或预防皮肤、软组织、呼吸系统、肺、消化道、眼、耳、齿、鼻咽、口、骨骼、阴道、菌血症的伤口和/或心内膜炎的感染,但不限于此。
在根据本发明的治疗(或预防)的方法中使用的剂量和施用的途径取决于待治疗的特定疾病/感染的部位。施用的途径可以是例如口服、局部、鼻咽、肠胃外、静脉内、直肠或任何其他施用的途径。
为了将本发明的多肽(例如本发明的融合蛋白)、核酸、载体、噬菌体、宿主细胞或组合物应用于感染的部位(或要被感染的危及部位),可以使用制剂,其保护活性化合物免受环境影响,例如蛋白酶、氧化、免疫应答等,直至其到达感染的部位。因此,制剂可以是胶囊、糖衣丸、丸剂、栓剂、可注射溶液或任何其他医学上合理的盖伦制剂。优选地,盖伦制剂可以包含合适的载体、稳定剂、调味剂、缓冲剂或其他合适的试剂。例如,对于局部施用,制剂可以是洗剂或膏药,对于鼻咽应用,制剂可以是盐水溶液,经由喷雾应用于鼻。
优选地,本发明的多肽(例如融合蛋白)、核酸、载体、噬菌体、宿主细胞或组合物与其他常规抗细菌剂(例如抗生素、羊毛硫抗生素、细菌素或内溶素等)组合使用。常规抗细菌剂的施用可以在施用本发明多肽(例如融合蛋白)、核酸、载体、噬菌体、宿主细胞或组合物之前、同时或之后发生。
在进一步的方面,本发明涉及本发明多肽、核酸、载体、噬菌体、宿主细胞或组合物,其在医学诊断学、食物诊断学、饲料诊断学或环境诊断学中用作诊断手段,特别是用作用于诊断细菌感染(特别是由革兰氏阴性细菌引起的那些细菌感染)的诊断手段。在这方面,本发明的多肽、核酸、载体、宿主细胞或组合物可以用作特异性降解病原性细菌,特别是革兰氏阴性病原性细菌的肽聚糖的工具。通过本发明的多肽、核酸、载体、宿主细胞或组合物对细菌细胞的降解可以通过添加去污剂,如Triton X-100或其他削弱细菌细胞包膜的添加剂,如多粘菌素B来支持。需要特异性细胞降解作为初始步骤,随后进行细菌的特异性检测,使用基于核酸的方法(如PCR、核酸杂交或NASBA(基于核酸序列的扩增))、免疫学方法(如IMS、免疫荧光或ELISA技术)或依赖于细菌细胞的细胞含量的其他方法(如使用对不同细菌群或物种具有特异性的蛋白质(例如针对肠杆菌的β-半乳糖苷酶、针对凝固酶阳性菌株的凝固酶)的酶促测定)进行。
在进一步的方面,本发明涉及本发明的多肽、本发明的核酸、本发明的载体、本发明的噬菌体、本发明的宿主细胞和/或本发明的组合物作为食物、饲料或化妆品中的抗微生物剂或者用作消毒剂的用途。它们特别是可以用于处理或预防以下各项的革兰氏阴性细菌污染:食品、食物加工设备、食物加工厂、与食品接触的(无生命)表面(如货架和食物存放区)、饲料、饲料加工设备、饲料加工厂、与饲料接触的(无生命)表面(如货架和饲料存放区)、医疗设备、医院、医生诊室和其他医疗设施中的(无生命)表面。
附图简述
在下文中,将给出附图的简要描述。该图旨在更详细地说明本发明。然而,并不意图将本发明的范围限制于这些具体实例。
图1显示了本发明优选序列基序的位置要求。对于长度为16(白色)至20(深灰色)个氨基酸的序列基序,该表指示可以不存在选自第一组的氨基酸的位置(相应位置用“X”标记)。在所述位置处(即用“X”标记的那些),仅可以存在选自第二组的,或者视情况而定,选自第三组的氨基酸。更优选地,在所述位置处仅存在选自第二组的氨基酸。序列基序的选自第一组的氨基酸仅可以存在于未用“X”标记的位置处。然而,在所述未标记的位置处,也可以存在第二组的,或者视情况而定,第三组的氨基酸。共提供18种备选,每种备选的长度为16、17、18、19或20个氨基酸。该表还清楚地说明了潜在可以存在第一组的三联体氨基酸的位置(没有“X”的成排的3个位置)。对于备选1,这将是位置8至10。如本发明的多肽的序列基序所需,相对于3个氨基酸区块的第一个氨基酸(即位置#8)的位置-5(即位置#3)、-4(即位置#4)、+6(即位置#14)、+7(即位置#15)和+10(即位置#18)处的氨基酸不选自第一组。在第一个备选中不能找到相对位置12、-11、-8、+13和+14,因此不予考虑。
图2更详细地显示了优选序列基序的位置要求。“X”表示序列基序在相应的位置处不展现出选自第一组的氨基酸。图2a:长度为16个氨基酸的序列基序的位置要求。图2b:长度为17个氨基酸的序列基序的位置要求。图2c:长度为18个氨基酸的序列基序的位置要求。图2d:长度为19个氨基酸的序列基序的位置要求。图2e:长度为20个氨基酸的序列基序的位置要求。
实施例
在下文中,呈现了说明本发明的各种实施方案和方面的特定实施例。然而,本发明的范围不应限于本文所述的具体实施方案。实际上,根据前面的描述、附图和下面的实施例,除了本文所述的那些之外,本发明的各种修改对于本领域技术人员而言将变得显而易见。所有此类修改都落入所附权利要求书的范围内。
实施例1:使抗微生物肽杀菌肽(Cecropin)A(埃及伊蚊(A.aegypti))适应本发明
的序列基序以增加抗细菌活性
本领域已经提出抗微生物肽杀菌肽A(埃及伊蚊)(GGLKKLGKKLEGAGKRVFNAAEKALPVVAGAKALRK;SEQ ID NO:44)作为与例如内溶素融合的候选物(参见WO 2010/149792)。然而,杀菌肽A(埃及伊蚊)与KZ144内溶素的融合物对于铜绿假单胞菌和大肠杆菌细菌的效果不如SMAP-29肽与KZ144的融合物。此外,杀菌肽A(埃及伊蚊)不遵从本发明的序列基序,因为杀菌肽A(埃及伊蚊)没有展现出满足要求的序列基序,所述要求即必须存在至少40%来自第一组的氨基酸。因此,发明人推断,引入所述组的其他氨基酸可能会增加抗细菌活性。
为了测试该假设,发明人将杀菌肽A(埃及伊蚊)与Lys394内溶素融合,产生包含SEQ ID NO:45的序列的融合蛋白。并行地,产生类似的融合蛋白,其中杀菌肽A(埃及伊蚊)肽序列在C端截短并在各个位置处突变(肽:GGLKKLGKKLKKAGKRVFKAAKKAL;SEQ ID NO:11)并与Lys394内溶素融合。所得的融合蛋白包含SEQ ID NO:32的序列。由于引入了另外的赖氨酸残基,经修饰的杀菌肽A(埃及伊蚊)序列现在遵从本发明的序列基序。测试这两种融合蛋白对肺炎克雷伯氏菌细菌的抗细菌活性。
在(Luria-Bertani)培养基中培养细菌,并在Mueller-Hinton培养基中以1:10稀释。在约0.6的光密度OD600下,将细菌在相同培养基中以1:10稀释,然后以1:500稀释。将蛋白质缓冲液(20mM HEPES,500mM NaCl,pH 7.4)和蛋白质移液到96孔板中,其使用不同浓度的蛋白质和20μl的终体积,包括500μM EDTA终浓度进行。将180μl的细菌细胞或培养基(Mueller-Hinton)对照给予96孔板并混合。将板在37℃温育18-22小时,并测量孔的OD600值来测定细菌生长。测定了MIC,其是显示与无细菌对照相同的OD600值的孔的蛋白质浓度。
表1:测试的融合蛋白的最小抑菌浓度
杀菌肽A(埃及伊蚊)与Lys394内溶素(SEQ ID NO:45)的融合物显示出抗细菌活性,MIC为25μg/ml。对于与突变的杀菌肽A(埃及伊蚊)序列(SEQ ID NO:32)的融合物,MIC低得多。≤5μg/ml意味着,已经处于(最低)起始浓度,未能观察到细菌生长。尚未测试较低浓度,即实际MIC甚至可以低于5μg/ml。因此,设计遵从本发明的序列基序的杀菌肽A(埃及伊蚊)变体改善了原始抗微生物肽的抗细菌活性。
实施例2:抗细菌活性的改善不依赖于内溶素部分
为了测试抗细菌活性的增加是否对于实施例1中利用的肽和内溶素的组合是独特的,发明人在与另一个内溶素OBPgpLys的融合物中测试了相同的肽(即SEQ ID NO:11和SEQID NO:44)。基本上如实施例1中所述但在铜绿假单胞菌PAO1细菌上测试所得的多肽(SEQID NO:46和SEQ ID NO:33)。
表2:测试的融合蛋白的最小抑菌浓度
杀菌肽A(埃及伊蚊)与OBPgpLys内溶素(SEQ ID NO:46)的融合物显示出抗细菌活性,MIC为17.5μg/ml。对于与突变的杀菌肽A(埃及伊蚊)序列(SEQ ID NO:33)的融合物,MIC显着更低(12.5μg/ml)。因此,抗细菌活性的改善不依赖于所用的内溶素的序列。
实施例3:使肽BMAP-28适应本发明的序列基序以增加抗细菌活性
将BMAP-28,抗菌肽家族的牛肽(GGLRSLGRKILRAWKKYGPIIVPIIRIG;SEQ ID NO:47)与KZ144内溶素(SEQ ID NO:25)融合,产生包含SEQ ID NO:48的融合蛋白。并行地,产生类似的融合蛋白,其中BMAP-28的肽序列在两个位置处突变(肽:RGLRRLGRKILRAWKKYGPIIVPIIRIG;SEQ ID NO:12)并与KZ144内溶素的相同衍生物融合(融合蛋白:SEQ ID NO:34)。由于在BMAP-28肽的N端区域中引入了两个精氨酸氨基酸,所述序列现在遵从本发明的序列基序。测试这两种融合蛋白对大肠杆菌细菌的抗细菌活性。
在(Luria-Bertani)培养基中培养细菌,并在Mueller-Hinton培养基中以1:10稀释。在约0.6的光密度OD600下,将细菌在相同培养基中以1:10稀释,然后以1:500稀释。将蛋白质缓冲液(20mM HEPES,500mM NaCl,pH 7.4)和蛋白质移液到96孔板中,其使用不同浓度的蛋白质和20μl的终体积,包括500μM EDTA终浓度进行。将180μl的细菌细胞或培养基(Mueller-Hinton)对照给予96孔板并混合。将板在37℃温育18-22小时,并测量孔的OD600值来测定细菌生长。测定了MIC,其是显示与无细菌对照相同的OD600值的孔的蛋白质浓度。
表3:测试的融合蛋白的最小抑菌浓度
“>30”意指对于具有原始BMAP-28肽(SEQ ID NO:48)的非突变融合蛋白,在高达30μg/ml的浓度未能观察到抗细菌活性。较高浓度下的抗细菌活性是可能的,但未经实验验证。相反,对于具有突变的BMAP-28肽片段的融合蛋白,观察到显著的抗细菌活性,MIC为10μg/ml。该结果强调了本发明人鉴定的序列基序的重要性,并表明设计相应的多肽将有助于生成新的抗细菌剂。
实施例4:序列基序中带正电荷的氨基酸的类型仅具有较小的意义
在进一步的实验中,发明人将由MSI 78(4-20)片段(KFLKKAKKFGKAFVKIL;SEQ IDNO:49)和Lys394内溶素构成的融合蛋白(具有SEQ ID NO:50的融合蛋白)与类似的融合蛋白进行了比较,在所述类似的融合蛋白中经修饰的MSI 78(4-20)肽(RFLRRARRFGRAFVRIL;SEQ ID NO:13)与Lys394内溶素融合(融合蛋白:SEQ ID NO:35)。在经修饰的MSI78(4-20)肽(SEQ ID NO:13)中,MSI 78(4-20)肽的赖氨酸残基已被精氨酸残基取代。测试这两种融合蛋白对大肠杆菌细菌的抗细菌活性。
在(Luria-Bertani)培养基中培养细菌,并在Mueller-Hinton培养基中以1:10稀释。在约0.6的光密度OD600下,将细菌在相同培养基中以1:10稀释,然后以1:500稀释。将蛋白质缓冲液(20mM HEPES,500mM NaCl,pH 7.4)和蛋白质移液到96孔板中,其使用不同浓度的蛋白质和20μl的终体积,包括500μM EDTA终浓度进行。将180μl的细菌细胞或培养基(Mueller-Hinton)对照给予96孔板并混合。将板在37℃温育18-22小时,并测量孔的OD600值来测定细菌生长。测定了MIC,其是显示与无细菌对照相同的OD600值的孔的蛋白质浓度。
表4:测试的融合蛋白的最小抑菌浓度
这两种融合蛋白显示出在基本相同的范围中的抗细菌活性。因此,选自本发明的序列基序中第一组的带正电荷的氨基酸的类型(例如K或R)具有较小的重要性。
实施例5:使肽爪蟾抗菌肽(magainin)适应本发明的序列基序以改善抗细菌活性
将爪蟾抗菌肽,来自非洲爪蟾的抗微生物肽(GIGKFLHSAKKFGKAFVGEIMNS;SEQ IDNO:51)与Lys394内溶素(SEQ ID NO:24)融合,产生包含SEQ ID NO:52的融合蛋白。并行地,产生类似的融合蛋白。将爪蟾抗菌肽的肽序列截短并与接头偶联(肽:GIKKFLKSAKKFGKAFKKVIRGGGGS;SEQ ID NO:14)。将所述肽与Lys394内溶素融合(融合蛋白:SEQ ID NO:36)。如实施例2中所述测试这两种融合蛋白对铜绿假单胞菌PAO1细菌的抗细菌活性。
表5:测试的融合蛋白的最小抑菌浓度
“>30”再次意指对于具有原始爪蟾抗菌肽肽(SEQ ID NO:52)的非突变融合蛋白,在高达30μg/ml的浓度未能观察到抗细菌活性。较高浓度下的抗细菌活性是可能的,但未经实验验证。相反,对于具有突变的爪蟾抗菌肽肽片段的融合蛋白,观察到显著的抗细菌活性,MIC≤5μg/ml。
实施例6:使肽HPA-NT3适应本发明的序列基序以增加抗细菌活性
将HPA-NT3,幽门螺杆菌衍生肽(FKRLKKLFKKIWNWK;SEQ ID NO:53)与KZ144内溶素的衍生物(SEQ ID NO:25)融合,产生包含SEQ ID NO:54的融合蛋白。并行地,产生类似的融合蛋白,其中HPA-NT3的肽序列适应于本发明的序列基序(肽:KRLKKLAKKIWKWGRRGPGS;SEQID NO:15)并与KZ144内溶素的相同衍生物融合(融合蛋白:SEQ ID NO:37)。如实施例2中所述测试两种融合蛋白对铜绿假单胞菌PAO1细菌的抗细菌活性。
表6:测试的融合蛋白的最小抑菌浓度
“>18”意指对于具有原始HPA-NT3肽(SEQ ID NO:54)的非突变融合蛋白,在高达18μg/ml的浓度未能观察到抗细菌活性。较高浓度下的抗细菌活性是可能的,但未经实验验证。相反,对于具有突变的HPA-NT3肽(SEQ ID NO:15)的融合蛋白,观察到抗细菌活性,MIC为12.5μg/ml。因此使抗微生物肽适应本发明的基序增加了融合蛋白的抗细菌活性。
实施例7:从stonustoxin的序列基序开始从头生成人工抗微生物肽
为了进一步验证所鉴定的序列基序的适合性,发明人试图将先前未知任何抗微生物活性的肽序列变为对于与内溶素融合有用的肽序列。为此目的,发明人依赖于stonustoxin的alpha亚基的氨基酸298-326(IPLIHDKISNFQQIFQDYMLTVQKKIAEK;SEQ IDNO:55)。Stonustoxin是礁石鱼毒液的组成部分。毒液的作用包括剧烈疼痛、休克、瘫痪(paralysis)和组织死亡。然而,抗微生物活性尚不清楚。
将SEQ ID NO:55与KZ144内溶素的衍生物融合,产生包含SEQ ID NO:56的融合蛋白。并行地,产生了类似的融合蛋白,其中石鱼序列在各个位置处突变(肽:IKLIKRVIKKFKKIFRKYPLTVKKGIAVG;SEQ ID NO:16)并与KZ144内溶素的相同衍生物融合(融合蛋白:SEQ ID NO:38)。由于石鱼序列中几种氨基酸的交换,所述序列的前18个氨基酸现在遵从本发明的序列基序。特别地,所述序列基序中带正电荷的氨基酸的百分比已经增加(具有赖氨酸和精氨酸残基)并且去除了脯氨酸残基。测试两种融合蛋白对铜绿假单胞菌细菌的抗细菌活性。
在(Luria-Bertani)培养基中培养细菌,并在Mueller-Hinton培养基中以1:10稀释。在约0.6的光密度OD600下,将细菌在相同培养基中以1:10稀释,然后以1:500稀释。将蛋白质缓冲液(20mM HEPES,500mM NaCl,pH 7.4)和蛋白质移液到96孔板中,其使用不同浓度的蛋白质和20μl的终体积,包括500μM EDTA终浓度进行。将180μl的细菌细胞或培养基(Mueller-Hinton)对照给予96孔板并混合。将板在37℃温育18-22小时,并测量孔的OD600值来测定细菌生长。测定了MIC,其是显示与无细菌对照相同的OD600值的孔的蛋白质浓度。
表7:测试的融合蛋白的最小抑菌浓度
>91意指对于具有原始stonustoxin肽(SEQ ID NO:55)的非突变融合蛋白(SEQ IDNO:56),在高达91μg/ml的浓度未能观察到抗细菌活性。不能排除较高浓度下的抗细菌活性,但未进行测试。这是预期的,因为在所述融合物中使用的stonustoxin片段在任何抗微生物活性上都是未知的,并且单独的KZ144内溶素原则上对铜绿假单胞菌无活性。相反,对于具有突变的stonustoxin肽片段的融合蛋白,观察到意想不到的从头抗细菌活性,MIC低至17。该结果强调了发明人鉴定的序列基序的重要性,并表明设计相应的多肽将有助于生成新的抗细菌剂。
实施例8:从CagL蛋白的序列基序开始从头生成人工抗微生物肽
本发明人基于幽门螺杆菌CagL蛋白的氨基酸26-48(GLKQLDSTYQETNQQVLKNLDE;SEQ ID NO:57)产生了两种其他的从头抗微生物肽。CagL蛋白是靶向菌毛表面的幽门螺杆菌的特化粘附素,在所述菌毛表面处其与整联蛋白α5β1结合并介导CagA蛋白的受体依赖性递送至胃上皮细胞中。尚未报道抗微生物活性。
将SEQ ID NO:57与KZ144内溶素的衍生物融合,产生包含SEQ ID NO:58的融合蛋白。并行地,产生了两种类似的融合蛋白,其中CagL序列在各个位置处突变(肽1:GLKKLKRVYRKWVKAVKKVLKLGGGGS;SEQ ID NO:17,包括C端接头;肽2:GLKVLKKAYRRIRKAVRKILKA;SEQ ID NO:18)以符合本发明的基序。将肽与KZ144内溶素的相同衍生物融合(融合蛋白:SEQ ID NO:39和SEQ ID NO:40)。如实施例2中所述测试两种融合蛋白对铜绿假单胞菌细菌的抗细菌活性。
表8:测试的融合蛋白的最小抑菌浓度
>90意指对于具有原始CagL肽(SEQ ID NO:57)的非突变融合蛋白(SEQ ID NO:58),在高达90μg/ml的浓度未能观察到抗细菌活性。这是预期的,因为在所述融合中使用的CagL片段在任何抗微生物活性上都是未知的,并且单独的KZ144内溶素对铜绿假单胞菌无活性。相反,对于具有突变的CagL肽片段的两种融合蛋白,观察到意想不到的从头抗细菌活性,MIC低至12.5和15μg/ml。
实施例9:从IE1蛋白的序列基序开始从头生成人工抗微生物肽
基于IE1蛋白的氨基酸178-198(YKEKFMVCLKQIVQYAVNS;SEQ ID NO:59)产生下一个从头抗微生物肽。IE1来源于人巨细胞病毒,并且抗细菌活性是未知的。
将SEQ ID NO:59再次与KZ144内溶素的衍生物融合,产生包含SEQ ID NO:60的融合蛋白。并行地,产生了融合蛋白,其中IE1序列在各个位置处突变(肽:YKRAFKKVLKRIRRYAKRS;SEQ ID NO:19)并与KZ144内溶素的相同衍生物融合(融合蛋白:SEQID NO:41)。如实施例2中所述测试两种融合蛋白对铜绿假单胞菌细菌的抗细菌活性。
表9:测试的融合蛋白的最小抑菌浓度
>30意指对于具有原始IE1肽(SEQ ID NO:59)的非突变融合蛋白(SEQ ID NO:60),在高达30μg/ml的浓度未能观察到抗细菌活性。不能排除较高浓度下的抗细菌活性,但未进行测试并将是不可预期的,因为在所述融合物中使用的IE1片段在任何抗微生物活性上都是未知的。相反,对于具有突变的IE1肽片段的融合蛋白,观察到意想不到的从头抗细菌活性,MIC在15和20μg/ml之间。
实施例10:生成包含具有本发明的序列基序的肽的另一种融合蛋白
本发明人还产生了另一种融合蛋白,其包含SEQ ID NO:42的序列。所述融合蛋白包含符合本发明的肽(SEQ ID NO:20)。如实施例2中所报道,测试融合蛋白对铜绿假单胞菌细菌的抗细菌活性。
表10:测试的融合蛋白的最小抑菌浓度
对于具有新肽的融合蛋白,观察到显著的抗细菌活性,MIC为≤5μg/ml。
实施例11:生成包含具有本发明的序列基序的肽的融合蛋白的其他变体
本发明人进一步产生了包含符合本发明的基序的肽的其他融合蛋白(SEQ ID NO:61、SEQ ID NO:62、SEQ ID NO:63、SEQ ID NO:64、SEQ ID NO:65、SEQ ID NO:66、SEQ IDNO:67、SEQ ID NO:68)。测试融合蛋白对大肠杆菌细菌的抗细菌活性。
表11:测试的融合蛋白的最小抑菌浓度
观察到所有融合蛋白的抗细菌活性。
实施例12:使Briers等人的肽MW2适应本发明的序列基序
Briers等人(MBio.2014;5(4):e01379-14)报道了各种融合蛋白的产生,尤其包括肽MW2(SEQ ID NO:69)。所述肽不遵从本发明的序列基序。从该肽开始,发明人产生了包含所述肽和KZ144内溶素的衍生物(SEQ ID NO:25)的融合蛋白,产生了根据SEQ ID NO:70的融合蛋白。此外,发明人产生了许多SEQ ID NO:69的肽的衍生物(SEQ ID NO:71、SEQ IDNO:72、SEQ ID NO:73和SEQ ID NO:74)。这些衍生物与本发明的序列基序匹配,而MW2则不匹配。所得的融合蛋白提供在SEQ ID NO:75、SEQ ID NO:76、SEQ ID NO:77和SEQ ID NO:78中。如实施例2中所述测试融合蛋白对铜绿假单胞菌PAO1细菌的抗细菌活性。
表12:测试的融合蛋白的最小抑菌浓度
观察到所有融合蛋白的抗菌活性。值得注意的是,与具有“野生型”MW2肽的融合蛋白相比,基于MW2肽的四种衍生物(即适应本发明的序列基序)的融合蛋白产生了改善的抗细菌活性。
实施例13:使用与另一个肽聚糖水解酶组合的肽爪蟾抗菌肽
本发明人还将实施例5的两种肽与另一个肽聚糖水解酶组合,即弧菌噬菌体ICP1的尾基片蛋白(SEQ ID NO:27)。得到的融合蛋白包含SEQ ID NO:79和SEQ ID NO:43的序列。测试融合蛋白对大肠杆菌细菌的抗细菌活性。
表13:测试的融合蛋白的最小抑菌浓度
所得的融合蛋白展现出两种抗细菌活性。遵从本发明的序列基序的肽(SEQ IDNO:14)再次提供比野生型肽(SEQ ID NO:51)更好的活性。
实施例14:其他肽
在最后一组实验中,发明人产生了三种其他融合蛋白,每种融合蛋白包含内溶素序列和遵从根据本发明的序列基序的肽。该三种肽是SEQ ID NO:21、SEQ ID NO:22和SEQID NO:23。所有三种所得的融合蛋白显示出优异的抗细菌活性。
序列表
<110> 萨西那帕斯有限公司
<120> 经修饰的肽
<130> [FILL IN]
<150> PCT/IB2016/057193
<151> 2016-11-30
<160> 79
<170> PatentIn version 3.5
<210> 1
<211> 29
<212> PRT
<213> 未知
<220>
<223> SMAP-29绵羊
<400> 1
Arg Gly Leu Arg Arg Leu Gly Arg Lys Ile Ala His Gly Val Lys Lys
1 5 10 15
Tyr Gly Pro Thr Val Leu Arg Ile Ile Arg Ile Ala Gly
20 25
<210> 2
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 序列,如果序列基序含有至少三个非相邻的组氨酸残基,则该序列不是序列基序的一部分
<400> 2
Ala Ala Leu Thr His
1 5
<210> 3
<211> 4
<212> PRT
<213> 人工序列
<220>
<223> 第一组的氨基酸的顺序内(intrasequential)成对区块的实例
<400> 3
Leu Lys Arg Glu
1
<210> 4
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 第一组的氨基酸的顺序内三联体区块的实例
<400> 4
Leu Lys Arg Lys Glu
1 5
<210> 5
<211> 4
<212> PRT
<213> 人工序列
<220>
<223> 第一组的氨基酸的N端三联体区块的实例
<400> 5
Lys Arg Lys Glu
1
<210> 6
<211> 4
<212> PRT
<213> 人工序列
<220>
<223> 第一组的氨基酸的C端三联体区块的实例
<400> 6
Leu Lys Arg Lys
1
<210> 7
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 如果存在第一组的氨基酸的三联体,则从序列基序排除的序列的实例
<400> 7
Arg Arg Arg Gly Leu Arg His
1 5
<210> 8
<211> 4
<212> PRT
<213> 人工序列
<220>
<223> 序列基序内不允许的序列的实例
<400> 8
Lys Arg Lys Lys
1
<210> 9
<211> 4
<212> PRT
<213> 人工序列
<220>
<223> 序列基序内不允许的序列的实例
<400> 9
Arg Arg Arg Arg
1
<210> 10
<211> 18
<212> PRT
<213> 未知
<220>
<223> SMAP-29绵羊;aa1-18
<400> 10
Arg Gly Leu Arg Arg Leu Gly Arg Lys Ile Ala His Gly Val Lys Lys
1 5 10 15
Tyr Gly
<210> 11
<211> 25
<212> PRT
<213> 人工序列
<220>
<223> 衍生自杀菌肽A(埃及伊蚊)的突变的肽
<400> 11
Gly Gly Leu Lys Lys Leu Gly Lys Lys Leu Lys Lys Ala Gly Lys Arg
1 5 10 15
Val Phe Lys Ala Ala Lys Lys Ala Leu
20 25
<210> 12
<211> 28
<212> PRT
<213> 人工序列
<220>
<223> 衍生自BMAP-28的突变的肽
<400> 12
Arg Gly Leu Arg Arg Leu Gly Arg Lys Ile Leu Arg Ala Trp Lys Lys
1 5 10 15
Tyr Gly Pro Ile Ile Val Pro Ile Ile Arg Ile Gly
20 25
<210> 13
<211> 17
<212> PRT
<213> 人工序列
<220>
<223> 衍生自MSI-78 (4-20)肽的突变的肽
<400> 13
Arg Phe Leu Arg Arg Ala Arg Arg Phe Gly Arg Ala Phe Val Arg Ile
1 5 10 15
Leu
<210> 14
<211> 26
<212> PRT
<213> 人工序列
<220>
<223> 衍生自爪蟾抗菌肽的突变的肽
<400> 14
Gly Ile Lys Lys Phe Leu Lys Ser Ala Lys Lys Phe Gly Lys Ala Phe
1 5 10 15
Lys Lys Val Ile Arg Gly Gly Gly Gly Ser
20 25
<210> 15
<211> 20
<212> PRT
<213> 人工序列
<220>
<223> 衍生自HPA-NT3肽的突变的肽
<400> 15
Lys Arg Leu Lys Lys Leu Ala Lys Lys Ile Trp Lys Trp Gly Arg Arg
1 5 10 15
Gly Pro Gly Ser
20
<210> 16
<211> 29
<212> PRT
<213> 人工序列
<220>
<223> 衍生自stonustoxin的alpha亚基的氨基酸298-326的突变的肽
<400> 16
Ile Lys Leu Ile Lys Arg Val Ile Lys Lys Phe Lys Lys Ile Phe Arg
1 5 10 15
Lys Tyr Pro Leu Thr Val Lys Lys Gly Ile Ala Val Gly
20 25
<210> 17
<211> 27
<212> PRT
<213> 人工序列
<220>
<223> 衍生自CagL蛋白的氨基酸26-48的突变的肽
<400> 17
Gly Leu Lys Lys Leu Lys Arg Val Tyr Arg Lys Trp Val Lys Ala Val
1 5 10 15
Lys Lys Val Leu Lys Leu Gly Gly Gly Gly Ser
20 25
<210> 18
<211> 22
<212> PRT
<213> 人工序列
<220>
<223> 衍生自CagL蛋白的氨基酸26-48的突变的肽
<400> 18
Gly Leu Lys Val Leu Lys Lys Ala Tyr Arg Arg Ile Arg Lys Ala Val
1 5 10 15
Arg Lys Ile Leu Lys Ala
20
<210> 19
<211> 19
<212> PRT
<213> 人工序列
<220>
<223> 衍生自IE1蛋白的氨基酸178-198的突变的肽
<400> 19
Tyr Lys Arg Ala Phe Lys Lys Val Leu Lys Arg Ile Arg Arg Tyr Ala
1 5 10 15
Lys Arg Ser
<210> 20
<211> 24
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 20
Gly Phe Phe Lys Lys Ala Trp Arg Lys Val Lys His Ala Gly Arg Arg
1 5 10 15
Val Leu Lys Thr Ala Lys Gly Val
20
<210> 21
<211> 20
<212> PRT
<213> 未知
<220>
<223> CAP18AA
<400> 21
Gly Leu Arg Lys Ala Leu Arg Lys Phe Arg Asn Lys Ile Lys Glu Ala
1 5 10 15
Leu Lys Lys Ile
20
<210> 22
<211> 20
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 22
Gly Leu Arg Lys Ala Leu Arg Lys Phe Arg Lys Lys Ile Lys Glu Ala
1 5 10 15
Leu Lys Lys Ile
20
<210> 23
<211> 29
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 23
Arg Gly Leu Arg Arg Leu Gly Arg Lys Ile Ala Arg Gly Val Lys Lys
1 5 10 15
Tyr Gly Pro Thr Val Leu Arg Ile Ile Arg Ile Ala Gly
20 25
<210> 24
<211> 136
<212> PRT
<213> 未知
<220>
<223> 没有N-末端甲硫氨酸的S394内溶素
<400> 24
Ser Phe Lys Phe Gly Lys Asn Ser Glu Lys Gln Leu Ala Thr Val Lys
1 5 10 15
Pro Glu Leu Gln Lys Val Ala Arg Arg Ala Leu Glu Leu Ser Pro Tyr
20 25 30
Asp Phe Thr Ile Val Gln Gly Ile Arg Thr Val Ala Gln Ser Ala Gln
35 40 45
Asn Ile Ala Asn Gly Thr Ser Phe Leu Lys Asp Pro Ser Lys Ser Lys
50 55 60
His Val Thr Gly Asp Ala Ile Asp Phe Ala Pro Tyr Ile Asn Gly Lys
65 70 75 80
Ile Asp Trp Lys Asp Leu Glu Ala Phe Trp Ala Val Lys Lys Ala Phe
85 90 95
Glu Gln Ala Gly Lys Glu Leu Gly Ile Lys Leu Arg Phe Gly Ala Asp
100 105 110
Trp Asn Ser Ser Gly Asp Tyr His Asp Glu Ile Asp Arg Gly Thr Tyr
115 120 125
Asp Gly Gly His Val Glu Leu Val
130 135
<210> 25
<211> 259
<212> PRT
<213> 人工序列
<220>
<223> 没有N-末端甲硫氨酸的具有C14S、C23S和C50S的突变的KZ144
<400> 25
Lys Val Leu Arg Lys Gly Asp Arg Gly Asp Glu Val Ser Gln Leu Gln
1 5 10 15
Thr Leu Leu Asn Leu Ser Gly Tyr Asp Val Gly Lys Pro Asp Gly Ile
20 25 30
Phe Gly Asn Asn Thr Phe Asn Gln Val Val Lys Phe Gln Lys Asp Asn
35 40 45
Ser Leu Asp Ser Asp Gly Ile Val Gly Lys Asn Thr Trp Ala Glu Leu
50 55 60
Phe Ser Lys Tyr Ser Pro Pro Ile Pro Tyr Lys Thr Ile Pro Met Pro
65 70 75 80
Thr Ala Asn Lys Ser Arg Ala Ala Ala Thr Pro Val Met Asn Ala Val
85 90 95
Glu Asn Ala Thr Gly Val Arg Ser Gln Leu Leu Leu Thr Phe Ala Ser
100 105 110
Ile Glu Ser Ala Phe Asp Tyr Glu Ile Lys Ala Lys Thr Ser Ser Ala
115 120 125
Thr Gly Trp Phe Gln Phe Leu Thr Gly Thr Trp Lys Thr Met Ile Glu
130 135 140
Asn Tyr Gly Met Lys Tyr Gly Val Leu Thr Asp Pro Thr Gly Ala Leu
145 150 155 160
Arg Lys Asp Pro Arg Ile Ser Ala Leu Met Gly Ala Glu Leu Ile Lys
165 170 175
Glu Asn Met Asn Ile Leu Arg Pro Val Leu Lys Arg Glu Pro Thr Asp
180 185 190
Thr Asp Leu Tyr Leu Ala His Phe Phe Gly Pro Gly Ala Ala Arg Arg
195 200 205
Phe Leu Thr Thr Gly Gln Asn Glu Leu Ala Ala Thr His Phe Pro Lys
210 215 220
Glu Ala Gln Ala Asn Pro Ser Ile Phe Tyr Asn Lys Asp Gly Ser Pro
225 230 235 240
Lys Thr Ile Gln Glu Val Tyr Asn Leu Met Asp Gly Lys Val Ala Ala
245 250 255
His Arg Lys
<210> 26
<211> 327
<212> PRT
<213> 未知
<220>
<223> OBPgpLYS片段
<400> 26
Lys Asn Ser Glu Lys Asn Ala Ser Ile Ile Met Ser Ile Gln Arg Thr
1 5 10 15
Leu Ala Ser Leu Ser Leu Tyr Gly Gly Arg Ile Asp Gly Leu Phe Gly
20 25 30
Glu Lys Cys Arg Gly Ala Ile Ile Leu Met Leu Asn Lys Val Tyr Pro
35 40 45
Asn Phe Ser Thr Asn Lys Leu Pro Ser Asn Thr Tyr Glu Ala Glu Ser
50 55 60
Val Phe Thr Phe Leu Gln Thr Ala Leu Ala Gly Val Gly Leu Tyr Thr
65 70 75 80
Ile Thr Ile Asp Gly Lys Trp Gly Gly Thr Ser Gln Gly Ala Ile Asp
85 90 95
Ala Leu Val Lys Ser Tyr Arg Gln Ile Thr Glu Ala Glu Arg Ala Gly
100 105 110
Ser Thr Leu Pro Leu Gly Leu Ala Thr Val Met Ser Lys His Met Ser
115 120 125
Ile Glu Gln Leu Arg Ala Met Leu Pro Thr Asp Arg Gln Gly Tyr Ala
130 135 140
Glu Val Tyr Ile Asp Pro Leu Asn Glu Thr Met Asp Ile Phe Glu Ile
145 150 155 160
Asn Thr Pro Leu Arg Ile Ala His Phe Met Ala Gln Ile Leu His Glu
165 170 175
Thr Ala Cys Phe Lys Tyr Thr Glu Glu Leu Ala Ser Gly Lys Ala Tyr
180 185 190
Glu Gly Arg Ala Asp Leu Gly Asn Thr Arg Pro Gly Asp Gly Pro Leu
195 200 205
Phe Lys Gly Arg Gly Leu Leu Gln Ile Thr Gly Arg Leu Asn Tyr Val
210 215 220
Lys Cys Gln Val Tyr Leu Arg Glu Lys Leu Lys Asp Pro Thr Phe Asp
225 230 235 240
Ile Thr Ser Ser Val Thr Cys Ala Gln Gln Leu Ser Glu Ser Pro Leu
245 250 255
Leu Ala Ala Leu Ala Ser Gly Tyr Phe Trp Arg Phe Ile Lys Pro Lys
260 265 270
Leu Asn Glu Thr Ala Asp Lys Asp Asp Ile Tyr Trp Val Ser Val Tyr
275 280 285
Val Asn Gly Tyr Ala Lys Gln Ala Asn Pro Tyr Tyr Pro Asn Arg Asp
290 295 300
Lys Glu Pro Asn His Met Lys Glu Arg Val Gln Met Leu Ala Val Thr
305 310 315 320
Lys Lys Ala Leu Gly Ile Val
325
<210> 27
<211> 237
<212> PRT
<213> 未知
<220>
<223> 没有N-末端甲硫氨酸的弧菌噬菌体ICP1的尾基片蛋白
<400> 27
Ile Leu Lys Arg Gly Ser Ser Gly Ala Asp Val Lys Asn Met Gln Glu
1 5 10 15
Tyr Leu Thr Ala Leu Gly Tyr Asp Thr Lys Gly Val Glu Gly Thr Phe
20 25 30
Glu Gly Gly Thr Glu Ser Ala Val Lys Ala Phe Gln Lys Asp Met Ser
35 40 45
Phe Thr Val Val Asp Gly Ile Ile Gly Asn Gln Thr Ala Lys His Leu
50 55 60
Val Asp Met Tyr Tyr Gly Lys Val Val Pro Phe Gly Tyr Val Thr Asn
65 70 75 80
Thr Pro Trp Val Ser Glu Ala Ile Glu Asp Tyr Phe Val Ser Glu Ile
85 90 95
Lys Gly Glu Lys His Asn Pro Arg Val Val Gln Tyr Phe Lys Asp Ala
100 105 110
His Ser Ser Trp Phe Thr Asp Asp Glu Thr Pro Trp Cys Ala Ala Ala
115 120 125
Val Ser Ser Trp Leu Glu Arg Ala Gly Ile Arg Ser Val Arg Ser Ala
130 135 140
Arg Ala Arg Asp His Ile Asn Phe Gly Thr Lys Leu Leu Glu Pro Arg
145 150 155 160
Phe Gly Ala Ile Val Val Leu Glu Arg Gly Ala Asn Ser Gly His Val
165 170 175
Gly Phe Val Asn Gly Val Thr Ala Asp Gly Lys Gln Ile Lys Val Leu
180 185 190
Gly Gly Asn Gln Ser Asp Ser Val Asn Glu Arg Met Phe Gln Val Thr
195 200 205
Arg Val Leu Gly Tyr Arg Gln Pro Glu Gly Phe Val Leu Pro Pro Cys
210 215 220
Pro Ile Val Gly Lys Gly Glu Leu Ser Lys Ser Glu Ala
225 230 235
<210> 28
<211> 4
<212> PRT
<213> 人工
<220>
<223> 接头
<400> 28
Gly Ala Gly Ala
1
<210> 29
<211> 8
<212> PRT
<213> 人工
<220>
<223> 接头
<400> 29
Gly Ala Gly Ala Gly Ala Gly Ala
1 5
<210> 30
<211> 12
<212> PRT
<213> 人工
<220>
<223> 接头
<400> 30
Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala
1 5 10
<210> 31
<211> 6
<212> PRT
<213> 人工序列
<220>
<223> His标签(6x)
<400> 31
His His His His His His
1 5
<210> 32
<211> 169
<212> PRT
<213> 人工序列
<220>
<223> 与S394内溶素连接的衍生自杀菌肽A(埃及伊蚊)的突变的肽
<400> 32
Met Gly Gly Leu Lys Lys Leu Gly Lys Lys Leu Lys Lys Ala Gly Lys
1 5 10 15
Arg Val Phe Lys Ala Ala Lys Lys Ala Leu Gly Gly Gly Ser Gly Ser
20 25 30
Met Ser Phe Lys Phe Gly Lys Asn Ser Glu Lys Gln Leu Ala Thr Val
35 40 45
Lys Pro Glu Leu Gln Lys Val Ala Arg Arg Ala Leu Glu Leu Ser Pro
50 55 60
Tyr Asp Phe Thr Ile Val Gln Gly Ile Arg Thr Val Ala Gln Ser Ala
65 70 75 80
Gln Asn Ile Ala Asn Gly Thr Ser Phe Leu Lys Asp Pro Ser Lys Ser
85 90 95
Lys His Val Thr Gly Asp Ala Ile Asp Phe Ala Pro Tyr Ile Asn Gly
100 105 110
Lys Ile Asp Trp Lys Asp Leu Glu Ala Phe Trp Ala Val Lys Lys Ala
115 120 125
Phe Glu Gln Ala Gly Lys Glu Leu Gly Ile Lys Leu Arg Phe Gly Ala
130 135 140
Asp Trp Asn Ser Ser Gly Asp Tyr His Asp Glu Ile Asp Arg Gly Thr
145 150 155 160
Tyr Asp Gly Gly His Val Glu Leu Val
165
<210> 33
<211> 355
<212> PRT
<213> 人工序列
<220>
<223> 与OBPgpLys内溶素连接的衍生自杀菌肽A(埃及伊蚊)的突变的肽
<400> 33
Met Arg Gly Leu Lys Lys Leu Gly Arg Lys Leu Lys Lys Ala Gly Lys
1 5 10 15
Arg Val Phe Lys Ala Ala Lys Lys Ala Leu Gly Ser Lys Asn Ser Glu
20 25 30
Lys Asn Ala Ser Ile Ile Met Ser Ile Gln Arg Thr Leu Ala Ser Leu
35 40 45
Ser Leu Tyr Gly Gly Arg Ile Asp Gly Leu Phe Gly Glu Lys Cys Arg
50 55 60
Gly Ala Ile Ile Leu Met Leu Asn Lys Val Tyr Pro Asn Phe Ser Thr
65 70 75 80
Asn Lys Leu Pro Ser Asn Thr Tyr Glu Ala Glu Ser Val Phe Thr Phe
85 90 95
Leu Gln Thr Ala Leu Ala Gly Val Gly Leu Tyr Thr Ile Thr Ile Asp
100 105 110
Gly Lys Trp Gly Gly Thr Ser Gln Gly Ala Ile Asp Ala Leu Val Lys
115 120 125
Ser Tyr Arg Gln Ile Thr Glu Ala Glu Arg Ala Gly Ser Thr Leu Pro
130 135 140
Leu Gly Leu Ala Thr Val Met Ser Lys His Met Ser Ile Glu Gln Leu
145 150 155 160
Arg Ala Met Leu Pro Thr Asp Arg Gln Gly Tyr Ala Glu Val Tyr Ile
165 170 175
Asp Pro Leu Asn Glu Thr Met Asp Ile Phe Glu Ile Asn Thr Pro Leu
180 185 190
Arg Ile Ala His Phe Met Ala Gln Ile Leu His Glu Thr Ala Cys Phe
195 200 205
Lys Tyr Thr Glu Glu Leu Ala Ser Gly Lys Ala Tyr Glu Gly Arg Ala
210 215 220
Asp Leu Gly Asn Thr Arg Pro Gly Asp Gly Pro Leu Phe Lys Gly Arg
225 230 235 240
Gly Leu Leu Gln Ile Thr Gly Arg Leu Asn Tyr Val Lys Cys Gln Val
245 250 255
Tyr Leu Arg Glu Lys Leu Lys Asp Pro Thr Phe Asp Ile Thr Ser Ser
260 265 270
Val Thr Cys Ala Gln Gln Leu Ser Glu Ser Pro Leu Leu Ala Ala Leu
275 280 285
Ala Ser Gly Tyr Phe Trp Arg Phe Ile Lys Pro Lys Leu Asn Glu Thr
290 295 300
Ala Asp Lys Asp Asp Ile Tyr Trp Val Ser Val Tyr Val Asn Gly Tyr
305 310 315 320
Ala Lys Gln Ala Asn Pro Tyr Tyr Pro Asn Arg Asp Lys Glu Pro Asn
325 330 335
His Met Lys Glu Arg Val Gln Met Leu Ala Val Thr Lys Lys Ala Leu
340 345 350
Gly Ile Val
355
<210> 34
<211> 290
<212> PRT
<213> 人工序列
<220>
<223> 与具有C14S、C23S和C50S的突变的KZ144连接的衍生自BMAP-28的突变的肽
<400> 34
Met Arg Gly Leu Arg Arg Leu Gly Arg Lys Ile Leu Arg Ala Trp Lys
1 5 10 15
Lys Tyr Gly Pro Ile Ile Val Pro Ile Ile Arg Ile Gly Gly Ser Lys
20 25 30
Val Leu Arg Lys Gly Asp Arg Gly Asp Glu Val Ser Gln Leu Gln Thr
35 40 45
Leu Leu Asn Leu Ser Gly Tyr Asp Val Gly Lys Pro Asp Gly Ile Phe
50 55 60
Gly Asn Asn Thr Phe Asn Gln Val Val Lys Phe Gln Lys Asp Asn Ser
65 70 75 80
Leu Asp Ser Asp Gly Ile Val Gly Lys Asn Thr Trp Ala Glu Leu Phe
85 90 95
Ser Lys Tyr Ser Pro Pro Ile Pro Tyr Lys Thr Ile Pro Met Pro Thr
100 105 110
Ala Asn Lys Ser Arg Ala Ala Ala Thr Pro Val Met Asn Ala Val Glu
115 120 125
Asn Ala Thr Gly Val Arg Ser Gln Leu Leu Leu Thr Phe Ala Ser Ile
130 135 140
Glu Ser Ala Phe Asp Tyr Glu Ile Lys Ala Lys Thr Ser Ser Ala Thr
145 150 155 160
Gly Trp Phe Gln Phe Leu Thr Gly Thr Trp Lys Thr Met Ile Glu Asn
165 170 175
Tyr Gly Met Lys Tyr Gly Val Leu Thr Asp Pro Thr Gly Ala Leu Arg
180 185 190
Lys Asp Pro Arg Ile Ser Ala Leu Met Gly Ala Glu Leu Ile Lys Glu
195 200 205
Asn Met Asn Ile Leu Arg Pro Val Leu Lys Arg Glu Pro Thr Asp Thr
210 215 220
Asp Leu Tyr Leu Ala His Phe Phe Gly Pro Gly Ala Ala Arg Arg Phe
225 230 235 240
Leu Thr Thr Gly Gln Asn Glu Leu Ala Ala Thr His Phe Pro Lys Glu
245 250 255
Ala Gln Ala Asn Pro Ser Ile Phe Tyr Asn Lys Asp Gly Ser Pro Lys
260 265 270
Thr Ile Gln Glu Val Tyr Asn Leu Met Asp Gly Lys Val Ala Ala His
275 280 285
Arg Lys
290
<210> 35
<211> 162
<212> PRT
<213> 人工序列
<220>
<223> 与S394内溶素连接的衍生自MSI-78 (4-20)肽的突变的肽
<400> 35
Met Arg Phe Leu Arg Arg Ala Arg Arg Phe Gly Arg Ala Phe Val Arg
1 5 10 15
Ile Leu Gly Gly Gly Gly Ser Gly Ser Met Ser Phe Lys Phe Gly Lys
20 25 30
Asn Ser Glu Lys Gln Leu Ala Thr Val Lys Pro Glu Leu Gln Lys Val
35 40 45
Ala Arg Arg Ala Leu Glu Leu Ser Pro Tyr Asp Phe Thr Ile Val Gln
50 55 60
Gly Ile Arg Thr Val Ala Gln Ser Ala Gln Asn Ile Ala Asn Gly Thr
65 70 75 80
Ser Phe Leu Lys Asp Pro Ser Lys Ser Lys His Val Thr Gly Asp Ala
85 90 95
Ile Asp Phe Ala Pro Tyr Ile Asn Gly Lys Ile Asp Trp Lys Asp Leu
100 105 110
Glu Ala Phe Trp Ala Val Lys Lys Ala Phe Glu Gln Ala Gly Lys Glu
115 120 125
Leu Gly Ile Lys Leu Arg Phe Gly Ala Asp Trp Asn Ser Ser Gly Asp
130 135 140
Tyr His Asp Glu Ile Asp Arg Gly Thr Tyr Asp Gly Gly His Val Glu
145 150 155 160
Leu Val
<210> 36
<211> 166
<212> PRT
<213> 人工序列
<220>
<223> 与S394内溶素连接的衍生自爪蟾抗菌肽肽的突变的肽
<400> 36
Met Gly Ile Lys Lys Phe Leu Lys Ser Ala Lys Lys Phe Gly Lys Ala
1 5 10 15
Phe Lys Lys Val Ile Arg Gly Gly Gly Gly Ser Gly Ser Met Ser Phe
20 25 30
Lys Phe Gly Lys Asn Ser Glu Lys Gln Leu Ala Thr Val Lys Pro Glu
35 40 45
Leu Gln Lys Val Ala Arg Arg Ala Leu Glu Leu Ser Pro Tyr Asp Phe
50 55 60
Thr Ile Val Gln Gly Ile Arg Thr Val Ala Gln Ser Ala Gln Asn Ile
65 70 75 80
Ala Asn Gly Thr Ser Phe Leu Lys Asp Pro Ser Lys Ser Lys His Val
85 90 95
Thr Gly Asp Ala Ile Asp Phe Ala Pro Tyr Ile Asn Gly Lys Ile Asp
100 105 110
Trp Lys Asp Leu Glu Ala Phe Trp Ala Val Lys Lys Ala Phe Glu Gln
115 120 125
Ala Gly Lys Glu Leu Gly Ile Lys Leu Arg Phe Gly Ala Asp Trp Asn
130 135 140
Ser Ser Gly Asp Tyr His Asp Glu Ile Asp Arg Gly Thr Tyr Asp Gly
145 150 155 160
Gly His Val Glu Leu Val
165
<210> 37
<211> 282
<212> PRT
<213> 人工序列
<220>
<223> 与具有C14S、C23S和C50S的突变的KZ144连接的衍生自HPA-NT3肽的突变的肽
<400> 37
Met Lys Arg Leu Lys Lys Leu Ala Lys Lys Ile Trp Lys Trp Gly Arg
1 5 10 15
Arg Gly Pro Gly Ser Gly Ser Lys Val Leu Arg Lys Gly Asp Arg Gly
20 25 30
Asp Glu Val Ser Gln Leu Gln Thr Leu Leu Asn Leu Ser Gly Tyr Asp
35 40 45
Val Gly Lys Pro Asp Gly Ile Phe Gly Asn Asn Thr Phe Asn Gln Val
50 55 60
Val Lys Phe Gln Lys Asp Asn Ser Leu Asp Ser Asp Gly Ile Val Gly
65 70 75 80
Lys Asn Thr Trp Ala Glu Leu Phe Ser Lys Tyr Ser Pro Pro Ile Pro
85 90 95
Tyr Lys Thr Ile Pro Met Pro Thr Ala Asn Lys Ser Arg Ala Ala Ala
100 105 110
Thr Pro Val Met Asn Ala Val Glu Asn Ala Thr Gly Val Arg Ser Gln
115 120 125
Leu Leu Leu Thr Phe Ala Ser Ile Glu Ser Ala Phe Asp Tyr Glu Ile
130 135 140
Lys Ala Lys Thr Ser Ser Ala Thr Gly Trp Phe Gln Phe Leu Thr Gly
145 150 155 160
Thr Trp Lys Thr Met Ile Glu Asn Tyr Gly Met Lys Tyr Gly Val Leu
165 170 175
Thr Asp Pro Thr Gly Ala Leu Arg Lys Asp Pro Arg Ile Ser Ala Leu
180 185 190
Met Gly Ala Glu Leu Ile Lys Glu Asn Met Asn Ile Leu Arg Pro Val
195 200 205
Leu Lys Arg Glu Pro Thr Asp Thr Asp Leu Tyr Leu Ala His Phe Phe
210 215 220
Gly Pro Gly Ala Ala Arg Arg Phe Leu Thr Thr Gly Gln Asn Glu Leu
225 230 235 240
Ala Ala Thr His Phe Pro Lys Glu Ala Gln Ala Asn Pro Ser Ile Phe
245 250 255
Tyr Asn Lys Asp Gly Ser Pro Lys Thr Ile Gln Glu Val Tyr Asn Leu
260 265 270
Met Asp Gly Lys Val Ala Ala His Arg Lys
275 280
<210> 38
<211> 291
<212> PRT
<213> 人工序列
<220>
<223> 与具有C14S、C23S和C50S的突变的KZ144连接的衍生自stonustoxin的alpha亚基的氨基酸298-326的突变的肽
<400> 38
Met Ile Lys Leu Ile Lys Arg Val Ile Lys Lys Phe Lys Lys Ile Phe
1 5 10 15
Arg Lys Tyr Pro Leu Thr Val Lys Lys Gly Ile Ala Val Gly Gly Ser
20 25 30
Lys Val Leu Arg Lys Gly Asp Arg Gly Asp Glu Val Ser Gln Leu Gln
35 40 45
Thr Leu Leu Asn Leu Ser Gly Tyr Asp Val Gly Lys Pro Asp Gly Ile
50 55 60
Phe Gly Asn Asn Thr Phe Asn Gln Val Val Lys Phe Gln Lys Asp Asn
65 70 75 80
Ser Leu Asp Ser Asp Gly Ile Val Gly Lys Asn Thr Trp Ala Glu Leu
85 90 95
Phe Ser Lys Tyr Ser Pro Pro Ile Pro Tyr Lys Thr Ile Pro Met Pro
100 105 110
Thr Ala Asn Lys Ser Arg Ala Ala Ala Thr Pro Val Met Asn Ala Val
115 120 125
Glu Asn Ala Thr Gly Val Arg Ser Gln Leu Leu Leu Thr Phe Ala Ser
130 135 140
Ile Glu Ser Ala Phe Asp Tyr Glu Ile Lys Ala Lys Thr Ser Ser Ala
145 150 155 160
Thr Gly Trp Phe Gln Phe Leu Thr Gly Thr Trp Lys Thr Met Ile Glu
165 170 175
Asn Tyr Gly Met Lys Tyr Gly Val Leu Thr Asp Pro Thr Gly Ala Leu
180 185 190
Arg Lys Asp Pro Arg Ile Ser Ala Leu Met Gly Ala Glu Leu Ile Lys
195 200 205
Glu Asn Met Asn Ile Leu Arg Pro Val Leu Lys Arg Glu Pro Thr Asp
210 215 220
Thr Asp Leu Tyr Leu Ala His Phe Phe Gly Pro Gly Ala Ala Arg Arg
225 230 235 240
Phe Leu Thr Thr Gly Gln Asn Glu Leu Ala Ala Thr His Phe Pro Lys
245 250 255
Glu Ala Gln Ala Asn Pro Ser Ile Phe Tyr Asn Lys Asp Gly Ser Pro
260 265 270
Lys Thr Ile Gln Glu Val Tyr Asn Leu Met Asp Gly Lys Val Ala Ala
275 280 285
His Arg Lys
290
<210> 39
<211> 289
<212> PRT
<213> 人工序列
<220>
<223> 与具有C14S、C23S和C50S的突变的KZ144连接的衍生自CagL蛋白的氨基酸26-48的突变的肽
<400> 39
Met Gly Leu Lys Lys Leu Lys Arg Val Tyr Arg Lys Trp Val Lys Ala
1 5 10 15
Val Lys Lys Val Leu Lys Leu Gly Gly Gly Gly Ser Gly Ser Lys Val
20 25 30
Leu Arg Lys Gly Asp Arg Gly Asp Glu Val Ser Gln Leu Gln Thr Leu
35 40 45
Leu Asn Leu Ser Gly Tyr Asp Val Gly Lys Pro Asp Gly Ile Phe Gly
50 55 60
Asn Asn Thr Phe Asn Gln Val Val Lys Phe Gln Lys Asp Asn Ser Leu
65 70 75 80
Asp Ser Asp Gly Ile Val Gly Lys Asn Thr Trp Ala Glu Leu Phe Ser
85 90 95
Lys Tyr Ser Pro Pro Ile Pro Tyr Lys Thr Ile Pro Met Pro Thr Ala
100 105 110
Asn Lys Ser Arg Ala Ala Ala Thr Pro Val Met Asn Ala Val Glu Asn
115 120 125
Ala Thr Gly Val Arg Ser Gln Leu Leu Leu Thr Phe Ala Ser Ile Glu
130 135 140
Ser Ala Phe Asp Tyr Glu Ile Lys Ala Lys Thr Ser Ser Ala Thr Gly
145 150 155 160
Trp Phe Gln Phe Leu Thr Gly Thr Trp Lys Thr Met Ile Glu Asn Tyr
165 170 175
Gly Met Lys Tyr Gly Val Leu Thr Asp Pro Thr Gly Ala Leu Arg Lys
180 185 190
Asp Pro Arg Ile Ser Ala Leu Met Gly Ala Glu Leu Ile Lys Glu Asn
195 200 205
Met Asn Ile Leu Arg Pro Val Leu Lys Arg Glu Pro Thr Asp Thr Asp
210 215 220
Leu Tyr Leu Ala His Phe Phe Gly Pro Gly Ala Ala Arg Arg Phe Leu
225 230 235 240
Thr Thr Gly Gln Asn Glu Leu Ala Ala Thr His Phe Pro Lys Glu Ala
245 250 255
Gln Ala Asn Pro Ser Ile Phe Tyr Asn Lys Asp Gly Ser Pro Lys Thr
260 265 270
Ile Gln Glu Val Tyr Asn Leu Met Asp Gly Lys Val Ala Ala His Arg
275 280 285
Lys
<210> 40
<211> 284
<212> PRT
<213> 人工序列
<220>
<223> 与具有C14S、C23S和C50S的突变的KZ144连接的衍生自CagL蛋白的氨基酸26-48的突变的肽
<400> 40
Met Gly Leu Lys Val Leu Lys Lys Ala Tyr Arg Arg Ile Arg Lys Ala
1 5 10 15
Val Arg Lys Ile Leu Lys Ala Gly Ser Lys Val Leu Arg Lys Gly Asp
20 25 30
Arg Gly Asp Glu Val Ser Gln Leu Gln Thr Leu Leu Asn Leu Ser Gly
35 40 45
Tyr Asp Val Gly Lys Pro Asp Gly Ile Phe Gly Asn Asn Thr Phe Asn
50 55 60
Gln Val Val Lys Phe Gln Lys Asp Asn Ser Leu Asp Ser Asp Gly Ile
65 70 75 80
Val Gly Lys Asn Thr Trp Ala Glu Leu Phe Ser Lys Tyr Ser Pro Pro
85 90 95
Ile Pro Tyr Lys Thr Ile Pro Met Pro Thr Ala Asn Lys Ser Arg Ala
100 105 110
Ala Ala Thr Pro Val Met Asn Ala Val Glu Asn Ala Thr Gly Val Arg
115 120 125
Ser Gln Leu Leu Leu Thr Phe Ala Ser Ile Glu Ser Ala Phe Asp Tyr
130 135 140
Glu Ile Lys Ala Lys Thr Ser Ser Ala Thr Gly Trp Phe Gln Phe Leu
145 150 155 160
Thr Gly Thr Trp Lys Thr Met Ile Glu Asn Tyr Gly Met Lys Tyr Gly
165 170 175
Val Leu Thr Asp Pro Thr Gly Ala Leu Arg Lys Asp Pro Arg Ile Ser
180 185 190
Ala Leu Met Gly Ala Glu Leu Ile Lys Glu Asn Met Asn Ile Leu Arg
195 200 205
Pro Val Leu Lys Arg Glu Pro Thr Asp Thr Asp Leu Tyr Leu Ala His
210 215 220
Phe Phe Gly Pro Gly Ala Ala Arg Arg Phe Leu Thr Thr Gly Gln Asn
225 230 235 240
Glu Leu Ala Ala Thr His Phe Pro Lys Glu Ala Gln Ala Asn Pro Ser
245 250 255
Ile Phe Tyr Asn Lys Asp Gly Ser Pro Lys Thr Ile Gln Glu Val Tyr
260 265 270
Asn Leu Met Asp Gly Lys Val Ala Ala His Arg Lys
275 280
<210> 41
<211> 281
<212> PRT
<213> 人工序列
<220>
<223> 与具有C14S、C23S和C50S的突变的KZ144连接的衍生自IE1蛋白的氨基酸178-198的突变的肽
<400> 41
Met Tyr Lys Arg Ala Phe Lys Lys Val Leu Lys Arg Ile Arg Arg Tyr
1 5 10 15
Ala Lys Arg Ser Gly Ser Lys Val Leu Arg Lys Gly Asp Arg Gly Asp
20 25 30
Glu Val Ser Gln Leu Gln Thr Leu Leu Asn Leu Ser Gly Tyr Asp Val
35 40 45
Gly Lys Pro Asp Gly Ile Phe Gly Asn Asn Thr Phe Asn Gln Val Val
50 55 60
Lys Phe Gln Lys Asp Asn Ser Leu Asp Ser Asp Gly Ile Val Gly Lys
65 70 75 80
Asn Thr Trp Ala Glu Leu Phe Ser Lys Tyr Ser Pro Pro Ile Pro Tyr
85 90 95
Lys Thr Ile Pro Met Pro Thr Ala Asn Lys Ser Arg Ala Ala Ala Thr
100 105 110
Pro Val Met Asn Ala Val Glu Asn Ala Thr Gly Val Arg Ser Gln Leu
115 120 125
Leu Leu Thr Phe Ala Ser Ile Glu Ser Ala Phe Asp Tyr Glu Ile Lys
130 135 140
Ala Lys Thr Ser Ser Ala Thr Gly Trp Phe Gln Phe Leu Thr Gly Thr
145 150 155 160
Trp Lys Thr Met Ile Glu Asn Tyr Gly Met Lys Tyr Gly Val Leu Thr
165 170 175
Asp Pro Thr Gly Ala Leu Arg Lys Asp Pro Arg Ile Ser Ala Leu Met
180 185 190
Gly Ala Glu Leu Ile Lys Glu Asn Met Asn Ile Leu Arg Pro Val Leu
195 200 205
Lys Arg Glu Pro Thr Asp Thr Asp Leu Tyr Leu Ala His Phe Phe Gly
210 215 220
Pro Gly Ala Ala Arg Arg Phe Leu Thr Thr Gly Gln Asn Glu Leu Ala
225 230 235 240
Ala Thr His Phe Pro Lys Glu Ala Gln Ala Asn Pro Ser Ile Phe Tyr
245 250 255
Asn Lys Asp Gly Ser Pro Lys Thr Ile Gln Glu Val Tyr Asn Leu Met
260 265 270
Asp Gly Lys Val Ala Ala His Arg Lys
275 280
<210> 42
<211> 168
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 42
Met Gly Phe Phe Lys Lys Ala Trp Arg Lys Val Lys His Ala Gly Arg
1 5 10 15
Arg Val Leu Lys Thr Ala Lys Gly Val Gly Gly Gly Ser Gly Ser Met
20 25 30
Ser Phe Lys Phe Gly Lys Asn Ser Glu Lys Gln Leu Ala Thr Val Lys
35 40 45
Pro Glu Leu Gln Lys Val Ala Arg Arg Ala Leu Glu Leu Ser Pro Tyr
50 55 60
Asp Phe Thr Ile Val Gln Gly Ile Arg Thr Val Ala Gln Ser Ala Gln
65 70 75 80
Asn Ile Ala Asn Gly Thr Ser Phe Leu Lys Asp Pro Ser Lys Ser Lys
85 90 95
His Val Thr Gly Asp Ala Ile Asp Phe Ala Pro Tyr Ile Asn Gly Lys
100 105 110
Ile Asp Trp Lys Asp Leu Glu Ala Phe Trp Ala Val Lys Lys Ala Phe
115 120 125
Glu Gln Ala Gly Lys Glu Leu Gly Ile Lys Leu Arg Phe Gly Ala Asp
130 135 140
Trp Asn Ser Ser Gly Asp Tyr His Asp Glu Ile Asp Arg Gly Thr Tyr
145 150 155 160
Asp Gly Gly His Val Glu Leu Val
165
<210> 43
<211> 267
<212> PRT
<213> 人工序列
<220>
<223> 与弧菌噬菌体ICP1的尾基片蛋白连接的衍生自爪蟾抗菌肽肽的突变的肽
<400> 43
Met Gly Ile Lys Lys Phe Leu Lys Ser Ala Lys Lys Phe Gly Lys Ala
1 5 10 15
Phe Lys Lys Val Ile Arg Gly Gly Gly Gly Ser Gly Ser Met Ile Leu
20 25 30
Lys Arg Gly Ser Ser Gly Ala Asp Val Lys Asn Met Gln Glu Tyr Leu
35 40 45
Thr Ala Leu Gly Tyr Asp Thr Lys Gly Val Glu Gly Thr Phe Glu Gly
50 55 60
Gly Thr Glu Ser Ala Val Lys Ala Phe Gln Lys Asp Met Ser Phe Thr
65 70 75 80
Val Val Asp Gly Ile Ile Gly Asn Gln Thr Ala Lys His Leu Val Asp
85 90 95
Met Tyr Tyr Gly Lys Val Val Pro Phe Gly Tyr Val Thr Asn Thr Pro
100 105 110
Trp Val Ser Glu Ala Ile Glu Asp Tyr Phe Val Ser Glu Ile Lys Gly
115 120 125
Glu Lys His Asn Pro Arg Val Val Gln Tyr Phe Lys Asp Ala His Ser
130 135 140
Ser Trp Phe Thr Asp Asp Glu Thr Pro Trp Cys Ala Ala Ala Val Ser
145 150 155 160
Ser Trp Leu Glu Arg Ala Gly Ile Arg Ser Val Arg Ser Ala Arg Ala
165 170 175
Arg Asp His Ile Asn Phe Gly Thr Lys Leu Leu Glu Pro Arg Phe Gly
180 185 190
Ala Ile Val Val Leu Glu Arg Gly Ala Asn Ser Gly His Val Gly Phe
195 200 205
Val Asn Gly Val Thr Ala Asp Gly Lys Gln Ile Lys Val Leu Gly Gly
210 215 220
Asn Gln Ser Asp Ser Val Asn Glu Arg Met Phe Gln Val Thr Arg Val
225 230 235 240
Leu Gly Tyr Arg Gln Pro Glu Gly Phe Val Leu Pro Pro Cys Pro Ile
245 250 255
Val Gly Lys Gly Glu Leu Ser Lys Ser Glu Ala
260 265
<210> 44
<211> 36
<212> PRT
<213> 埃及伊蚊
<400> 44
Gly Gly Leu Lys Lys Leu Gly Lys Lys Leu Glu Gly Ala Gly Lys Arg
1 5 10 15
Val Phe Asn Ala Ala Glu Lys Ala Leu Pro Val Val Ala Gly Ala Lys
20 25 30
Ala Leu Arg Lys
35
<210> 45
<211> 184
<212> PRT
<213> 人工序列
<220>
<223> 与S394内溶素连接的杀菌肽A(埃及伊蚊)
<400> 45
Met Gly Gly Leu Lys Lys Leu Gly Lys Lys Leu Glu Gly Ala Gly Lys
1 5 10 15
Arg Val Phe Asn Ala Ala Glu Lys Ala Leu Pro Val Val Ala Gly Ala
20 25 30
Lys Ala Leu Arg Lys Gly Ala Gly Ala Gly Ala Gly Ala Gly Ser Met
35 40 45
Ser Phe Lys Phe Gly Lys Asn Ser Glu Lys Gln Leu Ala Thr Val Lys
50 55 60
Pro Glu Leu Gln Lys Val Ala Arg Arg Ala Leu Glu Leu Ser Pro Tyr
65 70 75 80
Asp Phe Thr Ile Val Gln Gly Ile Arg Thr Val Ala Gln Ser Ala Gln
85 90 95
Asn Ile Ala Asn Gly Thr Ser Phe Leu Lys Asp Pro Ser Lys Ser Lys
100 105 110
His Val Thr Gly Asp Ala Ile Asp Phe Ala Pro Tyr Ile Asn Gly Lys
115 120 125
Ile Asp Trp Lys Asp Leu Glu Ala Phe Trp Ala Val Lys Lys Ala Phe
130 135 140
Glu Gln Ala Gly Lys Glu Leu Gly Ile Lys Leu Arg Phe Gly Ala Asp
145 150 155 160
Trp Asn Ser Ser Gly Asp Tyr His Asp Glu Ile Asp Arg Gly Thr Tyr
165 170 175
Asp Gly Gly His Val Glu Leu Val
180
<210> 46
<211> 366
<212> PRT
<213> 人工序列
<220>
<223> 与OBPgpLys内溶素连接的杀菌肽A(埃及伊蚊)
<400> 46
Met Gly Gly Leu Lys Lys Leu Gly Lys Lys Leu Glu Gly Ala Gly Lys
1 5 10 15
Arg Val Phe Asn Ala Ala Glu Lys Ala Leu Pro Val Val Ala Gly Ala
20 25 30
Lys Ala Leu Arg Lys Gly Ser Lys Asn Ser Glu Lys Asn Ala Ser Ile
35 40 45
Ile Met Ser Ile Gln Arg Thr Leu Ala Ser Leu Ser Leu Tyr Gly Gly
50 55 60
Arg Ile Asp Gly Leu Phe Gly Glu Lys Cys Arg Gly Ala Ile Ile Leu
65 70 75 80
Met Leu Asn Lys Val Tyr Pro Asn Phe Ser Thr Asn Lys Leu Pro Ser
85 90 95
Asn Thr Tyr Glu Ala Glu Ser Val Phe Thr Phe Leu Gln Thr Ala Leu
100 105 110
Ala Gly Val Gly Leu Tyr Thr Ile Thr Ile Asp Gly Lys Trp Gly Gly
115 120 125
Thr Ser Gln Gly Ala Ile Asp Ala Leu Val Lys Ser Tyr Arg Gln Ile
130 135 140
Thr Glu Ala Glu Arg Ala Gly Ser Thr Leu Pro Leu Gly Leu Ala Thr
145 150 155 160
Val Met Ser Lys His Met Ser Ile Glu Gln Leu Arg Ala Met Leu Pro
165 170 175
Thr Asp Arg Gln Gly Tyr Ala Glu Val Tyr Ile Asp Pro Leu Asn Glu
180 185 190
Thr Met Asp Ile Phe Glu Ile Asn Thr Pro Leu Arg Ile Ala His Phe
195 200 205
Met Ala Gln Ile Leu His Glu Thr Ala Cys Phe Lys Tyr Thr Glu Glu
210 215 220
Leu Ala Ser Gly Lys Ala Tyr Glu Gly Arg Ala Asp Leu Gly Asn Thr
225 230 235 240
Arg Pro Gly Asp Gly Pro Leu Phe Lys Gly Arg Gly Leu Leu Gln Ile
245 250 255
Thr Gly Arg Leu Asn Tyr Val Lys Cys Gln Val Tyr Leu Arg Glu Lys
260 265 270
Leu Lys Asp Pro Thr Phe Asp Ile Thr Ser Ser Val Thr Cys Ala Gln
275 280 285
Gln Leu Ser Glu Ser Pro Leu Leu Ala Ala Leu Ala Ser Gly Tyr Phe
290 295 300
Trp Arg Phe Ile Lys Pro Lys Leu Asn Glu Thr Ala Asp Lys Asp Asp
305 310 315 320
Ile Tyr Trp Val Ser Val Tyr Val Asn Gly Tyr Ala Lys Gln Ala Asn
325 330 335
Pro Tyr Tyr Pro Asn Arg Asp Lys Glu Pro Asn His Met Lys Glu Arg
340 345 350
Val Gln Met Leu Ala Val Thr Lys Lys Ala Leu Gly Ile Val
355 360 365
<210> 47
<211> 28
<212> PRT
<213> 未知
<220>
<223> BMAP-28, 牛
<400> 47
Gly Gly Leu Arg Ser Leu Gly Arg Lys Ile Leu Arg Ala Trp Lys Lys
1 5 10 15
Tyr Gly Pro Ile Ile Val Pro Ile Ile Arg Ile Gly
20 25
<210> 48
<211> 290
<212> PRT
<213> 人工序列
<220>
<223> 与具有C14S、C23S和C50S的突变的KZ144连接的BMAP-28
<400> 48
Met Gly Gly Leu Arg Ser Leu Gly Arg Lys Ile Leu Arg Ala Trp Lys
1 5 10 15
Lys Tyr Gly Pro Ile Ile Val Pro Ile Ile Arg Ile Gly Gly Ser Lys
20 25 30
Val Leu Arg Lys Gly Asp Arg Gly Asp Glu Val Ser Gln Leu Gln Thr
35 40 45
Leu Leu Asn Leu Ser Gly Tyr Asp Val Gly Lys Pro Asp Gly Ile Phe
50 55 60
Gly Asn Asn Thr Phe Asn Gln Val Val Lys Phe Gln Lys Asp Asn Ser
65 70 75 80
Leu Asp Ser Asp Gly Ile Val Gly Lys Asn Thr Trp Ala Glu Leu Phe
85 90 95
Ser Lys Tyr Ser Pro Pro Ile Pro Tyr Lys Thr Ile Pro Met Pro Thr
100 105 110
Ala Asn Lys Ser Arg Ala Ala Ala Thr Pro Val Met Asn Ala Val Glu
115 120 125
Asn Ala Thr Gly Val Arg Ser Gln Leu Leu Leu Thr Phe Ala Ser Ile
130 135 140
Glu Ser Ala Phe Asp Tyr Glu Ile Lys Ala Lys Thr Ser Ser Ala Thr
145 150 155 160
Gly Trp Phe Gln Phe Leu Thr Gly Thr Trp Lys Thr Met Ile Glu Asn
165 170 175
Tyr Gly Met Lys Tyr Gly Val Leu Thr Asp Pro Thr Gly Ala Leu Arg
180 185 190
Lys Asp Pro Arg Ile Ser Ala Leu Met Gly Ala Glu Leu Ile Lys Glu
195 200 205
Asn Met Asn Ile Leu Arg Pro Val Leu Lys Arg Glu Pro Thr Asp Thr
210 215 220
Asp Leu Tyr Leu Ala His Phe Phe Gly Pro Gly Ala Ala Arg Arg Phe
225 230 235 240
Leu Thr Thr Gly Gln Asn Glu Leu Ala Ala Thr His Phe Pro Lys Glu
245 250 255
Ala Gln Ala Asn Pro Ser Ile Phe Tyr Asn Lys Asp Gly Ser Pro Lys
260 265 270
Thr Ile Gln Glu Val Tyr Asn Leu Met Asp Gly Lys Val Ala Ala His
275 280 285
Arg Lys
290
<210> 49
<211> 17
<212> PRT
<213> 人工序列
<220>
<223> MSI-78 (4-20)肽
<400> 49
Lys Phe Leu Lys Lys Ala Lys Lys Phe Gly Lys Ala Phe Val Lys Ile
1 5 10 15
Leu
<210> 50
<211> 162
<212> PRT
<213> 人工序列
<220>
<223> 与S394内溶素连接的MSI-78 (4-20)肽
<400> 50
Met Lys Phe Leu Lys Lys Ala Lys Lys Phe Gly Lys Ala Phe Val Lys
1 5 10 15
Ile Leu Gly Gly Gly Gly Ser Gly Ser Met Ser Phe Lys Phe Gly Lys
20 25 30
Asn Ser Glu Lys Gln Leu Ala Thr Val Lys Pro Glu Leu Gln Lys Val
35 40 45
Ala Arg Arg Ala Leu Glu Leu Ser Pro Tyr Asp Phe Thr Ile Val Gln
50 55 60
Gly Ile Arg Thr Val Ala Gln Ser Ala Gln Asn Ile Ala Asn Gly Thr
65 70 75 80
Ser Phe Leu Lys Asp Pro Ser Lys Ser Lys His Val Thr Gly Asp Ala
85 90 95
Ile Asp Phe Ala Pro Tyr Ile Asn Gly Lys Ile Asp Trp Lys Asp Leu
100 105 110
Glu Ala Phe Trp Ala Val Lys Lys Ala Phe Glu Gln Ala Gly Lys Glu
115 120 125
Leu Gly Ile Lys Leu Arg Phe Gly Ala Asp Trp Asn Ser Ser Gly Asp
130 135 140
Tyr His Asp Glu Ile Asp Arg Gly Thr Tyr Asp Gly Gly His Val Glu
145 150 155 160
Leu Val
<210> 51
<211> 23
<212> PRT
<213> 非洲爪蟾
<400> 51
Gly Ile Gly Lys Phe Leu His Ser Ala Lys Lys Phe Gly Lys Ala Phe
1 5 10 15
Val Gly Glu Ile Met Asn Ser
20
<210> 52
<211> 163
<212> PRT
<213> 人工序列
<220>
<223> 与S394内溶素连接的爪蟾抗菌肽肽
<400> 52
Met Gly Ile Gly Lys Phe Leu His Ser Ala Lys Lys Phe Gly Lys Ala
1 5 10 15
Phe Val Gly Glu Ile Met Asn Ser Gly Ser Met Ser Phe Lys Phe Gly
20 25 30
Lys Asn Ser Glu Lys Gln Leu Ala Thr Val Lys Pro Glu Leu Gln Lys
35 40 45
Val Ala Arg Arg Ala Leu Glu Leu Ser Pro Tyr Asp Phe Thr Ile Val
50 55 60
Gln Gly Ile Arg Thr Val Ala Gln Ser Ala Gln Asn Ile Ala Asn Gly
65 70 75 80
Thr Ser Phe Leu Lys Asp Pro Ser Lys Ser Lys His Val Thr Gly Asp
85 90 95
Ala Ile Asp Phe Ala Pro Tyr Ile Asn Gly Lys Ile Asp Trp Lys Asp
100 105 110
Leu Glu Ala Phe Trp Ala Val Lys Lys Ala Phe Glu Gln Ala Gly Lys
115 120 125
Glu Leu Gly Ile Lys Leu Arg Phe Gly Ala Asp Trp Asn Ser Ser Gly
130 135 140
Asp Tyr His Asp Glu Ile Asp Arg Gly Thr Tyr Asp Gly Gly His Val
145 150 155 160
Glu Leu Val
<210> 53
<211> 15
<212> PRT
<213> 幽门螺杆菌
<400> 53
Phe Lys Arg Leu Lys Lys Leu Phe Lys Lys Ile Trp Asn Trp Lys
1 5 10 15
<210> 54
<211> 277
<212> PRT
<213> 人工序列
<220>
<223> 与具有C14S、C23S和C50S的突变的KZ144连接的HPA-NT3肽的氨基酸XXX
<400> 54
Met Phe Lys Arg Leu Lys Lys Leu Phe Lys Lys Ile Trp Asn Trp Lys
1 5 10 15
Gly Ser Lys Val Leu Arg Lys Gly Asp Arg Gly Asp Glu Val Ser Gln
20 25 30
Leu Gln Thr Leu Leu Asn Leu Ser Gly Tyr Asp Val Gly Lys Pro Asp
35 40 45
Gly Ile Phe Gly Asn Asn Thr Phe Asn Gln Val Val Lys Phe Gln Lys
50 55 60
Asp Asn Ser Leu Asp Ser Asp Gly Ile Val Gly Lys Asn Thr Trp Ala
65 70 75 80
Glu Leu Phe Ser Lys Tyr Ser Pro Pro Ile Pro Tyr Lys Thr Ile Pro
85 90 95
Met Pro Thr Ala Asn Lys Ser Arg Ala Ala Ala Thr Pro Val Met Asn
100 105 110
Ala Val Glu Asn Ala Thr Gly Val Arg Ser Gln Leu Leu Leu Thr Phe
115 120 125
Ala Ser Ile Glu Ser Ala Phe Asp Tyr Glu Ile Lys Ala Lys Thr Ser
130 135 140
Ser Ala Thr Gly Trp Phe Gln Phe Leu Thr Gly Thr Trp Lys Thr Met
145 150 155 160
Ile Glu Asn Tyr Gly Met Lys Tyr Gly Val Leu Thr Asp Pro Thr Gly
165 170 175
Ala Leu Arg Lys Asp Pro Arg Ile Ser Ala Leu Met Gly Ala Glu Leu
180 185 190
Ile Lys Glu Asn Met Asn Ile Leu Arg Pro Val Leu Lys Arg Glu Pro
195 200 205
Thr Asp Thr Asp Leu Tyr Leu Ala His Phe Phe Gly Pro Gly Ala Ala
210 215 220
Arg Arg Phe Leu Thr Thr Gly Gln Asn Glu Leu Ala Ala Thr His Phe
225 230 235 240
Pro Lys Glu Ala Gln Ala Asn Pro Ser Ile Phe Tyr Asn Lys Asp Gly
245 250 255
Ser Pro Lys Thr Ile Gln Glu Val Tyr Asn Leu Met Asp Gly Lys Val
260 265 270
Ala Ala His Arg Lys
275
<210> 55
<211> 29
<212> PRT
<213> 玫瑰毒鲉(Synanceia verrucosa)
<400> 55
Ile Pro Leu Ile His Asp Lys Ile Ser Asn Phe Gln Gln Ile Phe Gln
1 5 10 15
Asp Tyr Met Leu Thr Val Gln Lys Lys Ile Ala Glu Lys
20 25
<210> 56
<211> 291
<212> PRT
<213> 人工序列
<220>
<223> 与具有C14S、C23S和C50S的突变的KZ144连接的stonustoxin的alpha亚基的氨基酸298-326
<400> 56
Met Ile Pro Leu Ile His Asp Lys Ile Ser Asn Phe Gln Gln Ile Phe
1 5 10 15
Gln Asp Tyr Met Leu Thr Val Gln Lys Lys Ile Ala Glu Lys Gly Ser
20 25 30
Lys Val Leu Arg Lys Gly Asp Arg Gly Asp Glu Val Ser Gln Leu Gln
35 40 45
Thr Leu Leu Asn Leu Ser Gly Tyr Asp Val Gly Lys Pro Asp Gly Ile
50 55 60
Phe Gly Asn Asn Thr Phe Asn Gln Val Val Lys Phe Gln Lys Asp Asn
65 70 75 80
Ser Leu Asp Ser Asp Gly Ile Val Gly Lys Asn Thr Trp Ala Glu Leu
85 90 95
Phe Ser Lys Tyr Ser Pro Pro Ile Pro Tyr Lys Thr Ile Pro Met Pro
100 105 110
Thr Ala Asn Lys Ser Arg Ala Ala Ala Thr Pro Val Met Asn Ala Val
115 120 125
Glu Asn Ala Thr Gly Val Arg Ser Gln Leu Leu Leu Thr Phe Ala Ser
130 135 140
Ile Glu Ser Ala Phe Asp Tyr Glu Ile Lys Ala Lys Thr Ser Ser Ala
145 150 155 160
Thr Gly Trp Phe Gln Phe Leu Thr Gly Thr Trp Lys Thr Met Ile Glu
165 170 175
Asn Tyr Gly Met Lys Tyr Gly Val Leu Thr Asp Pro Thr Gly Ala Leu
180 185 190
Arg Lys Asp Pro Arg Ile Ser Ala Leu Met Gly Ala Glu Leu Ile Lys
195 200 205
Glu Asn Met Asn Ile Leu Arg Pro Val Leu Lys Arg Glu Pro Thr Asp
210 215 220
Thr Asp Leu Tyr Leu Ala His Phe Phe Gly Pro Gly Ala Ala Arg Arg
225 230 235 240
Phe Leu Thr Thr Gly Gln Asn Glu Leu Ala Ala Thr His Phe Pro Lys
245 250 255
Glu Ala Gln Ala Asn Pro Ser Ile Phe Tyr Asn Lys Asp Gly Ser Pro
260 265 270
Lys Thr Ile Gln Glu Val Tyr Asn Leu Met Asp Gly Lys Val Ala Ala
275 280 285
His Arg Lys
290
<210> 57
<211> 22
<212> PRT
<213> 幽门螺杆菌
<400> 57
Gly Leu Lys Gln Leu Asp Ser Thr Tyr Gln Glu Thr Asn Gln Gln Val
1 5 10 15
Leu Lys Asn Leu Asp Glu
20
<210> 58
<211> 284
<212> PRT
<213> 人工序列
<220>
<223> 与具有C14S、C23S和C50S的突变的KZ144连接的CagL蛋白的氨基酸26-48
<400> 58
Met Gly Leu Lys Gln Leu Asp Ser Thr Tyr Gln Glu Thr Asn Gln Gln
1 5 10 15
Val Leu Lys Asn Leu Asp Glu Gly Ser Lys Val Leu Arg Lys Gly Asp
20 25 30
Arg Gly Asp Glu Val Ser Gln Leu Gln Thr Leu Leu Asn Leu Ser Gly
35 40 45
Tyr Asp Val Gly Lys Pro Asp Gly Ile Phe Gly Asn Asn Thr Phe Asn
50 55 60
Gln Val Val Lys Phe Gln Lys Asp Asn Ser Leu Asp Ser Asp Gly Ile
65 70 75 80
Val Gly Lys Asn Thr Trp Ala Glu Leu Phe Ser Lys Tyr Ser Pro Pro
85 90 95
Ile Pro Tyr Lys Thr Ile Pro Met Pro Thr Ala Asn Lys Ser Arg Ala
100 105 110
Ala Ala Thr Pro Val Met Asn Ala Val Glu Asn Ala Thr Gly Val Arg
115 120 125
Ser Gln Leu Leu Leu Thr Phe Ala Ser Ile Glu Ser Ala Phe Asp Tyr
130 135 140
Glu Ile Lys Ala Lys Thr Ser Ser Ala Thr Gly Trp Phe Gln Phe Leu
145 150 155 160
Thr Gly Thr Trp Lys Thr Met Ile Glu Asn Tyr Gly Met Lys Tyr Gly
165 170 175
Val Leu Thr Asp Pro Thr Gly Ala Leu Arg Lys Asp Pro Arg Ile Ser
180 185 190
Ala Leu Met Gly Ala Glu Leu Ile Lys Glu Asn Met Asn Ile Leu Arg
195 200 205
Pro Val Leu Lys Arg Glu Pro Thr Asp Thr Asp Leu Tyr Leu Ala His
210 215 220
Phe Phe Gly Pro Gly Ala Ala Arg Arg Phe Leu Thr Thr Gly Gln Asn
225 230 235 240
Glu Leu Ala Ala Thr His Phe Pro Lys Glu Ala Gln Ala Asn Pro Ser
245 250 255
Ile Phe Tyr Asn Lys Asp Gly Ser Pro Lys Thr Ile Gln Glu Val Tyr
260 265 270
Asn Leu Met Asp Gly Lys Val Ala Ala His Arg Lys
275 280
<210> 59
<211> 19
<212> PRT
<213> 人巨细胞病毒
<400> 59
Tyr Lys Glu Lys Phe Met Val Cys Leu Lys Gln Ile Val Gln Tyr Ala
1 5 10 15
Val Asn Ser
<210> 60
<211> 281
<212> PRT
<213> 人工序列
<220>
<223> 与具有C14S、C23S和C50S的突变的KZ144连接的IE1蛋白的氨基酸178-198
<400> 60
Met Tyr Lys Glu Lys Phe Met Val Cys Leu Lys Gln Ile Val Gln Tyr
1 5 10 15
Ala Val Asn Ser Gly Ser Lys Val Leu Arg Lys Gly Asp Arg Gly Asp
20 25 30
Glu Val Ser Gln Leu Gln Thr Leu Leu Asn Leu Ser Gly Tyr Asp Val
35 40 45
Gly Lys Pro Asp Gly Ile Phe Gly Asn Asn Thr Phe Asn Gln Val Val
50 55 60
Lys Phe Gln Lys Asp Asn Ser Leu Asp Ser Asp Gly Ile Val Gly Lys
65 70 75 80
Asn Thr Trp Ala Glu Leu Phe Ser Lys Tyr Ser Pro Pro Ile Pro Tyr
85 90 95
Lys Thr Ile Pro Met Pro Thr Ala Asn Lys Ser Arg Ala Ala Ala Thr
100 105 110
Pro Val Met Asn Ala Val Glu Asn Ala Thr Gly Val Arg Ser Gln Leu
115 120 125
Leu Leu Thr Phe Ala Ser Ile Glu Ser Ala Phe Asp Tyr Glu Ile Lys
130 135 140
Ala Lys Thr Ser Ser Ala Thr Gly Trp Phe Gln Phe Leu Thr Gly Thr
145 150 155 160
Trp Lys Thr Met Ile Glu Asn Tyr Gly Met Lys Tyr Gly Val Leu Thr
165 170 175
Asp Pro Thr Gly Ala Leu Arg Lys Asp Pro Arg Ile Ser Ala Leu Met
180 185 190
Gly Ala Glu Leu Ile Lys Glu Asn Met Asn Ile Leu Arg Pro Val Leu
195 200 205
Lys Arg Glu Pro Thr Asp Thr Asp Leu Tyr Leu Ala His Phe Phe Gly
210 215 220
Pro Gly Ala Ala Arg Arg Phe Leu Thr Thr Gly Gln Asn Glu Leu Ala
225 230 235 240
Ala Thr His Phe Pro Lys Glu Ala Gln Ala Asn Pro Ser Ile Phe Tyr
245 250 255
Asn Lys Asp Gly Ser Pro Lys Thr Ile Gln Glu Val Tyr Asn Leu Met
260 265 270
Asp Gly Lys Val Ala Ala His Arg Lys
275 280
<210> 61
<211> 278
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 61
Met Arg Gly Leu Arg Arg Leu Gly Arg Lys Ile Ala His Gly Val Lys
1 5 10 15
Lys Gly Ser Lys Val Leu Arg Lys Gly Asp Arg Gly Asp Glu Val Ser
20 25 30
Gln Leu Gln Thr Leu Leu Asn Leu Ser Gly Tyr Asp Val Gly Lys Pro
35 40 45
Asp Gly Ile Phe Gly Asn Asn Thr Phe Asn Gln Val Val Lys Phe Gln
50 55 60
Lys Asp Asn Ser Leu Asp Ser Asp Gly Ile Val Gly Lys Asn Thr Trp
65 70 75 80
Ala Glu Leu Phe Ser Lys Tyr Ser Pro Pro Ile Pro Tyr Lys Thr Ile
85 90 95
Pro Met Pro Thr Ala Asn Lys Ser Arg Ala Ala Ala Thr Pro Val Met
100 105 110
Asn Ala Val Glu Asn Ala Thr Gly Val Arg Ser Gln Leu Leu Leu Thr
115 120 125
Phe Ala Ser Ile Glu Ser Ala Phe Asp Tyr Glu Ile Lys Ala Lys Thr
130 135 140
Ser Ser Ala Thr Gly Trp Phe Gln Phe Leu Thr Gly Thr Trp Lys Thr
145 150 155 160
Met Ile Glu Asn Tyr Gly Met Lys Tyr Gly Val Leu Thr Asp Pro Thr
165 170 175
Gly Ala Leu Arg Lys Asp Pro Arg Ile Ser Ala Leu Met Gly Ala Glu
180 185 190
Leu Ile Lys Glu Asn Met Asn Ile Leu Arg Pro Val Leu Lys Arg Glu
195 200 205
Pro Thr Asp Thr Asp Leu Tyr Leu Ala His Phe Phe Gly Pro Gly Ala
210 215 220
Ala Arg Arg Phe Leu Thr Thr Gly Gln Asn Glu Leu Ala Ala Thr His
225 230 235 240
Phe Pro Lys Glu Ala Gln Ala Asn Pro Ser Ile Phe Tyr Asn Lys Asp
245 250 255
Gly Ser Pro Lys Thr Ile Gln Glu Val Tyr Asn Leu Met Asp Gly Lys
260 265 270
Val Ala Ala His Arg Lys
275
<210> 62
<211> 286
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 62
Met Arg Gly Leu Arg Arg Leu Gly Arg Lys Ile Ala His Gly Val Lys
1 5 10 15
Lys Gly Ala Gly Ala Gly Ala Gly Ala Gly Ser Lys Val Leu Arg Lys
20 25 30
Gly Asp Arg Gly Asp Glu Val Ser Gln Leu Gln Thr Leu Leu Asn Leu
35 40 45
Ser Gly Tyr Asp Val Gly Lys Pro Asp Gly Ile Phe Gly Asn Asn Thr
50 55 60
Phe Asn Gln Val Val Lys Phe Gln Lys Asp Asn Ser Leu Asp Ser Asp
65 70 75 80
Gly Ile Val Gly Lys Asn Thr Trp Ala Glu Leu Phe Ser Lys Tyr Ser
85 90 95
Pro Pro Ile Pro Tyr Lys Thr Ile Pro Met Pro Thr Ala Asn Lys Ser
100 105 110
Arg Ala Ala Ala Thr Pro Val Met Asn Ala Val Glu Asn Ala Thr Gly
115 120 125
Val Arg Ser Gln Leu Leu Leu Thr Phe Ala Ser Ile Glu Ser Ala Phe
130 135 140
Asp Tyr Glu Ile Lys Ala Lys Thr Ser Ser Ala Thr Gly Trp Phe Gln
145 150 155 160
Phe Leu Thr Gly Thr Trp Lys Thr Met Ile Glu Asn Tyr Gly Met Lys
165 170 175
Tyr Gly Val Leu Thr Asp Pro Thr Gly Ala Leu Arg Lys Asp Pro Arg
180 185 190
Ile Ser Ala Leu Met Gly Ala Glu Leu Ile Lys Glu Asn Met Asn Ile
195 200 205
Leu Arg Pro Val Leu Lys Arg Glu Pro Thr Asp Thr Asp Leu Tyr Leu
210 215 220
Ala His Phe Phe Gly Pro Gly Ala Ala Arg Arg Phe Leu Thr Thr Gly
225 230 235 240
Gln Asn Glu Leu Ala Ala Thr His Phe Pro Lys Glu Ala Gln Ala Asn
245 250 255
Pro Ser Ile Phe Tyr Asn Lys Asp Gly Ser Pro Lys Thr Ile Gln Glu
260 265 270
Val Tyr Asn Leu Met Asp Gly Lys Val Ala Ala His Arg Lys
275 280 285
<210> 63
<211> 279
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 63
Met Arg Gly Leu Arg Arg Leu Gly Arg Lys Ile Ala His Gly Val Lys
1 5 10 15
Lys Tyr Gly Ser Lys Val Leu Arg Lys Gly Asp Arg Gly Asp Glu Val
20 25 30
Ser Gln Leu Gln Thr Leu Leu Asn Leu Ser Gly Tyr Asp Val Gly Lys
35 40 45
Pro Asp Gly Ile Phe Gly Asn Asn Thr Phe Asn Gln Val Val Lys Phe
50 55 60
Gln Lys Asp Asn Ser Leu Asp Ser Asp Gly Ile Val Gly Lys Asn Thr
65 70 75 80
Trp Ala Glu Leu Phe Ser Lys Tyr Ser Pro Pro Ile Pro Tyr Lys Thr
85 90 95
Ile Pro Met Pro Thr Ala Asn Lys Ser Arg Ala Ala Ala Thr Pro Val
100 105 110
Met Asn Ala Val Glu Asn Ala Thr Gly Val Arg Ser Gln Leu Leu Leu
115 120 125
Thr Phe Ala Ser Ile Glu Ser Ala Phe Asp Tyr Glu Ile Lys Ala Lys
130 135 140
Thr Ser Ser Ala Thr Gly Trp Phe Gln Phe Leu Thr Gly Thr Trp Lys
145 150 155 160
Thr Met Ile Glu Asn Tyr Gly Met Lys Tyr Gly Val Leu Thr Asp Pro
165 170 175
Thr Gly Ala Leu Arg Lys Asp Pro Arg Ile Ser Ala Leu Met Gly Ala
180 185 190
Glu Leu Ile Lys Glu Asn Met Asn Ile Leu Arg Pro Val Leu Lys Arg
195 200 205
Glu Pro Thr Asp Thr Asp Leu Tyr Leu Ala His Phe Phe Gly Pro Gly
210 215 220
Ala Ala Arg Arg Phe Leu Thr Thr Gly Gln Asn Glu Leu Ala Ala Thr
225 230 235 240
His Phe Pro Lys Glu Ala Gln Ala Asn Pro Ser Ile Phe Tyr Asn Lys
245 250 255
Asp Gly Ser Pro Lys Thr Ile Gln Glu Val Tyr Asn Leu Met Asp Gly
260 265 270
Lys Val Ala Ala His Arg Lys
275
<210> 64
<211> 285
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 64
Met Arg Gly Leu Arg Arg Leu Gly Arg Lys Ile Ala His Gly Val Lys
1 5 10 15
Lys Tyr Lys Pro Lys Pro Lys Pro Gly Ser Lys Val Leu Arg Lys Gly
20 25 30
Asp Arg Gly Asp Glu Val Ser Gln Leu Gln Thr Leu Leu Asn Leu Ser
35 40 45
Gly Tyr Asp Val Gly Lys Pro Asp Gly Ile Phe Gly Asn Asn Thr Phe
50 55 60
Asn Gln Val Val Lys Phe Gln Lys Asp Asn Ser Leu Asp Ser Asp Gly
65 70 75 80
Ile Val Gly Lys Asn Thr Trp Ala Glu Leu Phe Ser Lys Tyr Ser Pro
85 90 95
Pro Ile Pro Tyr Lys Thr Ile Pro Met Pro Thr Ala Asn Lys Ser Arg
100 105 110
Ala Ala Ala Thr Pro Val Met Asn Ala Val Glu Asn Ala Thr Gly Val
115 120 125
Arg Ser Gln Leu Leu Leu Thr Phe Ala Ser Ile Glu Ser Ala Phe Asp
130 135 140
Tyr Glu Ile Lys Ala Lys Thr Ser Ser Ala Thr Gly Trp Phe Gln Phe
145 150 155 160
Leu Thr Gly Thr Trp Lys Thr Met Ile Glu Asn Tyr Gly Met Lys Tyr
165 170 175
Gly Val Leu Thr Asp Pro Thr Gly Ala Leu Arg Lys Asp Pro Arg Ile
180 185 190
Ser Ala Leu Met Gly Ala Glu Leu Ile Lys Glu Asn Met Asn Ile Leu
195 200 205
Arg Pro Val Leu Lys Arg Glu Pro Thr Asp Thr Asp Leu Tyr Leu Ala
210 215 220
His Phe Phe Gly Pro Gly Ala Ala Arg Arg Phe Leu Thr Thr Gly Gln
225 230 235 240
Asn Glu Leu Ala Ala Thr His Phe Pro Lys Glu Ala Gln Ala Asn Pro
245 250 255
Ser Ile Phe Tyr Asn Lys Asp Gly Ser Pro Lys Thr Ile Gln Glu Val
260 265 270
Tyr Asn Leu Met Asp Gly Lys Val Ala Ala His Arg Lys
275 280 285
<210> 65
<211> 280
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 65
Met Arg Gly Leu Arg Arg Leu Gly Arg Lys Ile Ala His Gly Val Lys
1 5 10 15
Lys Tyr Gly Gly Ser Lys Val Leu Arg Lys Gly Asp Arg Gly Asp Glu
20 25 30
Val Ser Gln Leu Gln Thr Leu Leu Asn Leu Ser Gly Tyr Asp Val Gly
35 40 45
Lys Pro Asp Gly Ile Phe Gly Asn Asn Thr Phe Asn Gln Val Val Lys
50 55 60
Phe Gln Lys Asp Asn Ser Leu Asp Ser Asp Gly Ile Val Gly Lys Asn
65 70 75 80
Thr Trp Ala Glu Leu Phe Ser Lys Tyr Ser Pro Pro Ile Pro Tyr Lys
85 90 95
Thr Ile Pro Met Pro Thr Ala Asn Lys Ser Arg Ala Ala Ala Thr Pro
100 105 110
Val Met Asn Ala Val Glu Asn Ala Thr Gly Val Arg Ser Gln Leu Leu
115 120 125
Leu Thr Phe Ala Ser Ile Glu Ser Ala Phe Asp Tyr Glu Ile Lys Ala
130 135 140
Lys Thr Ser Ser Ala Thr Gly Trp Phe Gln Phe Leu Thr Gly Thr Trp
145 150 155 160
Lys Thr Met Ile Glu Asn Tyr Gly Met Lys Tyr Gly Val Leu Thr Asp
165 170 175
Pro Thr Gly Ala Leu Arg Lys Asp Pro Arg Ile Ser Ala Leu Met Gly
180 185 190
Ala Glu Leu Ile Lys Glu Asn Met Asn Ile Leu Arg Pro Val Leu Lys
195 200 205
Arg Glu Pro Thr Asp Thr Asp Leu Tyr Leu Ala His Phe Phe Gly Pro
210 215 220
Gly Ala Ala Arg Arg Phe Leu Thr Thr Gly Gln Asn Glu Leu Ala Ala
225 230 235 240
Thr His Phe Pro Lys Glu Ala Gln Ala Asn Pro Ser Ile Phe Tyr Asn
245 250 255
Lys Asp Gly Ser Pro Lys Thr Ile Gln Glu Val Tyr Asn Leu Met Asp
260 265 270
Gly Lys Val Ala Ala His Arg Lys
275 280
<210> 66
<211> 284
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 66
Met Arg Gly Leu Arg Arg Leu Gly Arg Lys Ile Ala His Gly Val Lys
1 5 10 15
Lys Tyr Gly Gly Gly Gly Ser Gly Ser Lys Val Leu Arg Lys Gly Asp
20 25 30
Arg Gly Asp Glu Val Ser Gln Leu Gln Thr Leu Leu Asn Leu Ser Gly
35 40 45
Tyr Asp Val Gly Lys Pro Asp Gly Ile Phe Gly Asn Asn Thr Phe Asn
50 55 60
Gln Val Val Lys Phe Gln Lys Asp Asn Ser Leu Asp Ser Asp Gly Ile
65 70 75 80
Val Gly Lys Asn Thr Trp Ala Glu Leu Phe Ser Lys Tyr Ser Pro Pro
85 90 95
Ile Pro Tyr Lys Thr Ile Pro Met Pro Thr Ala Asn Lys Ser Arg Ala
100 105 110
Ala Ala Thr Pro Val Met Asn Ala Val Glu Asn Ala Thr Gly Val Arg
115 120 125
Ser Gln Leu Leu Leu Thr Phe Ala Ser Ile Glu Ser Ala Phe Asp Tyr
130 135 140
Glu Ile Lys Ala Lys Thr Ser Ser Ala Thr Gly Trp Phe Gln Phe Leu
145 150 155 160
Thr Gly Thr Trp Lys Thr Met Ile Glu Asn Tyr Gly Met Lys Tyr Gly
165 170 175
Val Leu Thr Asp Pro Thr Gly Ala Leu Arg Lys Asp Pro Arg Ile Ser
180 185 190
Ala Leu Met Gly Ala Glu Leu Ile Lys Glu Asn Met Asn Ile Leu Arg
195 200 205
Pro Val Leu Lys Arg Glu Pro Thr Asp Thr Asp Leu Tyr Leu Ala His
210 215 220
Phe Phe Gly Pro Gly Ala Ala Arg Arg Phe Leu Thr Thr Gly Gln Asn
225 230 235 240
Glu Leu Ala Ala Thr His Phe Pro Lys Glu Ala Gln Ala Asn Pro Ser
245 250 255
Ile Phe Tyr Asn Lys Asp Gly Ser Pro Lys Thr Ile Gln Glu Val Tyr
260 265 270
Asn Leu Met Asp Gly Lys Val Ala Ala His Arg Lys
275 280
<210> 67
<211> 280
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 67
Met Gly Arg Lys Ile Ala His Gly Val Lys Lys Tyr Gly Arg Gly Leu
1 5 10 15
Arg Arg Leu Gly Ser Lys Val Leu Arg Lys Gly Asp Arg Gly Asp Glu
20 25 30
Val Ser Gln Leu Gln Thr Leu Leu Asn Leu Ser Gly Tyr Asp Val Gly
35 40 45
Lys Pro Asp Gly Ile Phe Gly Asn Asn Thr Phe Asn Gln Val Val Lys
50 55 60
Phe Gln Lys Asp Asn Ser Leu Asp Ser Asp Gly Ile Val Gly Lys Asn
65 70 75 80
Thr Trp Ala Glu Leu Phe Ser Lys Tyr Ser Pro Pro Ile Pro Tyr Lys
85 90 95
Thr Ile Pro Met Pro Thr Ala Asn Lys Ser Arg Ala Ala Ala Thr Pro
100 105 110
Val Met Asn Ala Val Glu Asn Ala Thr Gly Val Arg Ser Gln Leu Leu
115 120 125
Leu Thr Phe Ala Ser Ile Glu Ser Ala Phe Asp Tyr Glu Ile Lys Ala
130 135 140
Lys Thr Ser Ser Ala Thr Gly Trp Phe Gln Phe Leu Thr Gly Thr Trp
145 150 155 160
Lys Thr Met Ile Glu Asn Tyr Gly Met Lys Tyr Gly Val Leu Thr Asp
165 170 175
Pro Thr Gly Ala Leu Arg Lys Asp Pro Arg Ile Ser Ala Leu Met Gly
180 185 190
Ala Glu Leu Ile Lys Glu Asn Met Asn Ile Leu Arg Pro Val Leu Lys
195 200 205
Arg Glu Pro Thr Asp Thr Asp Leu Tyr Leu Ala His Phe Phe Gly Pro
210 215 220
Gly Ala Ala Arg Arg Phe Leu Thr Thr Gly Gln Asn Glu Leu Ala Ala
225 230 235 240
Thr His Phe Pro Lys Glu Ala Gln Ala Asn Pro Ser Ile Phe Tyr Asn
245 250 255
Lys Asp Gly Ser Pro Lys Thr Ile Gln Glu Val Tyr Asn Leu Met Asp
260 265 270
Gly Lys Val Ala Ala His Arg Lys
275 280
<210> 68
<211> 291
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 68
Met Gly Arg Lys Ile Ala His Gly Val Lys Lys Tyr Gly Arg Gly Leu
1 5 10 15
Arg Arg Leu Pro Thr Val Leu Arg Ile Ile Arg Ile Ala Gly Gly Ser
20 25 30
Lys Val Leu Arg Lys Gly Asp Arg Gly Asp Glu Val Ser Gln Leu Gln
35 40 45
Thr Leu Leu Asn Leu Ser Gly Tyr Asp Val Gly Lys Pro Asp Gly Ile
50 55 60
Phe Gly Asn Asn Thr Phe Asn Gln Val Val Lys Phe Gln Lys Asp Asn
65 70 75 80
Ser Leu Asp Ser Asp Gly Ile Val Gly Lys Asn Thr Trp Ala Glu Leu
85 90 95
Phe Ser Lys Tyr Ser Pro Pro Ile Pro Tyr Lys Thr Ile Pro Met Pro
100 105 110
Thr Ala Asn Lys Ser Arg Ala Ala Ala Thr Pro Val Met Asn Ala Val
115 120 125
Glu Asn Ala Thr Gly Val Arg Ser Gln Leu Leu Leu Thr Phe Ala Ser
130 135 140
Ile Glu Ser Ala Phe Asp Tyr Glu Ile Lys Ala Lys Thr Ser Ser Ala
145 150 155 160
Thr Gly Trp Phe Gln Phe Leu Thr Gly Thr Trp Lys Thr Met Ile Glu
165 170 175
Asn Tyr Gly Met Lys Tyr Gly Val Leu Thr Asp Pro Thr Gly Ala Leu
180 185 190
Arg Lys Asp Pro Arg Ile Ser Ala Leu Met Gly Ala Glu Leu Ile Lys
195 200 205
Glu Asn Met Asn Ile Leu Arg Pro Val Leu Lys Arg Glu Pro Thr Asp
210 215 220
Thr Asp Leu Tyr Leu Ala His Phe Phe Gly Pro Gly Ala Ala Arg Arg
225 230 235 240
Phe Leu Thr Thr Gly Gln Asn Glu Leu Ala Ala Thr His Phe Pro Lys
245 250 255
Glu Ala Gln Ala Asn Pro Ser Ile Phe Tyr Asn Lys Asp Gly Ser Pro
260 265 270
Lys Thr Ile Gln Glu Val Tyr Asn Leu Met Asp Gly Lys Val Ala Ala
275 280 285
His Arg Lys
290
<210> 69
<211> 25
<212> PRT
<213> 人工序列
<220>
<223> 合成序列; MW2
<400> 69
Gly Lys Pro Gly Trp Leu Ile Lys Val Ala Leu Lys Phe Lys Lys Leu
1 5 10 15
Ile Arg Arg Pro Leu Lys Arg Leu Ala
20 25
<210> 70
<211> 295
<212> PRT
<213> 人工序列
<220>
<223> 与具有C14S、C23S和C50S的突变的KZ144连接的MW2肽
<400> 70
Met Gly Lys Pro Gly Trp Leu Ile Lys Val Ala Leu Lys Phe Lys Lys
1 5 10 15
Leu Ile Arg Arg Pro Leu Lys Arg Leu Ala Gly Ser Lys Val Leu Arg
20 25 30
Lys Gly Asp Arg Gly Asp Glu Val Ser Gln Leu Gln Thr Leu Leu Asn
35 40 45
Leu Ser Gly Tyr Asp Val Gly Lys Pro Asp Gly Ile Phe Gly Asn Asn
50 55 60
Thr Phe Asn Gln Val Val Lys Phe Gln Lys Asp Asn Ser Leu Asp Ser
65 70 75 80
Asp Gly Ile Val Gly Lys Asn Thr Trp Ala Glu Leu Phe Ser Lys Tyr
85 90 95
Ser Pro Pro Ile Pro Tyr Lys Thr Ile Pro Met Pro Thr Ala Asn Lys
100 105 110
Ser Arg Ala Ala Ala Thr Pro Val Met Asn Ala Val Glu Asn Ala Thr
115 120 125
Gly Val Arg Ser Gln Leu Leu Leu Thr Phe Ala Ser Ile Glu Ser Ala
130 135 140
Phe Asp Tyr Glu Ile Lys Ala Lys Thr Ser Ser Ala Thr Gly Trp Phe
145 150 155 160
Gln Phe Leu Thr Gly Thr Trp Lys Thr Met Ile Glu Asn Tyr Gly Met
165 170 175
Lys Tyr Gly Val Leu Thr Asp Pro Thr Gly Ala Leu Arg Lys Asp Pro
180 185 190
Arg Ile Ser Ala Leu Met Gly Ala Glu Leu Ile Lys Glu Asn Met Asn
195 200 205
Ile Leu Arg Pro Val Leu Lys Arg Glu Pro Thr Asp Thr Asp Leu Tyr
210 215 220
Leu Ala His Phe Phe Gly Pro Gly Ala Ala Arg Arg Phe Leu Thr Thr
225 230 235 240
Gly Gln Asn Glu Leu Ala Ala Thr His Phe Pro Lys Glu Ala Gln Ala
245 250 255
Asn Pro Ser Ile Phe Tyr Asn Lys Asp Gly Ser Pro Lys Thr Ile Gln
260 265 270
Glu Val Tyr Asn Leu Met Asp Gly Lys Val Ala Ala His Arg Lys Leu
275 280 285
Glu His His His His His His
290 295
<210> 71
<211> 25
<212> PRT
<213> 人工序列
<220>
<223> 合成序列; MW2 mut (I7R;L11R;P20G)
<400> 71
Gly Lys Pro Gly Trp Leu Arg Lys Val Ala Arg Lys Phe Lys Lys Leu
1 5 10 15
Ile Arg Arg Gly Leu Lys Arg Leu Ala
20 25
<210> 72
<211> 25
<212> PRT
<213> 人工序列
<220>
<223> 合成序列; MW2 mut (P3V;I7R;L11R)
<400> 72
Gly Lys Val Gly Trp Leu Arg Lys Val Ala Arg Lys Phe Lys Lys Leu
1 5 10 15
Ile Arg Arg Pro Leu Lys Arg Leu Ala
20 25
<210> 73
<211> 25
<212> PRT
<213> 人工序列
<220>
<223> 合成序列; MW2 mut (I7R)
<400> 73
Gly Lys Pro Gly Trp Leu Arg Lys Val Ala Leu Lys Phe Lys Lys Leu
1 5 10 15
Ile Arg Arg Pro Leu Lys Arg Leu Ala
20 25
<210> 74
<211> 25
<212> PRT
<213> 人工序列
<220>
<223> 合成序列; MW2 mut (L11R)
<400> 74
Gly Lys Pro Gly Trp Leu Ile Lys Val Ala Arg Lys Phe Lys Lys Leu
1 5 10 15
Ile Arg Arg Pro Leu Lys Arg Leu Ala
20 25
<210> 75
<211> 295
<212> PRT
<213> 人工序列
<220>
<223> 与具有C14S、C23S和C50S的突变的KZ144连接的衍生自MW2肽的突变的肽
<400> 75
Met Gly Lys Pro Gly Trp Leu Arg Lys Val Ala Arg Lys Phe Lys Lys
1 5 10 15
Leu Ile Arg Arg Gly Leu Lys Arg Leu Ala Gly Ser Lys Val Leu Arg
20 25 30
Lys Gly Asp Arg Gly Asp Glu Val Ser Gln Leu Gln Thr Leu Leu Asn
35 40 45
Leu Ser Gly Tyr Asp Val Gly Lys Pro Asp Gly Ile Phe Gly Asn Asn
50 55 60
Thr Phe Asn Gln Val Val Lys Phe Gln Lys Asp Asn Ser Leu Asp Ser
65 70 75 80
Asp Gly Ile Val Gly Lys Asn Thr Trp Ala Glu Leu Phe Ser Lys Tyr
85 90 95
Ser Pro Pro Ile Pro Tyr Lys Thr Ile Pro Met Pro Thr Ala Asn Lys
100 105 110
Ser Arg Ala Ala Ala Thr Pro Val Met Asn Ala Val Glu Asn Ala Thr
115 120 125
Gly Val Arg Ser Gln Leu Leu Leu Thr Phe Ala Ser Ile Glu Ser Ala
130 135 140
Phe Asp Tyr Glu Ile Lys Ala Lys Thr Ser Ser Ala Thr Gly Trp Phe
145 150 155 160
Gln Phe Leu Thr Gly Thr Trp Lys Thr Met Ile Glu Asn Tyr Gly Met
165 170 175
Lys Tyr Gly Val Leu Thr Asp Pro Thr Gly Ala Leu Arg Lys Asp Pro
180 185 190
Arg Ile Ser Ala Leu Met Gly Ala Glu Leu Ile Lys Glu Asn Met Asn
195 200 205
Ile Leu Arg Pro Val Leu Lys Arg Glu Pro Thr Asp Thr Asp Leu Tyr
210 215 220
Leu Ala His Phe Phe Gly Pro Gly Ala Ala Arg Arg Phe Leu Thr Thr
225 230 235 240
Gly Gln Asn Glu Leu Ala Ala Thr His Phe Pro Lys Glu Ala Gln Ala
245 250 255
Asn Pro Ser Ile Phe Tyr Asn Lys Asp Gly Ser Pro Lys Thr Ile Gln
260 265 270
Glu Val Tyr Asn Leu Met Asp Gly Lys Val Ala Ala His Arg Lys Leu
275 280 285
Glu His His His His His His
290 295
<210> 76
<211> 295
<212> PRT
<213> 人工序列
<220>
<223> 与具有C14S、C23S和C50S的突变的KZ144连接的衍生自MW2肽的突变的肽
<400> 76
Met Gly Lys Val Gly Trp Leu Arg Lys Val Ala Arg Lys Phe Lys Lys
1 5 10 15
Leu Ile Arg Arg Pro Leu Lys Arg Leu Ala Gly Ser Lys Val Leu Arg
20 25 30
Lys Gly Asp Arg Gly Asp Glu Val Ser Gln Leu Gln Thr Leu Leu Asn
35 40 45
Leu Ser Gly Tyr Asp Val Gly Lys Pro Asp Gly Ile Phe Gly Asn Asn
50 55 60
Thr Phe Asn Gln Val Val Lys Phe Gln Lys Asp Asn Ser Leu Asp Ser
65 70 75 80
Asp Gly Ile Val Gly Lys Asn Thr Trp Ala Glu Leu Phe Ser Lys Tyr
85 90 95
Ser Pro Pro Ile Pro Tyr Lys Thr Ile Pro Met Pro Thr Ala Asn Lys
100 105 110
Ser Arg Ala Ala Ala Thr Pro Val Met Asn Ala Val Glu Asn Ala Thr
115 120 125
Gly Val Arg Ser Gln Leu Leu Leu Thr Phe Ala Ser Ile Glu Ser Ala
130 135 140
Phe Asp Tyr Glu Ile Lys Ala Lys Thr Ser Ser Ala Thr Gly Trp Phe
145 150 155 160
Gln Phe Leu Thr Gly Thr Trp Lys Thr Met Ile Glu Asn Tyr Gly Met
165 170 175
Lys Tyr Gly Val Leu Thr Asp Pro Thr Gly Ala Leu Arg Lys Asp Pro
180 185 190
Arg Ile Ser Ala Leu Met Gly Ala Glu Leu Ile Lys Glu Asn Met Asn
195 200 205
Ile Leu Arg Pro Val Leu Lys Arg Glu Pro Thr Asp Thr Asp Leu Tyr
210 215 220
Leu Ala His Phe Phe Gly Pro Gly Ala Ala Arg Arg Phe Leu Thr Thr
225 230 235 240
Gly Gln Asn Glu Leu Ala Ala Thr His Phe Pro Lys Glu Ala Gln Ala
245 250 255
Asn Pro Ser Ile Phe Tyr Asn Lys Asp Gly Ser Pro Lys Thr Ile Gln
260 265 270
Glu Val Tyr Asn Leu Met Asp Gly Lys Val Ala Ala His Arg Lys Leu
275 280 285
Glu His His His His His His
290 295
<210> 77
<211> 295
<212> PRT
<213> 人工序列
<220>
<223> 与具有C14S、C23S和C50S的突变的KZ144连接的衍生自MW2肽的突变的肽
<400> 77
Met Gly Lys Pro Gly Trp Leu Arg Lys Val Ala Leu Lys Phe Lys Lys
1 5 10 15
Leu Ile Arg Arg Pro Leu Lys Arg Leu Ala Gly Ser Lys Val Leu Arg
20 25 30
Lys Gly Asp Arg Gly Asp Glu Val Ser Gln Leu Gln Thr Leu Leu Asn
35 40 45
Leu Ser Gly Tyr Asp Val Gly Lys Pro Asp Gly Ile Phe Gly Asn Asn
50 55 60
Thr Phe Asn Gln Val Val Lys Phe Gln Lys Asp Asn Ser Leu Asp Ser
65 70 75 80
Asp Gly Ile Val Gly Lys Asn Thr Trp Ala Glu Leu Phe Ser Lys Tyr
85 90 95
Ser Pro Pro Ile Pro Tyr Lys Thr Ile Pro Met Pro Thr Ala Asn Lys
100 105 110
Ser Arg Ala Ala Ala Thr Pro Val Met Asn Ala Val Glu Asn Ala Thr
115 120 125
Gly Val Arg Ser Gln Leu Leu Leu Thr Phe Ala Ser Ile Glu Ser Ala
130 135 140
Phe Asp Tyr Glu Ile Lys Ala Lys Thr Ser Ser Ala Thr Gly Trp Phe
145 150 155 160
Gln Phe Leu Thr Gly Thr Trp Lys Thr Met Ile Glu Asn Tyr Gly Met
165 170 175
Lys Tyr Gly Val Leu Thr Asp Pro Thr Gly Ala Leu Arg Lys Asp Pro
180 185 190
Arg Ile Ser Ala Leu Met Gly Ala Glu Leu Ile Lys Glu Asn Met Asn
195 200 205
Ile Leu Arg Pro Val Leu Lys Arg Glu Pro Thr Asp Thr Asp Leu Tyr
210 215 220
Leu Ala His Phe Phe Gly Pro Gly Ala Ala Arg Arg Phe Leu Thr Thr
225 230 235 240
Gly Gln Asn Glu Leu Ala Ala Thr His Phe Pro Lys Glu Ala Gln Ala
245 250 255
Asn Pro Ser Ile Phe Tyr Asn Lys Asp Gly Ser Pro Lys Thr Ile Gln
260 265 270
Glu Val Tyr Asn Leu Met Asp Gly Lys Val Ala Ala His Arg Lys Leu
275 280 285
Glu His His His His His His
290 295
<210> 78
<211> 295
<212> PRT
<213> 人工序列
<220>
<223> 与具有C14S、C23S和C50S的突变的KZ144连接的衍生自MW2肽的突变的肽
<400> 78
Met Gly Lys Pro Gly Trp Leu Ile Lys Val Ala Arg Lys Phe Lys Lys
1 5 10 15
Leu Ile Arg Arg Pro Leu Lys Arg Leu Ala Gly Ser Lys Val Leu Arg
20 25 30
Lys Gly Asp Arg Gly Asp Glu Val Ser Gln Leu Gln Thr Leu Leu Asn
35 40 45
Leu Ser Gly Tyr Asp Val Gly Lys Pro Asp Gly Ile Phe Gly Asn Asn
50 55 60
Thr Phe Asn Gln Val Val Lys Phe Gln Lys Asp Asn Ser Leu Asp Ser
65 70 75 80
Asp Gly Ile Val Gly Lys Asn Thr Trp Ala Glu Leu Phe Ser Lys Tyr
85 90 95
Ser Pro Pro Ile Pro Tyr Lys Thr Ile Pro Met Pro Thr Ala Asn Lys
100 105 110
Ser Arg Ala Ala Ala Thr Pro Val Met Asn Ala Val Glu Asn Ala Thr
115 120 125
Gly Val Arg Ser Gln Leu Leu Leu Thr Phe Ala Ser Ile Glu Ser Ala
130 135 140
Phe Asp Tyr Glu Ile Lys Ala Lys Thr Ser Ser Ala Thr Gly Trp Phe
145 150 155 160
Gln Phe Leu Thr Gly Thr Trp Lys Thr Met Ile Glu Asn Tyr Gly Met
165 170 175
Lys Tyr Gly Val Leu Thr Asp Pro Thr Gly Ala Leu Arg Lys Asp Pro
180 185 190
Arg Ile Ser Ala Leu Met Gly Ala Glu Leu Ile Lys Glu Asn Met Asn
195 200 205
Ile Leu Arg Pro Val Leu Lys Arg Glu Pro Thr Asp Thr Asp Leu Tyr
210 215 220
Leu Ala His Phe Phe Gly Pro Gly Ala Ala Arg Arg Phe Leu Thr Thr
225 230 235 240
Gly Gln Asn Glu Leu Ala Ala Thr His Phe Pro Lys Glu Ala Gln Ala
245 250 255
Asn Pro Ser Ile Phe Tyr Asn Lys Asp Gly Ser Pro Lys Thr Ile Gln
260 265 270
Glu Val Tyr Asn Leu Met Asp Gly Lys Val Ala Ala His Arg Lys Leu
275 280 285
Glu His His His His His His
290 295
<210> 79
<211> 264
<212> PRT
<213> 人工序列
<220>
<223> 与弧菌噬菌体ICP1的尾基片蛋白连接的爪蟾抗菌肽肽
<400> 79
Met Gly Ile Gly Lys Phe Leu His Ser Ala Lys Lys Phe Gly Lys Ala
1 5 10 15
Phe Val Gly Glu Ile Met Asn Ser Gly Ser Met Ile Leu Lys Arg Gly
20 25 30
Ser Ser Gly Ala Asp Val Lys Asn Met Gln Glu Tyr Leu Thr Ala Leu
35 40 45
Gly Tyr Asp Thr Lys Gly Val Glu Gly Thr Phe Glu Gly Gly Thr Glu
50 55 60
Ser Ala Val Lys Ala Phe Gln Lys Asp Met Ser Phe Thr Val Val Asp
65 70 75 80
Gly Ile Ile Gly Asn Gln Thr Ala Lys His Leu Val Asp Met Tyr Tyr
85 90 95
Gly Lys Val Val Pro Phe Gly Tyr Val Thr Asn Thr Pro Trp Val Ser
100 105 110
Glu Ala Ile Glu Asp Tyr Phe Val Ser Glu Ile Lys Gly Glu Lys His
115 120 125
Asn Pro Arg Val Val Gln Tyr Phe Lys Asp Ala His Ser Ser Trp Phe
130 135 140
Thr Asp Asp Glu Thr Pro Trp Cys Ala Ala Ala Val Ser Ser Trp Leu
145 150 155 160
Glu Arg Ala Gly Ile Arg Ser Val Arg Ser Ala Arg Ala Arg Asp His
165 170 175
Ile Asn Phe Gly Thr Lys Leu Leu Glu Pro Arg Phe Gly Ala Ile Val
180 185 190
Val Leu Glu Arg Gly Ala Asn Ser Gly His Val Gly Phe Val Asn Gly
195 200 205
Val Thr Ala Asp Gly Lys Gln Ile Lys Val Leu Gly Gly Asn Gln Ser
210 215 220
Asp Ser Val Asn Glu Arg Met Phe Gln Val Thr Arg Val Leu Gly Tyr
225 230 235 240
Arg Gln Pro Glu Gly Phe Val Leu Pro Pro Cys Pro Ile Val Gly Lys
245 250 255
Gly Glu Leu Ser Lys Ser Glu Ala
260
Claims (25)
1.融合蛋白,其包含以下的序列:
a)肽聚糖水解酶,和
b)与所述肽聚糖水解酶异源的肽序列,其中所述异源肽序列包含序列基序,所述序列基序:
i)长度为16、17、18、19或20个氨基酸;
ii)包含至少40%并且至多60%选自由赖氨酸、精氨酸和组氨酸组成的第一组氨基酸的氨基酸,
其中每个氨基酸独立地选自所述第一组,
其中选自该第一组的每个氨基酸在所述序列基序中单独地排列、与选自所述第一组的另外的氨基酸一起成对地排列,或者与选自所述第一组的另外2个氨基酸成区块排列,但不与选自所述第一组的3个或更多个氨基酸成区块出现,其中至少2对的选自所述第一组的氨基酸存在于所述序列基序中,并且其中至多一个具有成排的选自所述第一组的3个氨基酸的区块存在于所述序列基序中,附加条件是如果此类具有所述第一组的3个氨基酸的区块存在于所述序列基序中,则在相对于所述3个氨基酸的区块的第一个氨基酸的位置-12、-11、-8、-5、-4、+6、+7、+10、+13和+14处的氨基酸不选自所述第一组,条件是相应的位置可以在所述序列基序中找到,
iii)包含至少40%并且至多60%选自由丙氨酸、甘氨酸、异亮氨酸、亮氨酸、苯丙氨酸、丝氨酸、苏氨酸、色氨酸、酪氨酸和缬氨酸组成的第二组氨基酸的氨基酸,
其中每个氨基酸独立地选自所述第二组,
其中如果选自所述第一组的和选自所述第二组的氨基酸的总和产生100%的所述序列基序,则至少三个不同的氨基酸选自该第二组;
其中如果所述序列基序含有至少两个单一非相邻的苯丙氨酸残基,并且这些苯丙氨酸残基中的至少一个直接在赖氨酸残基之后,则所述序列基序不包含序列AFV,并且
其中如果所述序列基序含有至少三个单一非相邻的组氨酸残基,则所述序列基序不包含序列AALTH(SEQ ID NO:2),
iv)其中如果所述基序中存在任何剩余的氨基酸,所述序列基序的剩余氨基酸选自由天冬酰胺、天冬氨酸、谷氨酰胺、谷氨酸、甲硫氨酸或半胱氨酸组成的第三组,其中所述氨基酸的每一个独立地选自所述第三组,并且其中谷氨酰胺可以仅选择一次并且此外其中所述选择可以不包含谷氨酰胺和谷氨酸的组合,并且
c)其中所述融合蛋白不包含SEQ ID NO:1的序列。
2.根据权利要求1的融合蛋白,其中所述序列基序遵从图1中描绘的序列基序之一,并且其中“X”表示所述序列基序在相应的位置处不展现出选自所述第一组的氨基酸。
3.根据权利要求2的融合蛋白,其中“X”表示所述序列基序在所述位置处展现出选自所述第二组的氨基酸。
4.根据前述权利要求中任一项的融合蛋白,其中所述序列基序长度为17、18或19个氨基酸。
5.根据前述权利要求中任一项的融合蛋白,其中选自所述第三组的氨基酸选自天冬酰胺、天冬氨酸、谷氨酰胺和谷氨酸。
6.根据前述权利要求中任一项的融合蛋白,其中所述序列基序不含有超过一个选自所述第三组的氨基酸,优选地其中所述序列基序不含有任何选自所述第三组的氨基酸。
7.根据前述权利要求中任一项的融合蛋白,其中所述序列基序不包含由所述第一组的3个氨基酸组成的区块。
8.根据前述权利要求中任一项的融合蛋白,其中所述肽序列是在自然界不存在的人工肽序列。
9.根据前述权利要求中任一项的融合蛋白,其中所述肽聚糖水解酶是Lys394、KZ144、OBPgpLys内溶素或弧菌噬菌体ICP1的尾基片(tail baseplate)蛋白,特别是其中所述融合蛋白包含SEQ ID NO:24、SEQ ID NO:25、SEQ ID NO:26或SEQ ID NO:27的序列。
10.根据前述权利要求中任一项的融合蛋白,其中所述序列基序是螺旋状的。
11.根据前述权利要求中任一项的融合蛋白,其中脯氨酸残基定位于所述序列基序的N端或C端的1至5个氨基酸残基内。
12.根据权利要求11的融合蛋白,其中所述脯氨酸残基定位于所述肽聚糖水解酶和所述序列基序之间。
13.根据前述权利要求中任一项的融合蛋白,其中所述序列基序位于所述肽聚糖水解酶的序列的N端。
14.根据前述权利要求中任一项的融合蛋白,其中所述融合蛋白包含选自下组的序列:SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ ID NO:21、SEQ IDNO:22、SEQ ID NO:23、SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:34、SEQ ID NO:35、SEQ IDNO:36、SEQ ID NO:37、SEQ ID NO:38、SEQ ID NO:39、SEQ ID NO:40、SEQ ID NO:41 SEQ IDNO:42和SEQ ID NO:43。
15.多肽,其包含SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ IDNO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19、SEQ ID NO:20、SEQ IDNO:21、SEQ ID NO:22或SEQ ID NO:23的序列。
16.核酸,其编码根据权利要求1至14中任一项的融合蛋白或根据权利要求15的多肽。
17.载体,其包含根据权利要求16的核酸。
18.噬菌体,其包含根据权利要求16的核酸。
19.宿主细胞,其包含根据权利要求1至14中任一项的融合蛋白、根据权利要求15的多肽、根据权利要求16的核酸、根据权利要求17的载体和/或根据权利要求18的噬菌体。
20.组合物,其包含根据权利要求1至14中任一项的融合蛋白、根据权利要求15的多肽、根据权利要求16的核酸、根据权利要求17的载体、根据权利要求18的噬菌体和/或根据权利要求19的宿主细胞。
21.根据权利要求20的组合物,其中所述组合物是包含药学上可接受的稀释剂、赋形剂或载体的药物组合物,或者其中所述组合物是包含可接受的稀释剂、赋形剂或载体的化妆品组合物。
22.试剂盒,其包含i)根据权利要求1至14中任一项的融合蛋白、根据权利要求15的多肽、根据权利要求16的核酸、根据权利要求17的载体、根据权利要求18的噬菌体、根据权利要求19的宿主细胞和/或根据权利要求20至21中任一项的组合物并且进一步包含ii)肽聚糖水解酶,或分别编码或包含所述肽聚糖水解酶的核酸、载体、噬菌体和/或宿主细胞。
23.根据权利要求1至14中任一项的融合蛋白、根据权利要求15的多肽、根据权利要求16的核酸、根据权利要求17的载体、根据权利要求18的噬菌体、根据权利要求19的宿主细胞、根据权利要求20至21中任一项的组合物或根据权利要求22的试剂盒,其用于通过手术或疗法治疗人或动物体的方法中或者用于在人或动物体上实施的诊断方法中,特别是用于治疗或预防细菌感染的方法中。
24.根据权利要求1至14中任一项的融合蛋白、根据权利要求15的多肽、根据权利要求16的核酸、根据权利要求17的载体、根据权利要求18的噬菌体、根据权利要求19的宿主细胞、根据权利要求20至21中任一项的组合物或根据权利要求22的试剂盒作为食物、饲料或化妆品中的抗微生物剂或者作为消毒剂的用途。
25.根据权利要求1至14中任一项的融合蛋白、根据权利要求15的多肽、根据权利要求16的核酸、根据权利要求17的载体、根据权利要求18的噬菌体、根据权利要求19的宿主细胞、根据权利要求20至21中任一项的组合物或根据权利要求22的试剂盒用于处理或防止以下各项的革兰氏阴性细菌污染的用途:食品、食物加工设备、食物加工厂、与食品接触的表面、饲料、饲料加工设备、饲料加工厂、与饲料接触的表面、医疗设备、医院、医生诊室和其他医疗设施中的无生命表面。
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