CN112204054A - 新抗微生物融合蛋白 - Google Patents
新抗微生物融合蛋白 Download PDFInfo
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- CN112204054A CN112204054A CN201980034505.0A CN201980034505A CN112204054A CN 112204054 A CN112204054 A CN 112204054A CN 201980034505 A CN201980034505 A CN 201980034505A CN 112204054 A CN112204054 A CN 112204054A
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Abstract
本发明涉及一种多肽,其包含革兰氏阴性内溶素和选自以下的肽:抗微生物肽、两亲性肽、阳离子肽、寿司肽或防御素,其中所述内溶素又包含根据SEQ ID NO:1或SEQ ID NO:2的序列。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、组合物和试剂盒在通过手术或疗法治疗人体或动物体的方法中或在人体或动物体上实践的诊断方法中的用途。还可以将根据本发明的多肽、核酸、载体、噬菌体、宿主细胞、组合物和试剂盒用作例如食品或饲料中,化妆品中的抗微生物剂,或用作消毒剂。
Description
本发明涉及一种多肽,其包含革兰氏阴性内溶素和选自以下的肽:抗微生物肽、两亲性肽、阳离子肽、寿司肽或防御素,其中所述内溶素又包含根据SEQ ID NO:1或SEQ IDNO:2的序列。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、组合物和试剂盒在通过手术或疗法治疗人体或动物体的方法中或在人体或动物体上实践的诊断方法中的用途。还可以将根据本发明的多肽、核酸、载体、噬菌体、宿主细胞、组合物和试剂盒用作例如食品或饲料中,化妆品中的抗微生物剂,或用作消毒剂。
对常规抗生素的抗药性正在成为人类日益增加的健康风险。新的抗生素抗药性机制正在出现并在全球迅速传播。因此,在不久的将来,治疗常见传染病的能力可能会越来越困难。在本领域中已经很容易理解这种危险,并且探索了对抗细菌感染因子的新方法。
其中,这些新方法是将内溶素与不同种类的肽组合形成融合蛋白。内溶素是由噬菌体(即,细菌病毒)编码的胞壁质水解酶(特别是肽聚糖水解酶)。其在噬菌体增殖的裂解周期的晚期基因表达过程中合成,并通过细菌肽聚糖的降解来介导后代病毒体从感染细胞的释放。就酶活性而言,其通常是β(1,4)-糖基化酶(溶解酶)、转糖基化酶、酰胺酶或内肽酶。在1991年已提出了内溶素的抗菌应用。尽管对内溶素的杀伤能力已经知晓了很长时间,但是由于抗生素的成功和优势,忽视了将这些酶作为抗菌剂的用途。仅在出现了多种抗生素耐药细菌之后,这种用内溶素对抗人类病原体的简单概念才引起人们的兴趣。迫切需要开发出新型抗菌剂,而作为“酶抗生素(enzybiotics)”(即“酶”和“抗生素”混合术语)的内溶素完全可以满足这一需求。在2001年,Fischetti及其同事首次证明了噬菌体C1内溶素对A组链球菌的治疗潜力。从那时起,很多出版物已将内溶素确立为控制细菌感染(尤其是革兰氏阳性细菌感染)的有吸引力和互补的替代方法。随后,已证明针对其他革兰氏阳性病原体(如肺炎链球菌、炭疽芽孢杆菌、无乳链球菌和金黄色葡萄球菌)的不同内溶素具有作为酶抗生素的功效。而长期以来,内溶素治疗的最重要挑战在于革兰氏阴性细菌对内溶素的外源性作用不敏感,因为外膜屏蔽了内溶素与肽聚糖的接触。
革兰氏阴性细菌具有外膜,以其特征性的不对称双层作为标志。外膜双层由包含磷脂(主要是磷脂酰乙醇胺)的内部单层和主要由单个糖脂脂多糖(LPS)组成的外部单层组成。细菌界中存在大量的LPS结构,并且LPS结构可能会因应当前的环境条件而被修改。LPS层的稳定性以及不同LPS分子之间的相互作用主要是通过二价离子(Mg2+、Ca2+)与LPS分子的阴离子组分(脂质A中的磷酸酯基团以及KDO的内核和羧基)的静电相互作用来实现的。此外,脂质A的疏水部分的致密和有序堆积,由于缺乏不饱和脂肪酸的青睐,因而形成具有高粘度的刚性结构。这使其对亲脂分子的渗透性降低,并赋予外膜(OM)额外的稳定性。
为了克服外膜的屏蔽效应,已成功地将革兰氏阴性细菌的内溶素与例如阳离子、两亲性、疏水性或抗微生物肽融合。与未加入的情况相比,当加入时,此类融合蛋白能够消除革兰氏阴性细菌(参见例如WO 2010/023207、WO 2010/149792、WO 2011/134998、WO2012/146738或WO 2015/121443)。然而,为了获得改善的抗菌活性,通常将所述融合蛋白与少量乙二胺四乙酸(EDTA)组合。EDTA是一种螯合剂,是已知的外膜透化剂(Vaara,M.Microbiol.Rev.1992Sep;56(3):395-411,通过引用并入本文)。通过从其结合位点除去二价阳离子,引起外膜破坏,这通常改善了上述融合蛋白的抗菌活性(参见例如WO 2010/023207,表6和表8)。
尽管在各种应用领域中,EDTA的使用是完全可以接受的(例如,在消毒剂中),但是在其他应用领域中,EDTA或其他外膜通透剂的并行使用是次优甚至是不理想的(例如,在动物饲料、食品安全、医疗设备领域,以及通常在制药领域),因为EDTA会与任何种类的阳离子(不仅是细菌膜上的阳离子)非特异性地形成复合物。
因此,本领域仍然需要进一步改进此类抗菌融合蛋白的设计,特别是对于不允许并行使用EDTA的应用。
因而,本发明要解决的问题是提供上述类型的新的抗菌剂,其表现出(特别是在生理条件下)抗菌活性,并且较少依赖于平行存在的EDTA或其他外膜透化物质。
该问题通过所附权利要求书中定义的和下面阐述的主题来解决。
本发明的发明人已令人吃惊的发现,当与阳离子、两亲性或抗微生物肽融合时,显示出某些序列基序(分别是SEQ ID NO:1或SEQ ID NO:2)的内溶素是特别有用的。与未加入的情况相比,当加入革兰氏阴性细菌(如大肠杆菌)中时,所得到的融合蛋白显示出显著的抗菌活性,而且令人吃惊的是,与此同时,与这种类型的其他抗菌融合蛋白相比,其对于EDTA作为革兰氏阴性菌外膜透化剂平行存在的依赖性要小得多。这种令人吃惊的性质使这些多肽特别适于在EDTA敏感的应用领域中使用。
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聚合物。多肽的氨基酸残基可以通过例如碳水化合物和磷酸酯等各种基团的共价附着来修饰。其他物质可以与多肽更松散地结合,例如血红素或脂质,产生缀合的多肽,其也包括在如本文所用的术语“多肽”中。如本文使用,该术语还旨在包括蛋白质。因此,术语“多肽”还包括例如两个或更多个氨基酸聚合物链的复合物。术语“多肽”确实包括多肽的实施方案,所述多肽任选表现出本领域通常使用的修饰,例如,生物素化、乙酰化、聚乙二醇化、氨基、SH-或羧基(例如,保护基团)的化学变化等。从下面的描述中可以明显看出,根据本发明的多肽是人工工程改造的多肽,其在自然界中不以这种形式存在。此类多肽可以例如相对于天然存在的多肽表现出人工突变,或者可以包含异源序列,或者可以是天然存在的多肽片段,该片段在自然界中不会以这种形式出现。此外,根据本发明的多肽是融合蛋白,即代表至少两个氨基酸序列的连接,所述氨基酸序列在该组合中自然不存在。如本文所用,术语“多肽”不限于氨基酸聚合物链的特定长度。通常但不是必须的,本发明的典型多肽的长度将不超过约1000个氨基酸。本发明的多肽可以例如为至多约750个氨基酸的长度、至多约500个氨基酸的长度或者至多约300个氨基酸的长度。因此,本发明多肽的可能的长度范围但不限于此,例如可以是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。
如本文所用,术语“胞壁质水解酶”是本领域中的通常理解。其指能够水解细菌(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促切割机制。就切割机制而言,胞壁质水解酶可以是例如内肽酶、几丁质酶、T4样胞壁质酶(muraminidase)、λ样胞壁质酶、N-乙酰基-胞壁酰-L-丙氨酸-酰胺酶(酰胺酶)、胞壁酰基-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-氨基葡糖苷酶或转糖基酶。此外,该术语涵盖天然存在的胞壁质水解酶,如真核、原核或病毒(特别是噬菌体)来源的胞壁质水解酶(例如,肽聚糖水解酶)。该术语包含例如脊椎动物的溶菌酶(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)、与病毒粒子相关的肽聚糖水解酶(VAPGH)、细菌素(例如,溶葡萄球菌素)和自溶素。“胞壁质水解酶”也可以是能够切割细菌的肽聚糖的合成或人工修饰的多肽。例如,其中两个或更多个内溶素的结构域已被调换/交换的酶促活性改组的内溶素的确像“胞壁质水解酶”一样,仅保留了酶促活性结构域。活性(特别是内溶素的)可以通过本领域技术人员熟知的测定法测量,例如抗菌测定,例如参见Briers等(J.Biochem.Biophys Methods;2007;70:531-533)或Donovan等(J.FEMS Microbiol Lett.2006Dec;265(1))(均通过引用并入本文)和类似的出版物。
如本文所用,术语“内溶素”是指来源于噬菌体的酶,其适于催化细菌细胞壁的裂解(特别是通过水解)。优选地,内溶素是噬菌体来源的酶,由病毒利用噬菌体繁殖的裂解周期中的晚期基因表达合成,并介导子代病毒体的释放。内溶素通常显示出下述活性的至少一种:内肽酶、几丁质酶、T4样胞壁质酶(muraminidase)、λ样胞壁质酶、N-乙酰基-胞壁酰-L-丙氨酸-酰胺酶(酰胺酶)、胞壁酰基-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-氨基葡糖苷酶或转糖基酶,如例如KZ144内溶素。在一些内溶素中,这种活性表现在单个的“酶活性结构域”(EAD)中。此外,内溶素还可以含有无酶促活性的区域,并且与宿主细菌的细胞壁结合,即所谓的CBD(细胞壁结合域)。术语“内溶素”还包括相对于天然存在的内溶素包含修饰和/或改变的酶。此类改变和/或修饰可包括氨基酸残基的突变,例如缺失、插入和添加、取代或其组合和/或化学变化。特别优选的化学变化是生物素化、乙酰化、聚乙二醇化、氨基、SH或羧基的化学变化。所述内溶素在一般水平上表现出各自野生型内溶素的裂解活性。然而,所述活性可以与相应的野生型内溶素的活性相同,更高或更低。所述活性可以是例如相应野生型内溶素活性的至少约50、60、70、80、90、100、110、120、130、140、150、160、170、180、190或至少约200%或者甚至更多。活性可以通过本领域技术人员熟知的测定法测量,例如板裂解测定或液相裂解测定,例如参见Briers等(J.Biochem.Biophys Methods;2007;70:531-533)或Donovan等(J.FEMS Microbiol Lett.2006Dec;265(1))(均通过引用并入本文)和类似的出版物。
术语“革兰氏阴性内溶素”指来源于靶向革兰氏阴性细胞的噬菌体的内溶素。这些内溶素能够降解相应(宿主)细菌的肽聚糖。
如本文所用,“模块化”内溶素是表现出至少两个不同的功能性结构域的内溶素,即至少一个“酶促活性结构域”(EAD)和至少一个“细胞壁结合结构域”(CBD)。前者提供实际的酶促活性,后者可以提供靶点结合。由于其结构域特征,这两种活性可以彼此分化。缺乏独特CBD的内溶素不属于术语“模块化内溶素”。
术语“噬菌体尾/基板蛋白”是本领域技术人员的通常理解。尾蛋白和基板蛋白是噬菌体的蛋白。位于噬菌体尾纤维和/或基板的结合结构域在介导将噬菌体基因组注射进入宿主细胞中起重要作用。尾纤维蛋白位于尾的末端,通过识别潜在宿主细菌并附着在其外表面上,从而与细胞表面结合。基板蛋白控制遗传物质的转移,并且还可以具有细胞结合性质。特别是对于革兰氏阴性细菌的肌病毒(例如,T4或P2噬菌体),描述了不同的基序,这些基序与肽聚糖结合域(如LysM)具有同源性。另一个例实例是ICP1弧菌噬菌体的gp5和噬菌体基因组中编码的相关蛋白,其感染不同物种,例如甲基杆菌属种。这些由肽聚糖结合结构域和酶促活性结构域组成,能够降解宿主细菌的胞壁质层。
如本文所用,术语“抗微生物肽”(AMP)优选是指对例如细菌、病毒、真菌、酵母、支原体和原生动物具有杀微生物和/或微生物抑制活性的任何肽。在一些实施方式中,肽将是天然存在的肽。在其他实施方式中,肽将是自然界中不存在的人工肽。例如,抗微生物肽可以是天然存在的肽的突变形式。如本文所用,术语“抗微生物肽”特别是指具有抗细菌、抗真菌、抗霉菌、抗寄生物、抗原生动物、抗病毒、抗感染、抗传染和/或杀菌、杀藻、杀阿米巴(amoebicidal)、杀微生物、杀细菌、杀真菌、杀寄生物、杀原生动物(protozoacidal,protozoicidal)特性的任何肽。优选的是抗细菌肽。抗微生物肽可以是RNA酶A超家族的成员、防御素、抗微生物肽(cathelicidin)、颗粒溶素(granulysin)、组氨素(histatin)、psoriasin、dermicidine或hepcidin。抗微生物肽可以天然存在于昆虫、鱼、植物、蛛形纲动物(arachnids)、脊椎动物或哺乳动物中。优选地,抗微生物肽可以天然存在于萝卜、蚕蛾、狼蛛、蛙中,优选在非洲爪蟾(Xenopus laevis),Rana蛙中,更优选在牛蛙(Ranacatesbeiana),蟾蜍,优选亚洲蟾蜍Bufo bufo gargarizans,蝇,优选在果蝇(Drosophila)中,更优选在黑腹果蝇(Drosophila melanogaster),埃及伊蚊(Aedes aegypti),蜜蜂,大黄蜂,优选在Bombus pascuorum,肉蝇(flesh fly)中,优选在Sarcophaga peregrine,蝎子,鲎,鲶鱼中,优选在Parasilurus asotus,奶牛,猪,绵羊,猪,牛,猴和人类中。如本文所用,“抗微生物肽”(AMP)可以特别是不作为阳离子肽、聚阳离子肽、两亲性肽、寿司肽或防御素,但是仍然表现出抗微生物活性的肽。抗微生物肽的实例可以在内布拉斯加大学医学中心的“抗微生物肽数据库”(Omaha,NE,USA;http://aps.unmc.edu/AP/main.php)中找到。
如本文所用,术语“两亲性肽”是指具有亲水和疏水官能团两者的合成肽。优选地,如本文所用,术语“两亲性肽”是指具有限定的亲水和疏水基团排列的肽。
如本文所用,术语“阳离子肽”指具有荷正电的氨基酸残基的肽。优选地,阳离子肽具有9.0或更高的pKa值。通常,所述阳离子肽的至少四个氨基酸残基可荷正电,例如为赖氨酸或精氨酸。“荷正电”指所述氨基酸残基的侧链在接近生理环境具有净的正电荷。如本文所用,术语“阳离子肽”还指聚阳离子肽,但也包括阳离子肽,其包含例如小于20%,优选小于10%的带正电荷的氨基酸残基。
如本文所用,术语“聚阳离子肽”优选指由通常带正电荷的氨基酸残基,特别是赖氨酸和/或精氨酸残基构成的肽。若至少约60、70、75、80、85、90、95或约100%的氨基酸残基是带正电荷的氨基酸残基,特别是赖氨酸和/或精氨酸残基,则肽由通常带正电荷的氨基酸残基构成。不带正电荷的氨基酸残基的氨基酸残基可以是带中性电荷的氨基酸残基和/或带负电荷的氨基酸残基和/或疏水氨基酸残基。优选地,不带正电荷的氨基酸残基的氨基酸残基是带中性电荷的氨基酸残基,特别是丝氨酸和/或甘氨酸。
如本文所用,术语“寿司肽”是指具有短共有重复的补体控制蛋白(CCP)。寿司肽的寿司模块在许多不同的蛋白质中起蛋白质-蛋白质相互作用域的作用。已显示含有寿司域的肽具有抗微生物活性。优选地,寿司肽是天然存在的肽。
如本文所用,术语“防御素”是指存在于动物,优选哺乳动物,更优选人类中的肽,其中防御素在先天宿主防御系统中起破坏外来物质如感染性细菌和/或感染性病毒和/或真菌的作用。防御素是非抗体杀微生物和/或杀肿瘤蛋白、肽或多肽。“防御素”的实例是“哺乳动物防御素”、α-防御素、β-防御素、吲哚菌肽和magainin。如本文所用,术语“防御素”是指从动物细胞的分离形式或合成产生的形式,并且还指基本上保留其亲本蛋白的细胞毒活性但其序列已通过插入或缺失一个或多个氨基酸残基而改变的变体。
如本文所用,术语“标签”是指下述氨基酸序列,其通常在本领域中与另一个氨基酸序列融合或包含在另一氨基酸序列中,用于a)改善总体氨基酸序列或多肽的表达,b)促进整个氨基酸序列或多肽的纯化,c)促进整个氨基酸序列或多肽的固定,和/或d)促进总氨基酸序列或多肽的检测。标签的实例是His标签,例如His5标签、His6标签、His7标签、His8标签、His9标签、His10标签、His11标签、His12标签、His16标签和His20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、HA标签、硫氧还蛋白或麦芽糖结合蛋白(MBP)、CAT、GFP、YFP等。本领域技术人员将知道适用于不同技术应用的大量标签。标签可以例如使此类带有标签的多肽适用于例如不同ELISA测定形式或其他技术应用中的抗体结合。
如本文所用,术语“%序列同一性”必须如下理解:比对两个待比较的序列以给出序列之间的最大相关性。这可以包括在一个或两个序列中插入“缺口”,以增强比对程度。然后,可以在所比较的每个序列的整个长度(所谓的全局比对)(其特别适合于相同或相似长度的序列),或者更短的限定长度的序列(所谓的局部比对)(其更适合不相等长度的序列上)确定%同一性。在上述背景中,与查询氨基酸序列具有至少例如95%的“序列同一性”的氨基酸序列意指主题氨基酸序列的序列与查询序列相同,只是主题氨基酸序列可以包括查询氨基酸序列的每100个氨基酸最多五个氨基酸改变。换言之,为了获得与查询氨基酸序列具有至少95%同一性序列的氨基酸序列,可以插入或用另一种氨基酸取代或缺失主题序列中多达5%(100中的5个)氨基酸残基。用于比较两种或更多种序列的同一性和同源性的方法是本领域熟知的。两个序列相同的百分比可以例如通过使用数学算法来确定。可以使用的数学算法的优选但非限制性的例子是Karlin等,(1993),PNAS USA,90:5873-5877的算法。此种算法集成在BLAST系列程序中,例如,BLAST或NBLAST程序(亦参见Altschul等,1990,J.Mol.Biol.215,403-410或Altschul等,(1997),Nucleic Acids Res,25:3389-3402)(可通过NCBI主页在万维网站点ncbi.nlm.nih.gov访问)和FASTA(Pearson(1990),Methods Enzymol.83,63-98;Pearson和Lipman(1988),Proc.Natl.Acad.Sci.U.S.A 85,2444-2448.)。通过这些程序可以鉴定在一定程度上与其他序列相同的序列。此外,Wisconsin序列分析包9.1版(Devereux等,1984,Nucleic Acids Res.,387-395)中可用的程序,例如BESTFIT和GAP程序,可用于确定两个多肽序列之间的%同一性。BESTFIT使用(Smith和Waterman(1981),J.Mol.Biol.147,195-197)的“局部同源性”算法,并找到两个序列之间最佳的单一相似性区域。若在本文中提及与参考序列共享特定序列同一性程度的氨基酸序列,则所述序列差异优选归因于保守氨基酸取代。优选地,此类序列保留参考序列的活性,例如,虽然可能速率较慢。另外,若在本文中提及在特定的序列同一性百分比下共享“至少”的序列,则优选不包括100%序列同一性。
如本文所用,术语“包含”不应解释为限于“由......组成”的含义(即排除存在另外的其他物质)。相反,“包含”意味着任选地可以存在另外的物质。术语“包含”涵盖落入其范围“由......组成”(即排除存在另外的其他物质)和“包括但不由......组成”(即,需要存在另外的其他物质)的特别设想的实施方案,前者是更优选的。
当在本文中使用时,单词“一个(a)”或“一种(an)”的使用可能表示“一个(one)”,但其也与“一个或多个”、“至少一个”以及“一个或一个以上”的含义一致。
本发明在第一个方面涉及一种多肽,其包含革兰氏阴性内溶素和选自以下的肽:抗微生物肽、两亲性肽、阳离子肽、疏水肽、寿司肽或防御素,其中所述内溶素又包含根据SEQ ID NO:1的序列,并且优选地另外限制条件是
a)所述多肽既不包含根据SEQ ID NO:3的序列,也不包含根据SEQ ID NO:4的序列,也不包含根据SEQ ID NO:5的序列,
b)所述内溶素既不是气单胞菌噬菌体Aeh1的Aeh1p339(例如,NP_944217.1),也不是肠杆菌噬菌体JS98的EpJS98_gp116(例如,YP_001595245.1),
c)如果所述多肽包含SEQ ID NO:6所示的序列,则肽选自以下:抗微生物肽、两亲性肽、寿司肽或防御素,
d)如果所述内溶素具有选自以下的序列:
宿主 | 噬菌体名称 | 蛋白ID |
气单胞菌 | 气单胞菌噬菌体PX29 | ADQ53036.1 |
气单胞菌 | 气单胞菌噬菌体phiAS4 | YP_003969055.1 |
气单胞菌 | 气单胞菌噬菌体44RR2.8t | NP_932578.1 |
气单胞菌 | 气单胞菌噬菌体25 | YP_656449.1 |
气单胞菌 | 气单胞菌噬菌体31 | YP_238949.1 |
气单胞菌 | 气单胞菌噬菌体65 | YP_004300997.1 |
气单胞菌 | 气单胞菌噬菌体phiAS5 | YP_003969406.1 |
大肠杆菌 | 大肠杆菌噬菌体wV7 | AEM00790.1 |
大肠杆菌 | 肠杆菌噬菌体vB_EcoM-VR7 | YP_004063811.1 |
大肠杆菌 | 肠杆菌噬菌体Bp7 | AEN93735.1 |
大肠杆菌 | 肠杆菌噬菌体AR1 | BAI83135.1 |
大肠杆菌 | 肠杆菌噬菌体JS10 | YP_002922463.1 |
大肠杆菌 | 肠杆菌噬菌体IME08 | YP_003734260.1 |
大肠杆菌 | 肠杆菌噬菌体CC31 | YP_004009990.1 |
大肠杆菌 | 肠杆菌噬菌体RB69 | NP_861818.1 |
大肠杆菌 | 肠杆菌噬菌体RB14 | YP_002854463.1 |
大肠杆菌 | 肠杆菌噬菌体RB32 | ABI94948.1 |
大肠杆菌 | 肠杆菌噬菌体RB51 | YP_002854084.1 |
沙雷氏菌 | 沙雷氏菌噬菌体Shfl2 | YP_004415022.1 |
和仅缺少N-末端甲硫氨酸的相应序列,则所述多肽不包含i)模块化革兰氏阴性内溶素或ii)噬菌体尾/基板蛋白的细胞壁结合结构域,
e)如果所述内溶素具有根据以下的氨基酸2-164的序列,则所述多肽不包含i)模块化革兰氏阴性内溶素或ii)噬菌体尾/基板蛋白的细胞壁结合结构域:
宿主 | 噬菌体名称 | 蛋白ID |
大肠杆菌 | 肠杆菌噬菌体T4 | NP_049736.1 |
f)如果所述内溶素具有根据以下的氨基酸2-165的序列,则所述多肽不包含i)模块化革兰氏阴性内溶素或ii)噬菌体尾/基板蛋白的细胞壁结合结构域:
宿主 | 噬菌体名称 | 蛋白ID |
气单胞菌 | 气单胞菌噬菌体Aeh1 | NP_944217.1 |
g)如果所述内溶素具有根据以下的氨基酸2-161的序列,则所述多肽不包含i)模块化革兰氏阴性内溶素或ii)噬菌体尾/基板蛋白的细胞壁结合结构域:
宿主 | 噬菌体名称 | 蛋白ID |
大肠杆菌 | 肠杆菌噬菌体JS98 | YP_001595245.1 |
本发明在第二个方面涉及一种多肽,其包含革兰氏阴性内溶素和选自以下的肽:抗微生物肽、两亲性肽、阳离子肽、疏水肽、寿司肽或防御素,其中所述内溶素又包含根据SEQ ID NO:2的序列(特别地包含根据SEQ ID NO:7的序列),并且优选地另外限制条件是
a)所述多肽既不包含根据SEQ ID NO:3的序列,也不包含根据SEQ ID NO:4的序列,也不包含根据SEQ ID NO:5的序列,
b)所述内溶素不是肠杆菌噬菌体JS98的EpJS98_gp116(例如,YP_001595245.1),
c)如果所述多肽包含SEQ ID NO:6所示的序列,则肽选自以下:抗微生物肽、两亲性肽、寿司肽或防御素,
d)如果所述内溶素具有选自以下的序列:
宿主 | 噬菌体名称 | 蛋白ID |
气单胞菌 | 气单胞菌噬菌体65 | YP_004300997.1 |
大肠杆菌 | 大肠杆菌噬菌体wV7 | AEM00790.1 |
大肠杆菌 | 肠杆菌噬菌体vB_EcoM-VR7 | YP_004063811.1 |
大肠杆菌 | 肠杆菌噬菌体Bp7 | AEN93735.1 |
大肠杆菌 | 肠杆菌噬菌体AR1 | BAI83135.1 |
大肠杆菌 | 肠杆菌噬菌体JS10 | YP_002922463.1 |
大肠杆菌 | 肠杆菌噬菌体IME08 | YP_003734260.1 |
大肠杆菌 | 肠杆菌噬菌体CC31 | YP_004009990.1 |
大肠杆菌 | 肠杆菌噬菌体RB69 | NP_861818.1 |
大肠杆菌 | 肠杆菌噬菌体RB14 | YP_002854463.1 |
大肠杆菌 | 肠杆菌噬菌体RB32 | ABI94948.1 |
大肠杆菌 | 肠杆菌噬菌体RB51 | YP_002854084.1 |
沙雷氏菌 | 沙雷氏菌噬菌体Shfl2 | YP_004415022.1 |
和仅缺少N-末端甲硫氨酸的相应序列,则所述多肽不包含i)模块化革兰氏阴性内溶素或ii)噬菌体尾/基板蛋白的细胞壁结合结构域,
e)如果所述内溶素具有根据以下的氨基酸2-164的序列,则所述多肽不包含i)模块化革兰氏阴性内溶素或ii)噬菌体尾/基板蛋白的细胞壁结合结构域:
宿主 | 噬菌体名称 | 蛋白ID |
大肠杆菌 | 肠杆菌噬菌体T4 | NP_049736.1 |
f)如果所述内溶素具有根据以下的氨基酸2-161的序列,则所述多肽不包含i)模块化革兰氏阴性内溶素或ii)噬菌体尾/基板蛋白的细胞壁结合结构域:
宿主 | 噬菌体名称 | 蛋白ID |
大肠杆菌 | 肠杆菌噬菌体JS98 | YP_001595245.1 |
在第三个方面,本发明涉及一种多肽,其包含革兰氏阴性内溶素和选自以下的肽:抗微生物肽、两亲性肽、阳离子肽、疏水肽、寿司肽或防御素,其中所述内溶素又包含根据SEQ ID NO:1的序列,且其中所述内溶素在其序列中不包含任何半胱氨酸残基,并且优选地另外限制条件是:
a)所述内溶素不是气单胞菌噬菌体Aeh1的Aeh1p339,
b)如果所述内溶素具有选自以下的序列:
宿主 | 噬菌体名称 | 蛋白ID |
气单胞菌 | 气单胞菌噬菌体PX29 | ADQ53036.1 |
气单胞菌 | 气单胞菌噬菌体65 | YP_004300997.1 |
气单胞菌 | 气单胞菌噬菌体phiAS5 | YP_003969406.1 |
和仅缺少N-末端甲硫氨酸的相应序列,则所述多肽不包含i)模块化革兰氏阴性内溶素或ii)噬菌体尾/基板蛋白的细胞壁结合结构域,
c)如果所述内溶素具有根据以下的氨基酸2-165的序列,则所述多肽不包含i)模块化革兰氏阴性内溶素或ii)噬菌体尾/基板蛋白的细胞壁结合结构域:
宿主 | 噬菌体名称 | 蛋白ID |
气单胞菌 | 气单胞菌噬菌体Aeh1 | NP_944217.1 |
在内溶素序列中不存在任何半胱氨酸残基可能是因为内溶素的野生型形式已经不包含此类半胱氨酸残基,或者因为在野生型序列中出现的任何半胱氨酸残基已经过技术修饰/突变(例如,C→S、或C→A或C→G,优选地C→S),例如以增加稳定性并减少聚集。
在第四个方面中,本发明涉及一种多肽,其包含革兰氏阴性内溶素和选自以下的肽:抗微生物肽、两亲性肽、阳离子肽、疏水肽、寿司肽或防御素,其中所述内溶素又包含根据SEQ ID NO:2的序列(特别地包含根据SEQ ID NO:7的序列),且其中所述内溶素在其序列中不包含任何半胱氨酸残基,并且优选地另外限制条件是:
如果所述内溶素具有选自以下的序列:
宿主 | 噬菌体名称 | 蛋白ID |
气单胞菌 | 气单胞菌噬菌体65 | YP_004300997.1 |
和仅缺少N-末端甲硫氨酸的相应序列,则所述多肽不包含i)模块化革兰氏阴性内溶素或ii)噬菌体尾/基板蛋白的细胞壁结合结构域。
在内溶素序列中不存在任何半胱氨酸残基也可能是因为内溶素的野生型形式已经不包含此类半胱氨酸残基,或者因为在野生型序列中出现的任何半胱氨酸残基已经过技术修饰/突变(例如,C→S、或C→A或C→G,优选地C→S),例如以增加稳定性并减少聚集。
在第五个方面中,本发明涉及一种多肽,其包含革兰氏阴性内溶素和选自以下的肽:抗微生物肽、两亲性肽、阳离子肽、寿司肽或防御素,其中所述内溶素又包含根据SEQ IDNO:1的序列,且其中所述肽位于内溶素N末端多肽内(例如,在其中内溶素构成多肽最C末端组分的实施方式中),并且优选地另外限制条件是所述多肽既不包含根据SEQ ID NO:4的序列,也不包含根据SEQ ID NO:5的序列。
在第六个方面中,本发明涉及一种多肽,其包含革兰氏阴性内溶素和选自以下的肽:抗微生物肽、两亲性肽、阳离子肽、寿司肽或防御素,其中所述内溶素又是包含根据SEQID NO:2的序列的内溶素(特别地包含根据SEQ ID NO:7的序列),且其中所述肽位于内溶素N末端多肽内(例如,在其中内溶素构成多肽最C末端组分的实施方式中),并且优选地另外限制条件是所述多肽既不包含根据SEQ ID NO:4的序列,也不包含根据SEQ ID NO:5的序列。
在下文中,将更详细地讨论本发明的多肽(现在是本发明的第一个、第二个、第三个、第四个、第五个或第六个方面)。应当理解,除非另有明确说明,否则以下阐述的任何内容均等同地适用于以上所有六个方面。
本发明多肽的内溶素组分包含根据SEQ ID NO:1或SEQ ID NO:2的序列(特别是包含根据SEQ ID NO:7的序列)。SEQ ID NO:1的长度为13个氨基酸,并且具有序列X1NRAX2RVX3X4X5X6X7X8,其中X1可以是P或T,X2可以是K、M、N或Q,X3可以是A、I或T,X4可以是A、D、E、K、Q、S或T,X5可以是T或V,X6可以是F、I、L或V、X7可以是E、K、L或R,X8可以是L或T。SEQID NO:7的长度也为13个氨基酸,并且具有序列X1NRAKRVX2X3X4X5X6X7,其中X1可以是P或T,X2可以是A、I或T,X3可以是A、D、E或S,X4可以是T或V,X5可以是F、I或L,X6可以是E、K或R,X7可以是L或T。内溶素可以是例如天然地在其序列中表现出此类序列元件的内溶素,或者是具有这种序列的修饰的(并且因此不再是天然存在的)内溶素(例如,来源于天然存在的内溶素的内溶素,但是已通过突变、截短等对其进行修饰,例如以增加活性、稳定性、表达、克隆原因等)。在任一种情况下,根据SEQ ID NO:1(或者分别为SEQ ID NO:2或SEQ ID NO:7)的序列是内溶素的组成部分。与此相反的是,短语“包含根据SEQ ID NO:1的序列的内溶素”(或者分别为SEQ ID NO:2或SEQ ID NO:7)优选地并非旨在涵盖其中包含SEQ ID NO:1(或者分别为SEQ ID NO:2或SEQ ID NO:7)的序列元件已人工地与原本完全不相关的内溶素或EAD组合的情形。换言之,根据SEQ ID NO:1(或者分别为SEQ ID NO:2或SEQ ID NO:7)的序列与实际的内溶素不是异源的。SEQ ID NO:1(分别类似于SEQ ID NO:7)是指示变异可能位置的共有序列。典型地,根据SEQ ID NO:1(或SEQ ID NO:7)的序列将存在于内溶素序列的C末端部分,例如在内溶素C末端的最后40个、最后30个或者甚至最后25个氨基酸范围内。根据SEQID NO:1的共有序列的优选形式是SEQ ID NO:8。甚至更优选的形式是SEQ ID NO:9。同样地,SEQ ID NO:1特别优选的形式是SEQ ID NO:10和SEQ ID NO:11。SEQ ID NO:1也可以采用SEQ ID NO:12、SEQ ID NO:13或SEQ ID NO:14的形式。然而,在优选的实施方式中,多肽不包含SEQ ID NO:12、SEQ ID NO:13和/或SEQ ID NO:14。SEQ ID NO:1和SEQ ID NO:7特别优选的实例分别是SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ IDNO:19和SEQ ID NO:20。来源于感染革兰氏阴性细菌的噬菌体的内溶素即是革兰氏阴性内溶素。包含根据SEQ ID NO:1(或者分别为SEQ ID NO:2或SEQ ID NO:7)的序列的内溶素的实例是例如克氏柠檬酸杆菌噬菌体CkP1(SEQ ID NO:21)、肠杆菌噬菌体CC31(SEQ ID NO:22)、沙雷氏菌噬菌体CHI14(SEQ ID NO:23)、气单胞菌噬菌体Ah1(SEQ ID NO:24)、沙雷氏菌噬菌体PS2(SEQ ID NO:25)和气单胞菌噬菌体AS-szw(SEQ ID NO:26)内溶素;或分别与SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:23、SEQ ID NO:24、SEQ ID NO:25和SEQ ID NO:26具有至少80%序列同一性、优选地至少85%序列同一性、更优选地至少90%序列同一性、更优选地至少95%序列同一性、更优选地至少96%序列同一性、更优选地至少97%序列同一性、更优选地至少98%序列同一性、更优选地至少99%序列同一性的序列。特别优选的是与SEQ ID NO:21和/或SEQ ID NO:22具有至少80%序列同一性、优选地至少85%序列同一性、更优选地至少90%序列同一性、更优选地至少95%序列同一性、更优选地至少96%序列同一性、更优选地至少97%序列同一性、更优选地至少98%序列同一性、更优选地至少99%序列同一性的序列。用于本发明多肽的另一种适宜的内溶素是T4内溶素的衍生物,例如根据SEQ ID NO:27的序列,或与SEQ ID NO:27具有至少80%序列同一性、优选地至少85%序列同一性、更优选地至少90%序列同一性、更优选地至少95%序列同一性、更优选地至少96%序列同一性、更优选地至少97%序列同一性、更优选地至少98%序列同一性、更优选地至少99%序列同一性的序列(遵守上述关于SEQ ID NO:3的条件)。例如,这些内溶素的修饰形式,其中半胱氨酸被例如丝氨酸(例如,分别为C54S或C122S)替代和/或缺乏N末端甲硫氨酸,是用作本发明多肽组分的内溶素的优选形式(参见例如SEQ ID NO:6,受以上免责声明的约束,SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:32、SEQID NO:33、SEQ ID NO:34、SEQ ID NO:35和SEQ ID NO:36,其是特别优选的实施方式)。因此,特别优选的内溶素序列也是SEQ ID NO:6(遵守上述条件)、SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:34、SEQ IDNO:35和SEQ ID NO:36,或者与SEQ ID NO:6(遵守上述条件)、SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:34、SEQ IDNO:35和/或SEQ ID NO:36(特别是SEQ ID NO:28和/或SEQ ID NO:30)具有至少80%序列同一性、优选地至少85%序列同一性、更优选地至少90%序列同一性、更优选地至少95%序列同一性、更优选地至少96%序列同一性、更优选地至少97%序列同一性、更优选地至少98%序列同一性、更优选地至少99%序列同一性的序列。
下表1中列出了适宜的内溶素组分(特别是对于本发明的第一个、第三个和第五个方面,并且总是要遵守上述条件,但不限于此)的其他实例。
表1:在本发明多肽中适宜的内溶素组分的实例:
例如,可以通过美国国家生物技术信息中心(NCBI;https://www.ncbi.nlm.nih.gov/)的蛋白质数据库来访问表1中的内溶素序列。应当理解的是,表1中列出的内溶素的序列也可以被修饰,例如可以缺乏N末端甲硫氨酸,以避免在相应核酸序列中再有一个起始密码子。使用这样的边缘修饰的序列也在本发明的范围内,并且应当理解,当在本文中参考表1的内溶素时,所述修饰的内溶素也被所述定义涵盖。此外,发明人还设想,与表1中的序列显示出至少80%序列同一性、优选地至少85%序列同一性、更优选地至少90%序列同一性、更优选地至少95%序列同一性、更优选地至少96%序列同一性、更优选地至少97%序列同一性、更优选地至少98%序列同一性、更优选地至少99%序列同一性的内溶素也可以用于实施本发明。
应当理解的是,本文所讨论的任何序列,因为例如分别与SEQ ID NO:21、SEQ IDNO:22、SEQ ID NO:27、SEQ ID NO:28或SEQ ID NO:30具有一定水平的同一性,因而需要保留根据SEQ ID NO:1(或者SEQ ID NO:2或SEQ ID NO:7)的序列,即序列中的偏差将在SEQID NO:1(或者分别为SEQ ID NO:2或SEQ ID NO:7)之外,不在对应于SEQ ID NO:1(或者分别为SEQ ID NO:2或SEQ ID NO:7)基序的序列内。
如前文所提及的,同时已确立了大约十年的时间,如果将内溶素与例如抗微生物肽、两亲性肽或阳离子肽融合,与未加入时相比,使用相应的革兰氏阴性内溶素能够杀死革兰氏阴性细菌(参见例如,WO 2010/023207、WO 2010/149792、WO 2011/134998、WO 2012/146738或WO 2015/121443,其均通过引用并入本文)。在下文中,将在本发明多肽内的该肽也称为“肽组分”。如本文所用,应当理解的是,本文使用的该肽组分不是像His标签(His5标签、His6标签、His7标签、His8标签、His9标签、His10标签、His11标签、His12标签、His16标签和His20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)那样的常规标签,也不是本领域公知的其他标签(如硫氧还蛋白或麦芽糖结合蛋白(MBP))。
优选的阳离子和/或聚阳离子肽是包含至少一个根据SEQ ID NO:37所示的主题(KRKKRK)的那些。特别地,包含至少2、3、4、5、6、7、8、9、10、11、12、13、14、15、16或17个根据SEQ ID NO:37所示的主题(KRKKRK)的阳离子氨基酸序列段是优选的。更优选的是包含至少一个KRK主题(lys-arg-lys),优选地至少2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32或33个KRK主题的阳离子肽段。
在本发明的另一个优选的实施方式中,除了带正电的氨基酸残基(特别是赖氨酸和/或精氨酸残基)外,阳离子肽还包含带中性电荷的氨基酸残基(特别是甘氨酸和/或丝氨酸残基)。优选的是由约70%至约100%、或约80%至约95%、或约85%至约90%的带正电荷的氨基酸残基(特别是赖氨酸、精氨酸和/或组氨酸残基,更优选地赖氨酸和/或精氨酸残基)和约0%至约30%、或约5%至约20%、或约10%至约20%的带中性电荷的氨基酸残基(特别是甘氨酸和/或丝氨酸残基)组成的阳离子氨基酸序列段。优选的是由约4%至约8%丝氨酸残基、约33%至约36%精氨酸残基和约56%至约63%赖氨酸残基组成的氨基酸序列段。特别优选的是包含至少一个根据SEQ ID NO:38所示的主题(KRXKR)的氨基酸序列段,其中X是赖氨酸、精氨酸和组氨酸以外的任何其他氨基酸。特别优选的是包含至少一个根据SEQ ID NO:39所示的主题(KRSKR)的多肽段。更优选的是包含至少约2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19个或至少约20个根据SEQ ID NO:38所示的主题(KRXKR)或根据SEQ ID NO:39所示的主题(KRSKR)的阳离子段。
也是优选的是由约9至约16%甘氨酸残基、约4至约11%丝氨酸残基、约26至约32%精氨酸残基和约47至约55%赖氨酸残基组成的阳离子氨基酸序列段。特别优选的是包含至少一个根据SEQ ID NO:40所示的主题(KRGSG)的氨基酸序列段。更优选的是包含至少约2、3、4、5、6、7、8、9、10、11、12、13、14、15、16、17、18、19个或至少约20个根据SEQ ID NO:40所示的主题(KRGSG)的阳离子段。
在本发明的另一个优选的实施方式中,除了带正电的氨基酸残基(特别是赖氨酸和/或精氨酸残基)外,此类阳离子氨基酸序列段包含疏水性氨基酸残基,特别是缬氨酸、异亮氨酸、亮氨酸、甲硫氨酸、苯丙氨酸、色氨酸、半胱氨酸、丙氨酸、酪氨酸、脯氨酸和甘氨酸残基,更优选地丙氨酸、缬氨酸、亮氨酸、异亮氨酸、苯丙氨酸和/或色氨酸残基。优选的是由约70%至约100%、或约80%至约95%、或约85%至约90%的带正电荷的氨基酸残基(特别是赖氨酸和/或精氨酸残基)和约0%至约30%、或约5%至约20%、或约10%至约20%疏水性氨基酸残基缬氨酸、异亮氨酸、亮氨酸、甲硫氨酸、苯丙氨酸、色氨酸、半胱氨酸、丙氨酸、酪氨酸、脯氨酸和甘氨酸残基(更优选地丙氨酸、缬氨酸、亮氨酸、异亮氨酸、苯丙氨酸和/或色氨酸残基)组成的阳离子氨基酸序列段。下表中列出了阳离子和聚阳离子氨基酸序列段的实例:
表2:
在本发明的另一个方面中,肽是抗微生物肽,其包含正净电荷和约50%疏水性氨基酸。抗微生物肽是两亲性的,具有约12个至约50个氨基酸残基的长度。抗微生物肽天然存在于昆虫、鱼类、植物、蜘蛛、脊椎动物或哺乳动物中。优选地,抗微生物肽可以天然存在于萝卜、蚕蛾、狼蛛、蛙中,优选在非洲爪蟾(Xenopus laevis),Rana蛙中,更优选在牛蛙(Ranacatesbeiana),蟾蜍,优选亚洲蟾蜍Bufo bufo gargarizans,蝇,优选在果蝇(Drosophila)中,更优选在黑腹果蝇(Drosophila melanogaster),埃及伊蚊(Aedes aegypti),蜜蜂,大黄蜂,优选在Bombus pascuorum,肉蝇(flesh fly)中,优选在Sarcophaga peregrine,蝎子,鲎,鲶鱼中,优选在Parasilurus asotus,奶牛,猪,绵羊,猪,牛,猴和人类中。
在本发明的另一个优选实施方式中,抗微生物肽由约10%至约35%、或约15%至约45%、或约20%至约45%带正电的氨基酸残基(特别是赖氨酸和/或精氨酸残基)和约50%至约80%、或约60%至约80%、或约55%至约75%、或约70%至约90%疏水性氨基酸残基缬氨酸、异亮氨酸、亮氨酸、甲硫氨酸、苯丙氨酸、色氨酸、半胱氨酸、丙氨酸、酪氨酸、脯氨酸和甘氨酸残基(更优选地丙氨酸、缬氨酸、亮氨酸、异亮氨酸、苯丙氨酸和/或色氨酸残基)组成。
在本发明的另一个优选实施方式中,抗微生物肽由约4%至约58%带正电的氨基酸残基(特别是赖氨酸和/或精氨酸残基)和约33%至约89%疏水性氨基酸残基缬氨酸、异亮氨酸、亮氨酸、甲硫氨酸、苯丙氨酸、色氨酸、半胱氨酸、丙氨酸、酪氨酸、脯氨酸和甘氨酸残基(更优选地丙氨酸、缬氨酸、亮氨酸、异亮氨酸、苯丙氨酸和/或色氨酸残基)组成。
下表中列出了可以用于实施本发明的抗微生物氨基酸序列的实例。
表3:
用于本发明的发明多肽的特别优选的抗微生物肽是SMAP-29(SEQ ID NO:63)。
其他特别优选的抗微生物肽是根据SEQ ID NO:96和SEQ ID NO:107的肽。
在其他实施方式中,发明多肽的肽组分可以是寿司肽,其如Ding JL,Li P,Ho BCell Mol Life Sci.2008Apr;65(7-8):1202-19,The Sushi peptides:structuralcharacterization and mode of action against Gram-negative bacteria中所述。特别优选的是根据SEQ ID NO:108的寿司1肽。优选的寿司肽是寿司肽S1和S3及其倍数;Tan等,FASEB J.2000Sep;14(12):1801-13。
在本发明的其他方面中,肽组分是两亲性肽,其包含一个或多个带正电的氨基酸残基赖氨酸、精氨酸和/或组氨酸与一个或多个疏水性氨基酸残基缬氨酸、异亮氨酸、亮氨酸、甲硫氨酸、苯丙氨酸、色氨酸、半胱氨酸、丙氨酸、酪氨酸、脯氨酸和/或甘氨酸的组合。氨基酸残基的侧链经过排列,以使阳离子和疏水表面聚集在肽的对侧。优选地,在所述肽中超过约30、40、50、60或70%的氨基酸是带正电的氨基酸。优选地,在所述肽中超过约30、40、50、60或70%的氨基酸残基是疏水性氨基酸残基。有利地,两亲性肽存在于根据本发明的多肽的N末端(最优选)或C末端。
在本发明的另一个实施方式中,两亲性肽由至少5个、更优选地至少6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49个或至少50个氨基酸残基组成。在优选的实施方式中,至少约30、40、50、60或70%的两亲性肽的所述氨基酸残基是精氨酸或赖氨酸残基和/或至少约30、40、50、60或70%的两亲性肽的所述氨基酸残基是疏水性氨基酸缬氨酸、异亮氨酸、亮氨酸、甲硫氨酸、苯丙氨酸、色氨酸、半胱氨酸、丙氨酸、酪氨酸、脯氨酸和/或甘氨酸。
在本发明的另一个优选实施方式中,除了带正电的氨基酸残基(特别是赖氨酸和/或精氨酸残基)外,两亲性肽段包含疏水性氨基酸残基,特别是缬氨酸、异亮氨酸、亮氨酸、甲硫氨酸、苯丙氨酸、色氨酸、半胱氨酸、丙氨酸、酪氨酸、脯氨酸和甘氨酸,更优选地丙氨酸、缬氨酸、亮氨酸、异亮氨酸、苯丙氨酸和/或色氨酸残基。优选的是由约10%至约50%、或约20%至约50%、或约30%至约45%或约5%至约30%带正电的氨基酸残基(特别是赖氨酸和/或精氨酸残基)和约50%至约85%、或约50%至约90%、或约55%至约90%、或约60%至约90%、或约65%至约90%疏水性氨基酸残基缬氨酸、异亮氨酸、亮氨酸、甲硫氨酸、苯丙氨酸、色氨酸、半胱氨酸、丙氨酸、酪氨酸、脯氨酸和甘氨酸残基(更优选地丙氨酸、缬氨酸、亮氨酸、异亮氨酸、苯丙氨酸和/或色氨酸残基)组成的两亲性肽段。在另一个优选的实施方式中,两亲性肽段由12%至约50%带正电的氨基酸残基(特别是赖氨酸和/或精氨酸残基)和约50%至约85%疏水性氨基酸残基缬氨酸、异亮氨酸、亮氨酸、甲硫氨酸、苯丙氨酸、色氨酸、半胱氨酸、丙氨酸、酪氨酸、脯氨酸和甘氨酸(更优选地丙氨酸、缬氨酸、亮氨酸、异亮氨酸、苯丙氨酸和/或色氨酸残基)组成。
优选的两亲性肽是具有根据SEQ ID NO:109的氨基酸序列的WLBU2变体和根据SEQID NO:110的Walmagh 2。
在本发明的一个特别优选的实施方式中,肽(在本发明的多肽内)包含以下序列基序:
i)长度为16、17、18、19或20个氨基酸;
ii)包含至少40%和至多60%的氨基酸选自第一组氨基酸,所述第一组氨基酸由赖氨酸、精氨酸和组氨酸组成,
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单独地,与选自所述第一组的一个其他氨基酸成对,或与选自所述第一组的2个其他氨基酸嵌段,但不发生与选自所述第一组的3个或更多个氨基酸嵌段排布在所述序列基序中,其中在所述基序中存在选自所述第一组的至少2对氨基酸,且其中在所述序列基序中存在具有连续选自所述第一组的3个所述氨基酸的至多一个嵌段,附加条件是,如果在所述序列基序中存在具有所述第一组的3个氨基酸的嵌段,则不从所述第一组中选择相对于3个氨基酸嵌段的所述第一氨基酸的在位置-12、-11、-8、-5、-4、+6、+7、+10、+13和+14的所述氨基酸,如果可以在所述序列基序中找到各自的位置的话,
iii)包含至少40%和至多60%的氨基酸选自第二组氨基酸,所述第二组氨基酸由丙氨酸、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,
其中每个氨基酸独立地选自所述第二组,
其中优选地,如果选自所述第一组合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3个不同氨基酸选自该第二组;
其中优选地,如果序列基序包含至少两个单一的不相邻的苯丙氨酸残基,且这些苯丙氨酸残基中的至少一个之前是赖氨酸残基,则该序列基序不包含序列AFV,和
其中如果序列基序含有至少3个不相邻的组氨酸残基,则该序列基序优选地不包含序列AALTH(SEQ ID NO:111),
iv)其中所述序列基序的其余氨基酸(如果在所述基序中存在任何氨基酸)选自第三组,所述第三组选自以下:天冬酰胺、天冬氨酸、谷氨酸、甲硫氨酸或半胱氨酸,其中每个所述氨基酸独立地选自所述第三组,且其中谷氨酰胺可以优选仅被选择一次,并且其中选择还可以优选地不包含谷氨酰胺和谷氨酸的组合。
上文在i)至iv)中定义的序列基序可能仅代表本发明多肽的肽组分的一部分,即本发明多肽的肽组分比序列基序更长。或者,序列基序可以是肽组分的序列,即本发明多肽中肽组分的序列与序列基序的序列相同。而且,以及从图2中提供的实施例将显而易见的是,本发明的多肽可能包含一个或多个这样的序列基序。例如,20mer基序可以固有地包括也符合上述标准的16mer基序。因此,可以不将包含如上文所定义的“一条”序列基序的发明多肽的肽组分的事实理解为是指本发明多肽仅可以包含“一条”序列而没有其他(例如,重叠)序列基序也符合具有上述限制。
本发明多肽的肽组分的序列基序的长度可以是16、17、18、19或20个氨基酸。优选地,序列基序的长度是17、18或19个氨基酸,甚至更优选地长度是17或18个氨基酸。
本发明多肽的肽组分的序列基序包含至少40%和至多60%的氨基酸选自第一组氨基酸。所述第一组由赖氨酸、精氨酸和组氨酸组成。如果序列基序的长度是16个氨基酸,则其将显示出选自该第一组的至少7个和至多9个氨基酸。如果序列基序的长度是17个氨基酸,则其将显示出选自该第一组的至少7个和至多10个氨基酸。如果序列基序的长度是18个氨基酸,则其将显示出选自该第一组的至少8个和至多10个氨基酸。如果序列基序的长度是19个氨基酸,则其将显示出选自该第一组的至少8个和至多11个氨基酸。如果序列基序的长度是20个氨基酸,则其将显示出选自该第一组的至少8个和至多12个氨基酸。
在该第一组中选择的优选的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式中,序列基序仅包含一个或者甚至不包含组氨酸残基。
选自第一组的氨基酸是独立地选择的。这意味着,例如,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中的每一个可以独立地选自所述第一组的先前或随后的选择。因此,选定的氨基酸可以包含全部三种类型的氨基酸(赖氨酸、精氨酸和组氨酸),其可以是相同的(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中的两种(例如,赖氨酸和精氨酸)。同样地,独立地选择并没有规定单独选择的氨基酸之间的任何特定比例。例如,但不限于,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个组氨酸残基或者3个精氨酸、4个赖氨酸和1个组氨酸残基。
选自序列基序内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单独地,与选自第一组的其他氨基酸配对,或者与选自第一组的2个另外的氨基酸在嵌段中排布在所需序列基序中。
“单独的”指选自所述第一组的氨基酸(例如,赖氨酸(K))既不在侧翼为来自所述第一组的另一氨基酸N末端也不在C末端。相邻氨基酸残基可以选自第二组,或者视情况而定可以选自第三组(例如,LKE、N-KE(在基序的N末端)、LK-C(在基序的C末端))。值得注意的是,在本发明多肽内但在序列基序之外的潜在其他氨基酸未考虑用于确定该位置。因此,即使序列基序之外的前一个(N末端)或后一个(C末端)氨基酸残基偶尔还有精氨酸、组氨酸或赖氨酸残基,仍将在序列基序两个末端之一的来自第一组的氨基酸认为是单独定位的。
“与选自第一组的其他氨基酸配对”指在序列基序内选自第一组的氨基酸与选自第一组的另一氨基酸直接相邻。因此,这两个氨基酸形成选自第一组的一对氨基酸。所述对依次在C末端和N末端侧接来自第二组或视情况而定来自第三组的氨基酸(例如,LKRE(SEQID NO:112)、N-KRE(在基序的N末端))、LKR-C(在基序的C末端))。在本发明多肽的肽组分内但在序列基序之外的其他潜在氨基酸也不考虑用于确定该位置。
“与选自第一组的2个另外的氨基酸在嵌段中”指选自第一组的3个氨基酸彼此之间直接相邻。所述嵌段(或三联体)在C末端和N末端侧接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:113)、N-KRKE(在基序的N末端;SEQ ID NO:114)、LKRK-C(在基序的C末端;SEQ ID NO:115))。在本发明多肽的肽组分内但在序列基序之外的其他潜在氨基酸也不考虑用于确定该位置。对于以这种方式排列的氨基酸(三联体;用第一组的3个氨基酸嵌段),必须满足其他位置要求,相对于3氨基酸嵌段的第一个氨基酸(只要在所述序列基序中可以找到各自的位置),在位置-12、-11、-8、-5、-4、+6、+7、+10、+13和+14的氨基酸均不是选自所述第一组的氨基酸。负值表示三联体第一个氨基酸的N末端位置;正值指三联体第一个氨基酸的C末端位置。位置计算的基础是三联体的第一个(N末端)氨基酸(例如,三联体氨基酸的直接N末端将是-1,三联体氨基酸的直接C末端将是+3)。因此,该限制排除了诸如RRRGLRH(SEQ ID NO:116)的序列,因为位置+6(H)是第一组的氨基酸。序列基序中是否存在相应位置(-12、-11、-8、-5、-4、+6、+7、+10、+13和+14)将取决于序列基序中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末端,则将所有负值淘汰(即,无需考虑)。如果三联体位于序列基序的C末端,则正值也是如此。然而,在优选的实施方式中,序列基序根本不包含第一组氨基酸的这种三联体嵌段,即不包含由选自第一组的3个氨基酸组成的嵌段。
应当理解的是,单独的,与选自第一组的另外的氨基酸配对,以及与具有选自第一组的另外两个氨基酸残基在嵌段中的位置要求不重叠,并且术语是互斥的(例如,三联体不是“单独的”和/或“配对在一起”等的情况)。
对于选自第一组的氨基酸的另一个位置要求是,序列基序必须包含至少两对选自第一组的氨基酸。然而,优选不是所有选自第一组的氨基酸都在序列基序中配对排列。
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)的嵌段(即,第一组的氨基酸不存在于具有选自第一组的3个或更多个氨基酸的嵌段中)。因此,序列基序可以例如不包含诸如“KRKK”(SEQ ID NO:117)或“RRRR”(SEQ IDNO:118)的序列。
由于第一组的氨基酸仅占序列基序的40%至60%,因而需要从其他氨基酸残基中选择其余的氨基酸。如上所述,序列基序还包含至少40%和至多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。与前面对于第一组氨基酸一样,原则上同样独立地选择第二组的每个氨基酸,即每个氨基酸独立地选自所述第二组的任何此前或随后的选择。
然而,对于第二组,优选地对该独立选择的一般原则有一些限制。如果选自第一组和选自第二组的氨基酸的综合产生序列基序的氨基酸的100%(即,序列基序中没有来自第三组的氨基酸),则优选应用第一限制。在这种情况下,优选地从第二组中选择至少3个氨基酸。在这种情况下,第二组的氨基酸可以例如优选不仅仅限于缬氨酸和色氨酸残基。
进一步优选的(位置)限制是,如果序列基序包含至少两个单一不相邻的苯丙氨酸残基和这些苯丙氨酸残基中的至少一个在(N末端)之前是赖氨酸残基(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其在序列中不连续出现但被一个或多个其他氨基酸隔开的苯丙氨酸残基。单个苯丙氨酸残基是指其不是一对苯丙氨酸残基的一部分,也不是几个苯丙氨酸残基嵌段的一部分,而是单独位于序列基序中。
下一个优选的限制是,如果序列基序包含至少3个单一不相邻的组氨酸残基,则该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)。不相邻的氨基酸残基是不连续出现但被一个或多个其他氨基酸隔开的组氨酸残基。单个组氨酸残基是指其不是一对组氨酸残基的一部分,也不是几个组氨酸残基嵌段的一部分,而是单独位于序列基序中。
在一个优选的实施方式中,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。
本发明多肽的肽组分的序列基序可能不仅仅由选自第一组和第二组的氨基酸组成(即,其表示在一起小于100%)。在这种情况下,所述序列基序的其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。与前面对于第一组和第二组氨基酸一样,原则上同样独立地选择第三组的每个氨基酸,即每个氨基酸独立地选自所述第三组的任何此前或随后的选择。然而,与前面对于第二组一样,从所述第三组中选择氨基酸有一些优选的限制:谷氨酰胺仅可以选择一次,并且也不允许并行选择谷氨酰胺和谷氨酸,即如果在序列基序中存在谷氨酰胺,则不可以存在谷氨酸,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。
在优选的实施方式中,序列基序仅包含来自第三组的单个氨基酸残基,或者甚至更优选地根本不包含氨基酸残基。
在本发明的优选实施方式中,序列基序中选择的氨基酸排布符合图2所示可能的序列基序替代之一中列出的要求。图2列举了在给定的16mer、17mer、18mer、19mer或20mer的特定位置上可能不存在选自第一组的氨基酸。在这些位置,仅可能存在选自第二组和/或第三组(如果有的话)的氨基酸。优选地,在所述位置存在第二组的氨基酸。第一组的氨基酸仅可能存在于序列基序的任何其余位置。这并不意味着在这些其余位置仅可能存在第一组的氨基酸。在这些其余位置也可能存在第二组和任选地第三组的氨基酸,只要仍满足第一组和第二组的总体百分比要求即可。
优选地,肽组分的序列基序具有螺旋结构。
肽组分的优选序列基序不包含除在第一组、第二组或第三组中定义的氨基酸残基以外的任何其他氨基酸残基。特别地,肽组分的优选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,则其也不包含甲硫氨酸和半胱氨酸。
然而,脯氨酸残基很可能在肽组分(或本发明多肽)中的其他地方存在。例如,如果脯氨酸残基位于序列基序的N末端或C末端(后者是优选的)的1至10个(优选地1至5个)氨基酸残基内,则其是优选的。如果在内溶素序列和序列基序之间存在此类脯氨酸残基,则是更优选的。优选地,序列基序是内溶素序列的N末端,脯氨酸残基位于两者之间的某个位置,通常靠近序列基序。
显示出上文讨论的优选序列基序的肽组分的实例是包含SEQ ID NO:63、SEQ IDNO:119、SEQ ID NO:120、SEQ ID NO:121、SEQ ID NO:122、SEQ ID NO:123、SEQ ID NO:124、SEQ ID NO:125、SEQ ID NO:126、SEQ ID NO:127、SEQ ID NO:128、SEQ ID NO:129、SEQ IDNO:130、SEQ ID NO:131、SEQ ID NO:132、SEQ ID NO:133、SEQ ID NO:134、SEQ ID NO:135和SEQ ID NO:136所示序列的肽。显示出上文提及的序列基序的特别优选的肽组分是SEQID NO:132。
本发明多肽的肽(组分)优选地由至少5个,更优选地至少6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29、30、31、32、33、34、35、36、37、38、39、40、41、42、43、44、45、46、47、48、49、50、51、52、53、54、55、56、57、58、59、60、61、62、63、64、65、66、67、68、69、70、71、72、73、74、75、76、77、78、79、80、81、82、83、84、85、86、87、88、89、90、91、92、93、94、95、96、97、98、99个或至少100个氨基酸残基组成。特别优选的是由约5至约100个氨基酸残基、约5至约50个或约5至约30个氨基酸残基组成的那些肽。更优选的是由约6至约42个氨基酸残基、约6至约39个氨基酸残基、约6至约38个氨基酸残基、约6至约31个氨基酸残基、约6至约25个氨基酸残基、约6至约24个氨基酸残基、约6至约22个氨基酸残基、约6至约21个氨基酸残基、约6至约20个氨基酸残基、约6至约19个氨基酸残基、约6至约16个氨基酸残基、约6至约14个氨基酸残基、约6至约12个氨基酸残基、约6至约10个氨基酸残基或约6至约9个氨基酸残基组成的肽段。
在优选的实施方式中,本发明的多肽包含选自以下的至少一个氨基酸序列:KRK和SEQ ID NO:37-136。
根据本发明的多肽的肽组分可以通过插入其他氨基酸残基(例如,由于克隆原因)连接至内溶素。或者,肽组分可以直接连接至内溶素序列,而无需任何插入的接头序列。
优选地,所述插入的其他氨基酸残基可以不被蛋白酶识别和/或切割。优选地,所述其他氨基酸残基彼此连接和/或通过至少1、2、3、4、5、6、7、8、9或10个其他插入的氨基酸残基连接至酶。
在优选的实施方式中,通过其他插入的氨基酸残基甘氨酸、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:137),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:138)或者甘氨酸、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:139),肽连接至本发明多肽的其余部分(优选地在根据本发明的多肽的N末端或C末端)。
优选地,肽组分位于本发明多肽内内溶素的N末端。在这种情况下(特别是对于本发明多肽的第五个和第六个方面,但不限于此),特别优选内溶素构成多肽的最C末端组分,即内溶素序列的C末端无其他功能性元件。优选地,在本发明的多肽中,内溶素序列的C末端具有10个或更少的,更优选地5个或更少的、更优选地4个或更少的、更优选地3个或更少的、更优选地2个或更少的、更优选地仅1个氨基酸和最优选地无氨基酸。
本发明的多肽的实例是包含例如作为内溶素的SEQ ID NO:28或SEQ ID NO:30(或与其具有至少80%序列同一性的序列),以及还包含作为肽组分的SEQ ID NO:63或SEQ IDNO:132的多肽。根据本发明的特别优选的多肽是包含SEQ ID NO:140或SEQ ID NO:141的多肽,以及与SEQ ID NO:140和/或SEQ ID NO:141具有至少80%序列同一性,优选地至少85%序列同一性,更优选地至少90%序列同一性,更优选地至少95%序列同一性,更优选地至少96%序列同一性,更优选地至少97%序列同一性,更优选地至少98%序列同一性,更优选地至少99%序列同一性的多肽。
根据本发明的多肽的其他实例是包含以下的多肽:像内溶素组分例如SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:34、SEQ ID NO:35或SEQ ID NO:36(或者与任何这些具有至少80%序列同一性的序列),以及像选自以下的肽组分:SEQ ID NO:96、SEQID NO:107、SEQ ID NO:132、SEQ ID NO:133、SEQ ID NO:134、SEQ ID NO:135或SEQ ID NO:136。针对此类本发明多肽的实例提供在SEQ ID NO:142、SEQ ID NO:143、SEQ ID NO:144、SEQ ID NO:145、SEQ ID NO:146、SEQ ID NO:147、SEQ ID NO:148、SEQ ID NO:149、SEQ IDNO:150、SEQ ID NO:151和SEQ ID NO:152,以及与任何这些序列具有至少80%序列同一性、优选地至少85%序列同一性、更优选地至少90%序列同一性、更优选地至少95%序列同一性、更优选地至少96%序列同一性、更优选地至少97%序列同一性、更优选地至少98%序列同一性、更优选地至少99%序列同一性的多肽中。
SEQ ID NO:140、SEQ ID NO:141、SEQ ID NO:143、SEQ ID NO:144、SEQ ID NO:145、SEQ ID NO:146、SEQ ID NO:147、SEQ ID NO:148、SEQ ID NO:151和SEQ ID NO:152在N末端显示出丙氨酸残基(例如,代替甲硫氨酸残基)。所述丙氨酸残基不是关键性的,仅在蛋白水解去除N末端的His标签后保留。在SEQ ID NO:115的情况下,在N末端蛋白水解除去His标签不留下另外的氨基酸,即多肽直接从根据SEQ ID NO:106的肽开始。
除了本文所定义的内溶素和肽之外,本发明的多肽当然还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标签或本领域公知的其他标签、硫氧还蛋白、麦芽糖结合蛋白(MBP)等。
在这种情况下,本发明的多肽可以另外包含例如用于纯化的标签。优选的是His6标签(SEQ ID NO:153),优选在根据本发明的多肽的C末端和/或N末端。所述标签可以通过其他氨基酸残基(例如,由于克隆原因)连接至多肽。优选地,所述标签可以通过至少1、2、3、4、5、6、7、8、9个或10个其他氨基酸残基连接至蛋白。优选地,所述其他氨基酸残基可以被蛋白酶识别和/或切割。在优选的实施方式中,本发明的多肽在其N末端包含His6标签。
在第七个方面中,本发明涉及一种多肽,其包含选自以下的肽序列:SEQ ID NO:107、SEQ ID NO:133、SEQ ID NO:134、SEQ ID NO:135和SEQ ID NO:136,以及任选地还包含胞壁质水解酶序列。根据本发明第七个方面的多肽的胞壁质水解酶可以是能够降解细菌肽聚糖的任何胞壁质水解酶(特别是肽聚糖水解酶)。就酶促活性而言,此类胞壁质水解酶可以是例如内肽酶、N-乙酰基-胞壁酰基-L-丙氨酸-酰胺酶(酰胺酶)、N-乙酰基-胞壁酸酶、N-乙酰基-葡糖胺酶或裂解转糖基酶,因此适用于降解细菌细胞壁的肽聚糖。优选地,胞壁质水解酶降解革兰氏阴性细菌(如大肠杆菌或铜绿假单胞菌)的肽聚糖。细菌细胞壁的肽聚糖结构总体上是保守的,仅作了较小的修饰(Schleifer&Kandler 1972)。细菌物种具有由不同氨基酸组成的肽间桥,甚至可能缺少肽间桥。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;代表赖氨酸的ε-羧基衍生物的氨基酸,是肽聚糖的典型组分)(二氨基庚二酸是取代了氨基酸L-Lys的残基,并直接交联至相反肽链的末端氨基酸D-Ala)。因此,可用的有限类型的化学键可以被胞壁质水解酶切割(例如,被肽聚糖水解酶水解)。胞壁质水解酶表现出具有上文列出的酶促活性的至少一个酶结构域。此外,在一些情况下,胞壁质水解酶含有至少一个适于结合肽聚糖并支持胞壁质水解酶的酶活性的结构域。通常将结合结构域称为细胞壁结合结构域(CBD)。胞壁质水解酶的实例是脊椎动物溶菌酶(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)、与病毒体相关的肽聚糖水解酶(VAPGH)、细菌素(例如,溶葡萄球菌素)和自溶素。最优选地,胞壁质水解酶是内溶素。特别优选的内溶素序列是上文针对本发明的第一个至第六个方面阐述的那些,例如SEQ ID NO:6、SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:34、SEQ ID NO:35和SEQ ID NO:36,或者与SEQID NO:6、SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:32、SEQID NO:33、SEQ ID NO:34、SEQ ID NO:35和/或SEQ ID NO:36(特别是SEQ ID NO:28和/或SEQ ID NO:30)具有至少80%序列同一性、优选地至少85%序列同一性、更优选地至少90%序列同一性、更优选地至少95%序列同一性、更优选地至少96%序列同一性、更优选地至少97%序列同一性、更优选地至少98%序列同一性、更优选地至少99%序列同一性的序列。一般而言:上文针对本发明多肽提出的(根据本发明的第一个至第六个方面)同样适用于(在适用的范围内)根据本发明第七个方面的本发明的多肽。
本发明的多肽包含或可以包含革兰氏阴性内溶素。因此,本发明的多肽将优选地能够至少在革兰氏阴性细菌(通常是各个亲代噬菌体的宿主物种)上降解肽聚糖。优选地,本发明的多肽将能够降解大肠杆菌和/或铜绿假单胞菌细菌的肽聚糖。最优选地,本发明的多肽降解大肠杆菌RKI 06-08410菌株的肽聚糖(来自Robert Koch-Institut,Berlin,Germany)。
革兰氏阴性细菌对肽聚糖的降解活性可以通过本领域熟知的测定法来测定,例如,通过溶胞壁质测定法,其中将革兰氏阴性细菌的外膜透化或除去(例如,使用氯仿),以使推定的酶接近肽聚糖层。如果酶具有活性,则肽聚糖层的降解将导致浊度下降,可以通过光度法对其进行测量(参见例如Briers等,J.Biochem.Biophys Methods 70:531-533,(2007)或Schmelcher等,Bacteriophage endolysins as novelantimicrobials.Schmelcher M,Donovan DM,Loessner MJ.Future Microbiol.2012Oct;7(10):1147-7)(这两个参考文献均通过引用并入本文)。
本发明的多肽通常不仅表现出肽聚糖降解酶的活性,即能够降解革兰氏阴性细菌肽聚糖。优选地,本发明的多肽将能够在不存在EDTA(或任何其他增加外膜通透性的辅助物质)的情况下降解革兰氏阴性细菌(例如,大肠杆菌和/或铜绿假单胞菌细菌)的肽聚糖。更优选地,在不存在其他外膜透化剂的情况下,本发明的多肽显示出40μg/ml或更低的最小抑菌浓度(MIC),优选地30μg/ml或更低,甚至更优选地25μg/ml或更低,甚至更优选地20μg/ml或更低,最优选地15μg/ml或更低。最优选地,在不存在其他外膜透化剂的情况下,多肽以40μg/ml或更低的最小抑菌浓度(MIC)降解大肠杆菌菌株RKI 06-08410的肽聚糖,优选地30μg/ml或更低,甚至更优选地25μg/ml或更低,甚至更优选地20μg/ml或更低,最优选地15μg/ml或更低。实施例1中示出了相应的适宜检测。对于铜绿假单胞菌,在实施例2中示出了适宜的检测,并且相应的检测菌株优选为PAO1(Pirnay等,Environmental Microbiology,2002,p.898-911)。
根据本发明的多肽可以通过本领域已知的标准方法生产,例如,通过在合适的宿主细胞中重组表达编码相应多肽的核酸来实现。如果本发明的多肽例如另外包含氨基酸序列延伸或标签等,则可以通过使用如在例如Sambrook等2001,Molecular Cloning:ALaboratory Manual中描述的标准克隆技术连接所需的单个核酸序列来生产这种融合蛋白。可以用本领域已知的方法(例如,在重组DNA表达系统中)类似地产生这种多肽。
本发明还涉及编码一种或多种本发明的本发明多肽的核酸。本发明的核酸可以采用核酸可想到的所有形式。特别地,根据本发明的核酸可以是RNA、DNA或其杂交体。其可以是单链的或双链的。其可以具有小转录本或整个基因组(例如,噬菌体基因组)的尺寸。如本文所用,编码一种或多种本发明的本发明多肽的核酸可以是反映有意义链的核酸。同样,也包含反义链。核酸可包含用于表达本发明多肽的异源启动子。
在另一个方面中,本发明涉及包含根据本发明的核酸的载体。此类载体可以例如是允许表达本发明多肽的表达载体。所述表达载体可以是组成型的或诱导型的。载体也可以是包含用于克隆目的的本发明核酸序列的克隆载体。
本发明确实还涉及包含本发明核酸的噬菌体,特别是包含编码根据本发明的融合蛋白的本发明的核酸。
本发明确实还涉及(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。宿主细胞可以特别地选自细菌细胞和酵母细胞。在适当的情况下,其他适宜的宿主细胞可以是永生化细胞系,例如哺乳动物(特别是人)来源的。
在另一个方面中,本发明涉及一种组合物,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。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。特别优选的是包含根据本发明的多肽但无EDTA的组合物。优选地,本发明的组合物无任何其他外膜透化物质。
在甚至另一个方面中,根据本发明的组合物是化妆品组合物。几种细菌会刺激患者身体暴露在环境中的表面(如皮肤)。为了防止这种刺激或为了消除所述细胞病原体的次要病候(minor manifestation),可以使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,以达到消除粉刺(comedolytic)的作用。优选地,将本发明的多肽、核酸、载体、噬菌体、宿主细胞或组合物用于本文中,而不使用任何其他外膜透化物质。
在另一个方面中,本发明涉及一种试剂盒,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,以及至少一种其他抗微生物剂,如本发明的其他多肽、抗生素或抗微生物肽。
在另一个方面中,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在通过手术或疗法治疗人体或动物体的方法中或在人体或动物体上实践的诊断方法中的用途。在这种情况下,可以利用本发明多肽的抗菌活性。
此类方法通常包括向对象施用有效量的本发明的多肽、核酸、载体、噬菌体、宿主细胞或组合物。优选地,多肽、核酸、载体、噬菌体、宿主细胞或组合物在不添加其他外膜透化物质(如EDTA)的情况下施用。对象可以是例如人或动物,其中人类对象是更优选的。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(如革兰氏阴性细菌感染)的方法中。不限于此,治疗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、牙齿、鼻腔、嘴、骨骼、阴道、菌血症伤口和/或心内膜炎的感染。
在根据本发明的治疗(或预防)方法中使用的剂量和施用途径取决于要治疗的特定疾病/感染部位。施用途径可以是例如口服、局部、鼻咽、胃肠外、静脉内、直肠或任何其他施用途径。
为了将本发明的多肽、核酸、载体、噬菌体、宿主细胞或组合物应用于感染部位(或濒临感染的部位),可以使用保护活性化合物不受环境影响(如蛋白酶、氧化、免疫应答等)的制剂,直至其到达感染部位。因此,制剂可为胶囊、锭剂、丸剂、栓剂、可注射溶液或任何其他医药上合理的盖仑制剂。优选地,所述盖仑制剂可包含合适的载体、稳定剂、调味剂、缓冲液或其他合适的试剂。例如,对于局部施用,所述制剂可为洗液或硬膏(plaster),对于鼻咽施用所述制剂可为通过喷雾施与鼻部的盐水溶液。优选地,制剂不包含任何其他外膜透化物质(本发明的多肽除外)。
优选地,本发明的多肽、核酸、载体、噬菌体、宿主细胞或组合物用于与其他常规抗菌剂(如抗生素、羊毛硫抗生素、细菌素或内溶素等)组合使用。可以在本发明的多肽(例如,融合蛋白)、核酸、载体、噬菌体、宿主细胞或组合物施用之前、同时或施用之后施用常规抗菌剂。
在另一个方面中,本发明涉及本发明的多肽、核酸、载体、噬菌体、宿主细胞或组合物用作医学诊断、食品诊断、饲料诊断或环境诊断中的诊断手段,特别是作为细菌感染(尤其是由革兰氏阴性细菌引起的那些感染)诊断的诊断手段。在这一方面,可以将本发明的多肽、核酸、载体、宿主细胞或组合物用作特异性降解革兰氏阴性病原细菌肽聚糖的工具。需要特定的细胞降解作为初始步骤,以便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技术或其他依赖细菌细胞的细胞内容物的方法如使用特异性针对不同细菌群或物种的蛋白的酶促测定(例如,针对肠球菌的β-半乳糖苷酶,针对凝固酶阳性菌株的凝固酶)进行随后的特异性检测。优选地,将本发明的多肽、核酸、载体、噬菌体、宿主细胞或组合物用于本文中,而不使用任何其他外膜透化物质。
在另一个方面中,本发明涉及将本发明的多肽、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品、饲料或化妆品中的抗微生物剂,或作为(例如,非治疗性)消毒剂的用途。本发明的多肽可以用于处理或预防食品、食品加工设备、食品加工工厂、与食品接触的(无生命)表面(如货架和食品存放区)、饲料、饲料加工设备、饲料加工工厂、与饲料接触的(无生命)表面(如货架或饲料存放区)、医疗设备或者医院、医生办公司和其他医疗设备的(无生命)表面的革兰氏阴性细菌污染。优选地,将本发明的多肽、核酸、载体、噬菌体、宿主细胞或组合物用于本文中,而不使用任何其他外膜透化物质。
附图说明
在下文中,将给出附图的简要说明。附图旨在更详细地说明本发明的一个方面。然而,并非旨在将本发明的主题仅限制在这种主题。
图1:示出了在优选的本发明多肽的内溶素组分中发现的SEQ ID NO:2的共同基序(框中),即克氏柠檬酸杆菌噬菌体CkP1(SEQ ID NO:21)、肠杆菌噬菌体CC31(SEQ ID NO:22)、沙雷氏菌噬菌体CHI14(SEQ ID NO:21)、气单胞菌噬菌体Ah1(SEQ ID NO:24)、沙雷氏菌噬菌体PS2(SEQ ID NO:25)和气单胞菌噬菌体AS-szw(SEQ ID NO:26)的内溶素,从而产生SEQ ID NO:7所示的共有序列。
图2:说明了本发明多肽的选定肽组分的优选序列基序的位置要求。该表指示了在长度位置上16个(白色)至20个(深灰色)氨基酸的序列基序,在该位置上可能不存在选自第一组的氨基酸(各个位置用“X”标记)。在所述位置(即,用“X”标记的那些位置),仅可能存在选自第二组或视情况而定选自第三组的氨基酸。更优选地,在所述位置仅存在选自第二组的氨基酸。选自序列基序第一组的氨基酸可能仅存在于未用“X”标记的位置。然而,在所述未标记的位置,也可以存在第二组或视情况而定第三组的氨基酸。提供了共计18个替代物,每个替代物的长度为16、17、18、19或20个氨基酸。表中还明确规定了其中潜在的第一组三联体氨基酸可能存在的位置(不含“X”的连续三个位置)。对于替代物1,其位置为8至10。根据本发明的肽组分多肽的优选序列基序的要求,相对于3氨基酸嵌段的第一个氨基酸(即,位置#8),在位置-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)的氨基酸不选自第一组。在第一替代物中未见相对位置12、-11、-8、+13和+14,因此未考虑。
实施例
在下文中,呈现了说明本发明的各种实施方式和方面的具体实施例。然而,本发明不应限于本文所述的具体实施方式的范围。实际上,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从前面的描述和以下实施例将变得显而易见。所有这些修改都落入所附权利要求书的范围内。
实施例1:在存在和不存在EDTA的情况下,几种抗菌多肽对大肠杆菌的最小抑菌浓
度
在存在和不存在EDTA的情况下,在大肠杆菌上评估了以下融合蛋白的抗菌活性:
-SEQ ID NO:154,天蚕素A(埃及伊蚊)肽(SEQ ID NO:69)与弧菌噬菌体VvAW1(YP_007518361.1)内溶素的融合物
-SEQ ID NO:155,天蚕素A(埃及伊蚊)肽与模块化KZ144内溶素和Lys68内溶素的突变的细胞壁结合结构域的融合物
-SEQ ID NO:156,符合优选的肽组分序列基序的经修饰的肽(SEQ ID NO:131)与假单胞菌噬菌体vB_PsyM_KIL1(见YP_009276009.1)的融合物
-SEQ ID NO:140,SMAP-29肽(SEQ ID NO:63)与克氏柠檬酸杆菌噬菌体CkP1内溶素的融合物,后者进行了C54S突变的另外的技术修饰以减少聚集(SEQ ID NO:28),和
-SEQ ID NO:141,包含优选的肽组分序列基序的肽(SEQ ID NO:132)与肠杆菌噬菌体CC31内溶素的融合物,后者进行了C54S突变的另外的技术修饰以减少聚集(SEQ IDNO:30)
-SEQ ID NO:142,根据SEQ ID NO:133的肽与克氏柠檬酸杆菌噬菌体CkP1内溶素的融合物,后者进行了C54S突变的另外的技术修饰以减少聚集(SEQ ID NO:28),
-SEQ ID NO:143,包含根据SEQ ID NO:134的优选序列的肽与肠杆菌噬菌体CC31内溶素的融合物,后者另外进行了C54S突变的另外的技术修饰以减少聚集(SEQ ID NO:30),
-SEQ ID NO:145,包含根据SEQ ID NO:107的优选序列的肽与沙雷氏菌属CHI14内溶素的融合物,后者另外进行了C54S突变的另外的技术修饰以减少聚集(SEQ ID NO:32),
-SEQ ID NO:146,包含根据SEQ ID NO:132的优选序列的肽与气单胞菌噬菌体Ah1内溶素的融合物,后者另外进行了半胱氨酸残基的另外的技术修饰(此处位于位置122)以减少聚集(SEQ ID NO:34),
-SEQ ID NO:147,包含根据SEQ ID NO:107的优选序列的肽与气单胞菌噬菌体Ah1内溶素的融合物,后者另外进行了半胱氨酸残基的另外的技术修饰(此处位于位置122)以减少聚集(SEQ ID NO:34),
-SEQ ID NO:148,包含根据SEQ ID NO:132的优选序列的肽与沙雷氏菌噬菌体PS2内溶素(SEQ ID NO:25)的融合物,
-SEQ ID NO:149,包含根据SEQ ID NO:96的优选序列的肽与沙雷氏菌噬菌体PS2内溶素(SEQ ID NO:25)的融合物,
-SEQ ID NO:150,包含根据SEQ ID NO:135的优选序列的肽与克氏柠檬酸杆菌噬菌体CkP1内溶素的融合物,后者进行了C54S突变的另外的技术修饰以减少聚集(SEQ IDNO:28),和
-SEQ ID NO:152,包含根据SEQ ID NO:132的优选序列的肽与气单胞菌噬菌体AS-szw内溶素(SEQ ID NO:36)的融合物。
SEQ ID NO:140所示的多肽和SEQ ID NO:141所示多肽的内溶素组分(见SEQ IDNO:28和SEQ ID NO:30)具有显著水平的序列同一性(80%)。值得注意的是,这两种内溶素在C末端均具有相对较好的保守螺旋基序(aa145-157),这在其他任何测试的融合蛋白中均不存在。
在(Luria-Bertani)培养基中培养大肠杆菌(大肠杆菌菌株RKI 06-08410;获自Robert Koch-Institut,Berlin,Germany),并在Mueller-Hinton培养基中以1:10稀释。在光密度OD600为约0.6时,将细菌在相同培养基中以1:10稀释,然后以1:500稀释。将蛋白缓冲液(20mM HEPES,150mM NaCl,pH 7.4)和蛋白以20μl的终体积(含有或不含终浓度为500μM的EDTA)的不同浓度吸取到96孔板中。将180μl细菌细胞悬液或培养基(Mueller-Hinton)作为对照,加入96孔板中并混合。将板在37℃下孵育18-22小时,并通过测量孔的OD600值确定细菌生长情况。确定最小抑菌浓度(MIC),其是显示出与无细菌对照相同OD600值的孔中的蛋白浓度。
最小抑菌浓度(MIC,以μg/ml计)的结果显示在下表4中。
表4:在存在和不存在EDTA情况下的抗菌活性
“≤”(例如,≤5、≤3.3等)指在所检测的第一浓度(例如,分别为5μg/ml和3.3μg/ml)下已观察到抗菌活性。因此,MIC至少是所检测的第一浓度(例如,分别为5μg/ml和3.3μg/ml)并且可能更低。>50表示浓度达到50μg/ml时未观察到抗菌活性。
在存在外膜透化剂EDTA的情况下,检测的所有多肽均显示出对大肠杆菌良好的抗菌活性。然而,在不存在EDTA的情况下,三种常规融合蛋白的抗菌活性显著下降。相反的是,即使在不存在EDTA的情况下,根据本发明的多肽仍保留了显著水平的抗微生物活性。
实施例2:在存在和不存在EDTA的情况下,几种抗菌多肽对铜绿假单胞菌的最小抑
菌浓度
在存在和不存在EDTA的情况下,还在铜绿假单胞菌细菌上评估了抗菌活性。使用了以下多肽:
-SEQ ID NO:154,天蚕素A(埃及伊蚊)肽(SEQ ID NO:69)与弧菌噬菌体VvAW1(YP_007518361.1)内溶素的融合物
-SEQ ID NO:155,天蚕素A(埃及伊蚊)肽与模块化KZ144内溶素和Lys68内溶素的突变的细胞壁结合结构域的融合物
-SEQ ID NO:156,符合优选的肽组分序列基序的经修饰的肽(SEQ ID NO:131)与假单胞菌噬菌体vB_PsyM_KIL1(见YP_009276009.1)的融合物-SEQ ID NO:140,SMAP-29肽(SEQ ID NO:63)与克氏柠檬酸杆菌噬菌体CkP1内溶素的融合物,后者进行了C54S突变的另外的技术修饰以减少聚集(SEQ ID NO:28),和
SEQ ID NO:141,包含优选的肽组分序列基序的肽(SEQ ID NO:132)与肠杆菌噬菌体CC31内溶素的融合物,后者进行了C54S突变的另外的技术修饰以减少聚集(SEQ ID NO:30)
-SEQ ID NO:144,包含根据SEQ ID NO:132的优选序列的肽与沙雷氏菌属CHI14内溶素的融合物,后者另外进行了C54S突变的另外的技术修饰以减少聚集(SEQ ID NO:32),
-SEQ ID NO:145,包含根据SEQ ID NO:107的优选序列的肽与沙雷氏菌属CHI14内溶素的融合物,后者另外进行了C54S突变的另外的技术修饰以减少聚集(SEQ ID NO:32),
-SEQ ID NO:146,包含根据SEQ ID NO:132的优选序列的肽与气单胞菌噬菌体Ah1内溶素的融合物,后者另外进行了半胱氨酸残基的另外的技术修饰(此处位于位置122)以减少聚集(SEQ ID NO:34),
-SEQ ID NO:147,包含根据SEQ ID NO:107的优选序列的肽与气单胞菌噬菌体Ah1内溶素的融合物,后者另外进行了半胱氨酸残基的另外的技术修饰(此处位于位置122)以减少聚集(SEQ ID NO:34),
-SEQ ID NO:148,包含根据SEQ ID NO:132的优选序列的肽与沙雷氏菌噬菌体PS2内溶素(SEQ ID NO:25)的融合物,
-SEQ ID NO:149,包含根据SEQ ID NO:96的优选序列的肽与沙雷氏菌噬菌体PS2内溶素(SEQ ID NO:25)的融合物,
-SEQ ID NO:150,包含根据SEQ ID NO:135的优选序列的肽与克氏柠檬酸杆菌噬菌体CkP1内溶素的融合物,后者进行了C54S突变的另外的技术修饰以减少聚集(SEQ IDNO:28),
-SEQ ID NO:151,包含根据SEQ ID NO:136的优选序列的肽与克氏柠檬酸杆菌噬菌体CkP1内溶素的融合物,后者进行了C54S突变的另外的技术修饰以减少聚集(SEQ IDNO:28),
-SEQ ID NO:152,包含根据SEQ ID NO:132的优选序列的肽与气单胞菌噬菌体AS-szw内溶素(SEQ ID NO:36)的融合物。
在(Luria-Bertani)培养基中培养细菌(铜绿假单胞菌PAO1),并在Mueller-Hinton培养基中以1:10稀释。在光密度OD600为约0.6时,将细菌在相同培养基中以1:10稀释,然后以1:500稀释。将蛋白缓冲液(20mM HEPES,150mM NaCl,pH 7.4)和蛋白以20μl的终体积(含有或不含终浓度为500μM的EDTA)的不同浓度吸取到96孔板中。将180μl细菌细胞悬液或培养基(Mueller-Hinton)作为对照,加入96孔板中并混合。将板在37℃下孵育18-22小时,并通过测量孔的OD600值确定细菌生长情况。确定MIC,其是显示出与无细菌对照相同OD600值的孔中的蛋白浓度。
最小抑菌浓度(MIC,以μg/ml计)的结果显示在下表5中。
表5:在存在和不存在EDTA情况下的抗菌活性
“≤”(例如,≤5、≤1.5等)指在所检测的第一浓度(例如,分别为5μg/ml和1.5μg/ml)下已观察到抗菌活性。因此,MIC至少是所检测的第一浓度(例如,分别为5μg/ml和1.5μg/ml)并且可能更低。>50表示浓度达到50μg/ml时未观察到抗菌活性。
在存在外膜透化剂EDTA的情况下,检测的所有多肽均显示出对铜绿假单胞菌良好的抗菌活性。然而,在不存在EDTA的情况下,三种常规融合蛋白的抗菌活性显著下降。相反的是,即使在不存在EDTA的情况下,根据本发明的多肽保留了显著水平的抗微生物活性。
实施例3:在本发明第一个、第三个和第五个方面的优选实施方式
1.一种多肽,其包含革兰氏阴性内溶素和选自以下的肽:抗微生物肽、两亲性肽、阳离子肽、寿司肽或防御素,
其中所述内溶素又是包含根据SEQ ID NO:1的序列的内溶素,
限制条件是:
a)所述多肽既不包含根据SEQ ID NO:3的序列,也不包含根据SEQ ID NO:4的序列,也不包含根据SEQ ID NO:5的序列,
b)所述内溶素不是气单胞菌噬菌体Aeh1的Aeh1p339,也不是肠杆菌噬菌体JS98的EpJS98_gp116,
c)如果所述多肽包含SEQ ID NO:6所示的序列,则肽选自以下:抗微生物肽、两亲性肽、寿司肽或防御素,
d)如果所述内溶素具有选自以下的序列:
和仅缺少N-末端甲硫氨酸的相应序列,则所述多肽不包含i)模块化革兰氏阴性内溶素或ii)噬菌体尾/基板蛋白的细胞壁结合结构域,
e)如果所述内溶素具有根据以下的氨基酸2-164的序列,则所述多肽不包含i)模块化革兰氏阴性内溶素或ii)噬菌体尾/基板蛋白的细胞壁结合结构域:
宿主 | 噬菌体名称 | 蛋白ID |
大肠杆菌 | 肠杆菌噬菌体T4 | NP_049736.1 |
f)如果所述内溶素具有根据以下的氨基酸2-165的序列,则所述多肽不包含i)模块化革兰氏阴性内溶素或ii)噬菌体尾/基板蛋白的细胞壁结合结构域:
宿主 | 噬菌体名称 | 蛋白ID |
气单胞菌 | 气单胞菌噬菌体Aeh1 | NP_944217.1 |
g)如果所述内溶素具有根据以下的氨基酸2-161的序列,则所述多肽不包含i)模块化革兰氏阴性内溶素或ii)噬菌体尾/基板蛋白的细胞壁结合结构域:
宿主 | 噬菌体名称 | 蛋白ID |
大肠杆菌 | 肠杆菌噬菌体JS98 | YP_001595245.1 |
2.根据项目1所述的多肽,其中所述内溶素选自以下:SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:28、SEQ ID NO:30,以及与SEQ ID NO:21、SEQ ID NO:22、SEQ ID NO:28和/或SEQ ID NO:30具有至少80%序列同一性的序列。
3.根据项目1所述的多肽,其中将SEQ ID NO:1进一步定义为具有选自以下的序列:SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO:10和11。
4.根据前述项目中任一项所述的多肽,其中所述肽是抗微生物肽或两亲性肽。
5.根据前述项目中任一项所述的多肽,其中所述肽包含以下序列基序:
i)长度为16、17、18、19或20个氨基酸;
ii)包含至少40%和至多60%的氨基酸选自第一组氨基酸,所述第一组氨基酸由赖氨酸、精氨酸和组氨酸组成,
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单独地,与选自所述第一组的一个其他氨基酸成对,或与选自所述第一组的2个其他氨基酸嵌段,但不发生与选自所述第一组的3个或更多个氨基酸嵌段排布在所述序列基序中,其中在所述基序中存在选自所述第一组的至少2对氨基酸,且其中在所述序列基序中存在具有连续选自所述第一组的3个所述氨基酸的至多一个嵌段,附加条件是,如果在所述序列基序中存在具有所述第一组的3个氨基酸的嵌段,则不从所述第一组中选择相对于3个氨基酸嵌段的所述第一氨基酸的在位置-12、-11、-8、-5、-4、+6、+7、+10、+13和+14的所述氨基酸,如果可以在所述序列基序中找到各自的位置的话,
iii)包含至少40%和至多60%的氨基酸选自第二组氨基酸,所述第二组氨基酸由丙氨酸、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,
其中每个氨基酸独立地选自所述第二组,
其中优选地,如果选自所述第一组合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3个不停氨基酸选自该第二组;
iv)其中所述序列基序的其余氨基酸(如果在所述基序中存在任何氨基酸)选自第三组,所述第三组选自以下:天冬酰胺、天冬氨酸、谷氨酸、甲硫氨酸或半胱氨酸,其中每个所述氨基酸独立地选自所述第三组。
6.根据项目5所述的多肽,其中肽包含根据SEQ ID NO:63或根据SEQ ID NO:132的所述序列。
7.根据前述项目中任一项所述的多肽,其中所述肽包含SEQ ID NO:140或SEQ IDNO:141所述的氨基酸序列,或与SEQ ID NO:140和/或SEQ ID NO:141具有至少80%序列同一性的序列。
8.根据前述项目中任一项所述的多肽,其中所述多肽降解至少一种革兰氏阴性细菌物种的肽聚糖,特别是其中所述多肽降解大肠杆菌细菌和/或铜绿假单胞菌细菌的肽聚糖。
9.根据项目8所述的多肽,其中在不存在其他外膜透化物质的情况下所述多肽降解至少一种革兰氏阴性细菌的肽聚糖,特别是其中在不存在外膜透化物质的情况下所述多肽降解大肠杆菌细菌和/或铜绿假单胞菌细菌的肽聚糖。
10.根据项目8所述的多肽,其中在不存在外膜透化物质的情况下所述多肽对大肠杆菌菌株RKI 06-08410的最小抑制浓度(MIC)为20μg/ml或更低。
11.编码项目1至10中任一项所述的多肽的核酸。
12.包含根据项目11所述的核酸的载体。
13.宿主细胞,其包含根据项目1至10中任一项所述的多肽,根据项目11所述的核酸,和/或根据项目12所述的载体。
14.根据项目1至10中任一项所述的多肽用于通过手术或疗法治疗人体或动物体的方法中或者用于在人体或动物体上实践的诊断方法中的用途,其中在不添加其他外膜透化物质的情况下施用所述多肽。
15.根据项目1至10中任一项所述的多肽作为非治疗性消毒剂的用途,其中在不添加其他外膜透化物质的情况下施用所述多肽。
序列表
<110> 莱桑多公司
<120> 新抗微生物融合蛋白
<130> LYS-049 PCT
<150> EP 18175064.7
<151> 2018-05-30
<160> 156
<170> PatentIn 3.5版
<210> 1
<211> 13
<212> PRT
<213> 人工序列
<220>
<223> 共有序列
<220>
<221> MISC_FEATURE
<222> (1)..(1)
<223> Xaa可以是Pro或Thr
<220>
<221> MISC_FEATURE
<222> (5)..(5)
<223> Xaa可以是Lys, Met, Asn或Gln
<220>
<221> MISC_FEATURE
<222> (8)..(8)
<223> Xaa可以是Ala, Ile或Thr
<220>
<221> MISC_FEATURE
<222> (9)..(9)
<223> Xaa可以是Ala, Asp, Glu, Lys, Gln, Ser或Thr
<220>
<221> MISC_FEATURE
<222> (10)..(10)
<223> Xaa可以是Val或Thr
<220>
<221> MISC_FEATURE
<222> (11)..(11)
<223> Xaa可以是Phe, Ile, Val或Leu
<220>
<221> MISC_FEATURE
<222> (12)..(12)
<223> Xaa可以是Glu, Lys, Leu或Arg
<220>
<221> MISC_FEATURE
<222> (13)..(13)
<223> Xaa可以是 Leu或Thr
<400> 1
Xaa Asn Arg Ala Xaa Arg Val Xaa Xaa Xaa Xaa Xaa Xaa
1 5 10
<210> 2
<211> 6
<212> PRT
<213> 肠球菌噬菌体CC31
<400> 2
Asn Arg Ala Lys Arg Val
1 5
<210> 3
<211> 163
<212> PRT
<213> 未知
<220>
<223> 肠球菌噬菌体T4内溶素
<400> 3
Met Asn Ile Phe Glu Met Leu Arg Ile Asp Glu Gly Leu Arg Leu Lys
1 5 10 15
Ile Tyr Lys Asp Thr Glu Gly Tyr Tyr Thr Ile Gly Ile Gly His Leu
20 25 30
Leu Thr Lys Ser Pro Ser Leu Asn Ala Ala Lys Ser Glu Leu Asp Lys
35 40 45
Ala Ile Gly Arg Asn Cys Asn Gly Val Ile Thr Lys Asp Glu Ala Glu
50 55 60
Lys Leu Phe Asn Gln Asp Val Asp Ala Ala Val Arg Gly Ile Leu Arg
65 70 75 80
Asn Ala Lys Leu Lys Pro Val Tyr Asp Ser Leu Asp Ala Val Arg Arg
85 90 95
Cys Ala Leu Ile Asn Met Val Phe Gln Met Gly Glu Thr Gly Val Ala
100 105 110
Gly Phe Thr Asn Ser Leu Arg Met Leu Gln Gln Lys Arg Trp Asp Glu
115 120 125
Ala Ala Val Asn Leu Ala Lys Ser Arg Trp Tyr Asn Gln Thr Pro Asn
130 135 140
Arg Ala Lys Arg Val Ile Thr Thr Phe Arg Thr Gly Thr Trp Asp Ala
145 150 155 160
Tyr Lys Asn
<210> 4
<211> 176
<212> PRT
<213> 未知
<220>
<223> smi02_KRK9
<400> 4
Ala Met Gly Ser Lys Arg Lys Lys Arg Lys Lys Arg Lys Gly Asn Ile
1 5 10 15
Phe Glu Met Leu Arg Ile Asp Glu Gly Leu Arg Leu Lys Ile Tyr Lys
20 25 30
Asp Thr Glu Gly Tyr Tyr Thr Ile Gly Ile Gly His Leu Leu Thr Lys
35 40 45
Ser Pro Ser Leu Asn Ala Ala Lys Ser Glu Leu Asp Lys Ala Ile Gly
50 55 60
Arg Asn Cys Asn Gly Val Ile Thr Lys Asp Glu Ala Glu Lys Leu Phe
65 70 75 80
Asn Gln Asp Val Asp Ala Ala Val Arg Gly Ile Leu Arg Asn Ala Lys
85 90 95
Leu Lys Pro Val Tyr Asp Ser Leu Asp Ala Val Arg Arg Cys Ala Leu
100 105 110
Ile Asn Met Val Phe Gln Met Gly Glu Thr Gly Val Ala Gly Phe Thr
115 120 125
Asn Ser Leu Arg Met Leu Gln Gln Lys Arg Trp Asp Glu Ala Ala Val
130 135 140
Asn Leu Ala Lys Ser Arg Trp Tyr Asn Gln Thr Pro Asn Arg Ala Lys
145 150 155 160
Arg Val Ile Thr Thr Phe Arg Thr Gly Thr Trp Asp Ala Tyr Lys Asn
165 170 175
<210> 5
<211> 184
<212> PRT
<213> 人工序列
<220>
<223> 来源于包含N末端His标签的克氏柠檬酸杆菌噬菌体CkP1的重组内溶素
<400> 5
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Asn Ile Phe Lys Met Leu Arg Ile Asp Glu Gly
20 25 30
Tyr Asp Ser Lys Ile Tyr Lys Asp Thr Glu Gly Phe Trp Thr Ile Gly
35 40 45
Ile Gly His Leu Leu Thr Arg Asp Pro Ser Leu Glu Val Ala Lys Arg
50 55 60
Glu Leu Asp Lys Leu Val Gly Arg Lys Cys Asn Gly Gln Ile Thr Gln
65 70 75 80
Ser Glu Ala Glu Lys Ile Phe Ala Asp Asp Val Asp Lys Ala Ile Asn
85 90 95
Gly Ile Lys Lys Asn Ala Ser Leu Lys Pro Val Tyr Asp Ser Leu Asp
100 105 110
Gly Asp Asp Pro Arg Gln Ala Ala Leu Ile Asn Met Val Phe Gln Met
115 120 125
Gly Val Ala Gly Val Ala Gly Phe Thr Asn Ser Met Arg Met Val Lys
130 135 140
Glu Lys Arg Trp Ala Asp Ala Ala Val Asn Leu Ala Gln Ser Lys Trp
145 150 155 160
Tyr Arg Gln Thr Pro Asn Arg Ala Lys Arg Val Ile Glu Thr Phe Arg
165 170 175
Thr Gly Thr Trp Asn Ala Tyr Lys
180
<210> 6
<211> 162
<212> PRT
<213> 未知
<220>
<223> 来源于无N末端甲硫氨酸的克氏柠檬酸杆菌噬菌体CkP1的内溶素的催化结构域
<400> 6
Asn Ile Phe Lys Met Leu Arg Ile Asp Glu Gly Tyr Asp Ser Lys Ile
1 5 10 15
Tyr Lys Asp Thr Glu Gly Phe Trp Thr Ile Gly Ile Gly His Leu Leu
20 25 30
Thr Arg Asp Pro Ser Leu Glu Val Ala Lys Arg Glu Leu Asp Lys Leu
35 40 45
Val Gly Arg Lys Cys Asn Gly Gln Ile Thr Gln Ser Glu Ala Glu Lys
50 55 60
Ile Phe Ala Asp Asp Val Asp Lys Ala Ile Asn Gly Ile Lys Lys Asn
65 70 75 80
Ala Ser Leu Lys Pro Val Tyr Asp Ser Leu Asp Gly Asp Asp Pro Arg
85 90 95
Gln Ala Ala Leu Ile Asn Met Val Phe Gln Met Gly Val Ala Gly Val
100 105 110
Ala Gly Phe Thr Asn Ser Met Arg Met Val Lys Glu Lys Arg Trp Ala
115 120 125
Asp Ala Ala Val Asn Leu Ala Gln Ser Lys Trp Tyr Arg Gln Thr Pro
130 135 140
Asn Arg Ala Lys Arg Val Ile Glu Thr Phe Arg Thr Gly Thr Trp Asn
145 150 155 160
Ala Tyr
<210> 7
<211> 13
<212> PRT
<213> 人工序列
<220>
<223> 共有序列
<220>
<221> MISC_FEATURE
<222> (1)..(1)
<223> Xaa可以是Pro或Thr
<220>
<221> MISC_FEATURE
<222> (8)..(8)
<223> Xaa可以是Ala, Ile或Thr
<220>
<221> MISC_FEATURE
<222> (9)..(9)
<223> Xaa可以是Ala, Asp, Glu,或Ser
<220>
<221> MISC_FEATURE
<222> (10)..(10)
<223> Xaa可以是Val或Thr
<220>
<221> MISC_FEATURE
<222> (11)..(11)
<223> Xaa可以是Phe, Ile或Leu
<220>
<221> MISC_FEATURE
<222> (12)..(12)
<223> Xaa可以是Glu, Lys或Arg
<220>
<221> MISC_FEATURE
<222> (13)..(13)
<223> Xaa可以是 Leu或Thr
<400> 7
Xaa Asn Arg Ala Lys Arg Val Xaa Xaa Xaa Xaa Xaa Xaa
1 5 10
<210> 8
<211> 13
<212> PRT
<213> 人工序列
<220>
<223> 共有序列
<220>
<221> MISC_FEATURE
<222> (1)..(1)
<223> Xaa可以是Pro或Thr
<220>
<221> MISC_FEATURE
<222> (9)..(9)
<223> Xaa可以是Ala或 Ser
<220>
<221> MISC_FEATURE
<222> (12)..(12)
<223> Xaa可以是Lys或Arg
<400> 8
Xaa Asn Arg Ala Lys Arg Val Ile Xaa Thr Phe Xaa Thr
1 5 10
<210> 9
<211> 13
<212> PRT
<213> 人工序列
<220>
<223> 共有序列
<220>
<221> MISC_FEATURE
<222> (9)..(9)
<223> Xaa可以是Ala或Ser
<220>
<221> MISC_FEATURE
<222> (12)..(12)
<223> Xaa可以是Lys或Arg
<400> 9
Pro Asn Arg Ala Lys Arg Val Ile Xaa Thr Phe Xaa Thr
1 5 10
<210> 10
<211> 13
<212> PRT
<213> 人工序列
<220>
<223> 共有序列
<220>
<221> MISC_FEATURE
<222> (1)..(1)
<223> Xaa可以是Pro或Thr
<220>
<221> MISC_FEATURE
<222> (9)..(9)
<223> Xaa可以是Ala, Glu或Ser
<220>
<221> MISC_FEATURE
<222> (12)..(12)
<223> Xaa可以是Lys或Arg
<400> 10
Xaa Asn Arg Ala Lys Arg Val Ile Xaa Thr Phe Xaa Thr
1 5 10
<210> 11
<211> 13
<212> PRT
<213> 人工序列
<220>
<223> 共有序列
<220>
<221> MISC_FEATURE
<222> (9)..(9)
<223> Xaa可以是Ala或Glu
<220>
<221> MISC_FEATURE
<222> (12)..(12)
<223> Xaa可以是Lys或Arg
<400> 11
Pro Asn Arg Ala Lys Arg Val Ile Xaa Thr Phe Xaa Thr
1 5 10
<210> 12
<211> 13
<212> PRT
<213> 气单胞菌噬菌体Aeh1
<400> 12
Pro Asn Arg Ala Asn Arg Val Ala Ser Val Leu Lys Leu
1 5 10
<210> 13
<211> 13
<212> PRT
<213> 气单胞菌噬菌体phiAS4
<400> 13
Pro Asn Arg Ala Met Arg Val Ala Lys Val Val Leu Thr
1 5 10
<210> 14
<211> 13
<212> PRT
<213> 气单胞菌噬菌体44RR2.8t
<400> 14
Pro Asn Arg Ala Gln Arg Val Ala Gln Val Ile Leu Thr
1 5 10
<210> 15
<211> 13
<212> PRT
<213> 未知
<220>
<223> 来源于克氏柠檬酸杆菌噬菌体CkP1的内溶素的序列基序
<400> 15
Pro Asn Arg Ala Lys Arg Val Ile Glu Thr Phe Arg Thr
1 5 10
<210> 16
<211> 13
<212> PRT
<213> 肠球菌噬菌体CC31
<400> 16
Pro Asn Arg Ala Lys Arg Val Ile Ala Thr Phe Lys Thr
1 5 10
<210> 17
<211> 13
<212> PRT
<213> 沙雷氏菌噬菌体CHI14
<400> 17
Thr Asn Arg Ala Lys Arg Val Ile Ser Thr Phe Lys Thr
1 5 10
<210> 18
<211> 13
<212> PRT
<213> 气单胞菌噬菌体Ah1
<400> 18
Pro Asn Arg Ala Lys Arg Val Ala Ser Val Leu Lys Leu
1 5 10
<210> 19
<211> 13
<212> PRT
<213> 沙雷氏菌噬菌体PS2
<400> 19
Pro Asn Arg Ala Lys Arg Val Ile Ser Val Phe Glu Thr
1 5 10
<210> 20
<211> 13
<212> PRT
<213> 气单胞菌噬菌体As-szw
<400> 20
Pro Asn Arg Ala Lys Arg Val Thr Asp Val Ile Glu Thr
1 5 10
<210> 21
<211> 164
<212> PRT
<213> 未知
<220>
<223> 来源于克氏柠檬酸杆菌噬菌体CkP1的内溶素
<400> 21
Met Asn Ile Phe Lys Met Leu Arg Ile Asp Glu Gly Tyr Asp Ser Lys
1 5 10 15
Ile Tyr Lys Asp Thr Glu Gly Phe Trp Thr Ile Gly Ile Gly His Leu
20 25 30
Leu Thr Arg Asp Pro Ser Leu Glu Val Ala Lys Arg Glu Leu Asp Lys
35 40 45
Leu Val Gly Arg Lys Cys Asn Gly Gln Ile Thr Gln Ser Glu Ala Glu
50 55 60
Lys Ile Phe Ala Asp Asp Val Asp Lys Ala Ile Asn Gly Ile Lys Lys
65 70 75 80
Asn Ala Ser Leu Lys Pro Val Tyr Asp Ser Leu Asp Gly Asp Asp Pro
85 90 95
Arg Gln Ala Ala Leu Ile Asn Met Val Phe Gln Met Gly Val Ala Gly
100 105 110
Val Ala Gly Phe Thr Asn Ser Met Arg Met Val Lys Glu Lys Arg Trp
115 120 125
Ala Asp Ala Ala Val Asn Leu Ala Gln Ser Lys Trp Tyr Arg Gln Thr
130 135 140
Pro Asn Arg Ala Lys Arg Val Ile Glu Thr Phe Arg Thr Gly Thr Trp
145 150 155 160
Asn Ala Tyr Lys
<210> 22
<211> 164
<212> PRT
<213> 肠球菌噬菌体CC31
<400> 22
Met Asp Ile Phe Gly Met Leu Arg Ile Asp Glu Gly Tyr Asp Ser Lys
1 5 10 15
Ile Tyr Lys Asp Thr Glu Gly Phe Trp Thr Ile Gly Ile Gly His Leu
20 25 30
Leu Thr Arg Asp Pro Ser Leu Asp Val Ala Lys Arg Glu Leu Asp Lys
35 40 45
Leu Val Gly Arg Pro Cys Asn Gly Gln Ile Thr Lys Ala Glu Ala Glu
50 55 60
Ala Ile Phe Ala Lys Asp Val Asp Lys Ala Thr Arg Gly Ile Leu Gly
65 70 75 80
Asn Ala Val Leu Lys Pro Val Tyr Asp Val Leu Asp Gly Val Arg Arg
85 90 95
Ala Ala Leu Ile Asn Met Val Phe Gln Met Gly Val Ala Gly Val Ala
100 105 110
Ser Phe Pro Ala Ser Met Arg Leu Leu Lys Ser Lys Gln Trp Glu Ala
115 120 125
Ala Ala Lys Glu Leu Ala Asn Ser Lys Trp Tyr Arg Gln Thr Pro Asn
130 135 140
Arg Ala Lys Arg Val Ile Ala Thr Phe Lys Thr Gly Thr Trp Lys Ala
145 150 155 160
Tyr Glu Asn Leu
<210> 23
<211> 161
<212> PRT
<213> 沙雷氏菌噬菌体CHI14
<400> 23
Met Asp Ile Phe Gly Met Leu Arg Ile Asp Glu Gly Tyr Asp Ser Lys
1 5 10 15
Ile Tyr Lys Asp Thr Glu Gly Tyr Trp Thr Ile Gly Ile Gly His Leu
20 25 30
Leu Thr Lys Asn Pro Ser Leu Ser Val Ala Lys Ala Glu Leu Asp Lys
35 40 45
Leu Val Gly Arg Ser Cys Asn Gly Gln Ile Thr Gln Asp Glu Ala Glu
50 55 60
Ser Ile Phe Ala Lys Asp Val Glu Lys Ala Val Lys Gly Ile Gln Gly
65 70 75 80
Asn Ser Val Leu Lys Pro Val Tyr Asp Ser Leu Asp Glu Ile Arg Arg
85 90 95
Ala Ala Leu Ile Asn Met Val Phe Gln Met Gly Val Ala Gly Val Ala
100 105 110
Gly Phe Thr Asn Ser Met Arg Met Leu Lys Glu Lys Arg Trp Asp Glu
115 120 125
Ala Ala Val Asn Leu Ala Lys Ser Arg Trp Tyr Asn Gln Thr Thr Asn
130 135 140
Arg Ala Lys Arg Val Ile Ser Thr Phe Lys Thr Gly Thr Trp Gly Ala
145 150 155 160
Tyr
<210> 24
<211> 165
<212> PRT
<213> 气单胞菌噬菌体Ah1
<400> 24
Met Leu Ala Gln Met Leu Lys Gln Asp Glu Gly Tyr Lys Glu Thr Val
1 5 10 15
Tyr Trp Asp Thr Glu Gly Tyr Pro Thr Ile Gly Ile Gly His Leu Ile
20 25 30
Leu Lys Lys Arg Thr Lys Asp Met Gly Glu Ile Asn Arg Glu Leu Ser
35 40 45
Ser His Val Gly Arg Val Val Lys Asp Gly Lys Ile Thr Gly Glu Glu
50 55 60
Val Leu Ala Leu Phe Glu Arg Asp Leu Ser Val Leu Lys Arg Ser Ile
65 70 75 80
Met Ser Leu Pro Asn Leu Ala Asp Val Tyr Val Ser Leu Asp Met Val
85 90 95
Arg Gln Thr Ala Ile Glu Asn Met Val Phe Gln Met Gly Ala Val Gly
100 105 110
Val Ser Lys Phe Pro Gly Met Leu Arg Cys Leu Lys Ala Lys Asp Trp
115 120 125
Asp Gly Ala Tyr Arg Asn Ala Leu Asp Ser Ala Trp Ala Arg Gln Thr
130 135 140
Pro Asn Arg Ala Lys Arg Val Ala Ser Val Leu Lys Leu Gly Ser Tyr
145 150 155 160
Ala Pro Tyr Gly Phe
165
<210> 25
<211> 162
<212> PRT
<213> 沙雷氏菌噬菌体PS2
<400> 25
Met Thr Ile Phe Glu Met Leu Ala Phe Asp Glu Gly Leu Lys Leu Thr
1 5 10 15
Val Tyr Leu Asp Thr Glu Gly Phe Trp Thr Val Gly Ile Gly His Leu
20 25 30
Leu Thr Lys Asn Pro Ser Lys Ala Val Ala Ile Ala Glu Leu Asp Lys
35 40 45
Leu Val Gly Arg Ser Thr Gly Gly Thr Ile Thr Lys Ala Glu Ala Glu
50 55 60
Arg Ile Phe Ala Gln Asp Val Ala Lys Ser Glu Lys Gly Ile Gln Gly
65 70 75 80
Asn Ala Val Leu Gly Pro Val Tyr Ala Gly Leu Asp Ala Thr Arg Lys
85 90 95
Met Ala Leu Val Asn Met Thr Phe Gln Leu Gly Val Ala Gly Ala Ala
100 105 110
Gly Phe Thr Asn Ser Met Lys Leu Leu Ala Ala Lys Gln Trp Lys Glu
115 120 125
Ala Ala Ile Asn Leu Ala Lys Ser Lys Trp Tyr Asn Gln Thr Pro Asn
130 135 140
Arg Ala Lys Arg Val Ile Ser Val Phe Glu Thr Gly Thr Leu Ala Ala
145 150 155 160
Tyr Lys
<210> 26
<211> 166
<212> PRT
<213> 噬菌体As-szw
<400> 26
Met Leu Glu Lys Met Leu Lys Phe Asp Glu Gly Ser Lys Leu Ser Val
1 5 10 15
Tyr Trp Asp Thr Glu Gly Tyr Pro Thr Ile Gly Ile Gly His Leu Ile
20 25 30
Lys Arg Leu Arg Thr Lys Asp Met Gly Glu Ile Asn Arg Glu Leu Ser
35 40 45
Ser His Val Gly Arg Val Ile Thr Asp Gly Lys Ile Thr Gln Ser Glu
50 55 60
Glu Ser Gln Leu Phe Ala Lys Asp Leu Glu Val Val Arg Asn Ser Met
65 70 75 80
Lys Gly Tyr Val Asp Leu Trp Ser Thr Tyr Val Gly Leu Asp Glu Val
85 90 95
Arg Lys Thr Ala Leu Glu Asn Met Val Phe Gln Met Gly Ala Lys Gly
100 105 110
Val Asn Gly Phe Pro Ser Met Leu Arg Ala Met Arg Ser Lys Asn Trp
115 120 125
Val Glu Ala Lys Lys His Gly Leu Ala Ser Ala Trp Ser Arg Gln Thr
130 135 140
Pro Asn Arg Ala Lys Arg Val Thr Asp Val Ile Glu Thr Gly Thr Tyr
145 150 155 160
Lys Gly Tyr Pro Phe Ala
165
<210> 27
<211> 162
<212> PRT
<213> 未知
<220>
<223> 无N末端甲硫氨酸的肠球菌噬菌体T4内溶素
<400> 27
Asn Ile Phe Glu Met Leu Arg Ile Asp Glu Gly Leu Arg Leu Lys Ile
1 5 10 15
Tyr Lys Asp Thr Glu Gly Tyr Tyr Thr Ile Gly Ile Gly His Leu Leu
20 25 30
Thr Lys Ser Pro Ser Leu Asn Ala Ala Lys Ser Glu Leu Asp Lys Ala
35 40 45
Ile Gly Arg Asn Cys Asn Gly Val Ile Thr Lys Asp Glu Ala Glu Lys
50 55 60
Leu Phe Asn Gln Asp Val Asp Ala Ala Val Arg Gly Ile Leu Arg Asn
65 70 75 80
Ala Lys Leu Lys Pro Val Tyr Asp Ser Leu Asp Ala Val Arg Arg Cys
85 90 95
Ala Leu Ile Asn Met Val Phe Gln Met Gly Glu Thr Gly Val Ala Gly
100 105 110
Phe Thr Asn Ser Leu Arg Met Leu Gln Gln Lys Arg Trp Asp Glu Ala
115 120 125
Ala Val Asn Leu Ala Lys Ser Arg Trp Tyr Asn Gln Thr Pro Asn Arg
130 135 140
Ala Lys Arg Val Ile Thr Thr Phe Arg Thr Gly Thr Trp Asp Ala Tyr
145 150 155 160
Lys Asn
<210> 28
<211> 163
<212> PRT
<213> 人工序列
<220>
<223> 来源于无N末端甲硫氨酸和具有C54S的克氏柠檬酸杆菌噬菌体CkP1的内溶素
<400> 28
Asn Ile Phe Lys Met Leu Arg Ile Asp Glu Gly Tyr Asp Ser Lys Ile
1 5 10 15
Tyr Lys Asp Thr Glu Gly Phe Trp Thr Ile Gly Ile Gly His Leu Leu
20 25 30
Thr Arg Asp Pro Ser Leu Glu Val Ala Lys Arg Glu Leu Asp Lys Leu
35 40 45
Val Gly Arg Lys Ser Asn Gly Gln Ile Thr Gln Ser Glu Ala Glu Lys
50 55 60
Ile Phe Ala Asp Asp Val Asp Lys Ala Ile Asn Gly Ile Lys Lys Asn
65 70 75 80
Ala Ser Leu Lys Pro Val Tyr Asp Ser Leu Asp Gly Asp Asp Pro Arg
85 90 95
Gln Ala Ala Leu Ile Asn Met Val Phe Gln Met Gly Val Ala Gly Val
100 105 110
Ala Gly Phe Thr Asn Ser Met Arg Met Val Lys Glu Lys Arg Trp Ala
115 120 125
Asp Ala Ala Val Asn Leu Ala Gln Ser Lys Trp Tyr Arg Gln Thr Pro
130 135 140
Asn Arg Ala Lys Arg Val Ile Glu Thr Phe Arg Thr Gly Thr Trp Asn
145 150 155 160
Ala Tyr Lys
<210> 29
<211> 163
<212> PRT
<213> 肠球菌噬菌体CC31
<400> 29
Asp Ile Phe Gly Met Leu Arg Ile Asp Glu Gly Tyr Asp Ser Lys Ile
1 5 10 15
Tyr Lys Asp Thr Glu Gly Phe Trp Thr Ile Gly Ile Gly His Leu Leu
20 25 30
Thr Arg Asp Pro Ser Leu Asp Val Ala Lys Arg Glu Leu Asp Lys Leu
35 40 45
Val Gly Arg Pro Cys Asn Gly Gln Ile Thr Lys Ala Glu Ala Glu Ala
50 55 60
Ile Phe Ala Lys Asp Val Asp Lys Ala Thr Arg Gly Ile Leu Gly Asn
65 70 75 80
Ala Val Leu Lys Pro Val Tyr Asp Val Leu Asp Gly Val Arg Arg Ala
85 90 95
Ala Leu Ile Asn Met Val Phe Gln Met Gly Val Ala Gly Val Ala Ser
100 105 110
Phe Pro Ala Ser Met Arg Leu Leu Lys Ser Lys Gln Trp Glu Ala Ala
115 120 125
Ala Lys Glu Leu Ala Asn Ser Lys Trp Tyr Arg Gln Thr Pro Asn Arg
130 135 140
Ala Lys Arg Val Ile Ala Thr Phe Lys Thr Gly Thr Trp Lys Ala Tyr
145 150 155 160
Glu Asn Leu
<210> 30
<211> 163
<212> PRT
<213> 人工序列
<220>
<223> 来源于无N末端甲硫氨酸和具有C54S的肠球菌噬菌体CC31的内溶素
<400> 30
Asp Ile Phe Gly Met Leu Arg Ile Asp Glu Gly Tyr Asp Ser Lys Ile
1 5 10 15
Tyr Lys Asp Thr Glu Gly Phe Trp Thr Ile Gly Ile Gly His Leu Leu
20 25 30
Thr Arg Asp Pro Ser Leu Asp Val Ala Lys Arg Glu Leu Asp Lys Leu
35 40 45
Val Gly Arg Pro Ser Asn Gly Gln Ile Thr Lys Ala Glu Ala Glu Ala
50 55 60
Ile Phe Ala Lys Asp Val Asp Lys Ala Thr Arg Gly Ile Leu Gly Asn
65 70 75 80
Ala Val Leu Lys Pro Val Tyr Asp Val Leu Asp Gly Val Arg Arg Ala
85 90 95
Ala Leu Ile Asn Met Val Phe Gln Met Gly Val Ala Gly Val Ala Ser
100 105 110
Phe Pro Ala Ser Met Arg Leu Leu Lys Ser Lys Gln Trp Glu Ala Ala
115 120 125
Ala Lys Glu Leu Ala Asn Ser Lys Trp Tyr Arg Gln Thr Pro Asn Arg
130 135 140
Ala Lys Arg Val Ile Ala Thr Phe Lys Thr Gly Thr Trp Lys Ala Tyr
145 150 155 160
Glu Asn Leu
<210> 31
<211> 160
<212> PRT
<213> 沙雷氏菌噬菌体CHI14
<400> 31
Asp Ile Phe Gly Met Leu Arg Ile Asp Glu Gly Tyr Asp Ser Lys Ile
1 5 10 15
Tyr Lys Asp Thr Glu Gly Tyr Trp Thr Ile Gly Ile Gly His Leu Leu
20 25 30
Thr Lys Asn Pro Ser Leu Ser Val Ala Lys Ala Glu Leu Asp Lys Leu
35 40 45
Val Gly Arg Ser Cys Asn Gly Gln Ile Thr Gln Asp Glu Ala Glu Ser
50 55 60
Ile Phe Ala Lys Asp Val Glu Lys Ala Val Lys Gly Ile Gln Gly Asn
65 70 75 80
Ser Val Leu Lys Pro Val Tyr Asp Ser Leu Asp Glu Ile Arg Arg Ala
85 90 95
Ala Leu Ile Asn Met Val Phe Gln Met Gly Val Ala Gly Val Ala Gly
100 105 110
Phe Thr Asn Ser Met Arg Met Leu Lys Glu Lys Arg Trp Asp Glu Ala
115 120 125
Ala Val Asn Leu Ala Lys Ser Arg Trp Tyr Asn Gln Thr Thr Asn Arg
130 135 140
Ala Lys Arg Val Ile Ser Thr Phe Lys Thr Gly Thr Trp Gly Ala Tyr
145 150 155 160
<210> 32
<211> 160
<212> PRT
<213> 人工序列
<220>
<223> 来源于无N末端甲硫氨酸和具有C54S的沙雷氏菌噬菌体CHI14的内溶素
<400> 32
Asp Ile Phe Gly Met Leu Arg Ile Asp Glu Gly Tyr Asp Ser Lys Ile
1 5 10 15
Tyr Lys Asp Thr Glu Gly Tyr Trp Thr Ile Gly Ile Gly His Leu Leu
20 25 30
Thr Lys Asn Pro Ser Leu Ser Val Ala Lys Ala Glu Leu Asp Lys Leu
35 40 45
Val Gly Arg Ser Ser Asn Gly Gln Ile Thr Gln Asp Glu Ala Glu Ser
50 55 60
Ile Phe Ala Lys Asp Val Glu Lys Ala Val Lys Gly Ile Gln Gly Asn
65 70 75 80
Ser Val Leu Lys Pro Val Tyr Asp Ser Leu Asp Glu Ile Arg Arg Ala
85 90 95
Ala Leu Ile Asn Met Val Phe Gln Met Gly Val Ala Gly Val Ala Gly
100 105 110
Phe Thr Asn Ser Met Arg Met Leu Lys Glu Lys Arg Trp Asp Glu Ala
115 120 125
Ala Val Asn Leu Ala Lys Ser Arg Trp Tyr Asn Gln Thr Thr Asn Arg
130 135 140
Ala Lys Arg Val Ile Ser Thr Phe Lys Thr Gly Thr Trp Gly Ala Tyr
145 150 155 160
<210> 33
<211> 164
<212> PRT
<213> 噬菌体Ah1
<400> 33
Leu Ala Gln Met Leu Lys Gln Asp Glu Gly Tyr Lys Glu Thr Val Tyr
1 5 10 15
Trp Asp Thr Glu Gly Tyr Pro Thr Ile Gly Ile Gly His Leu Ile Leu
20 25 30
Lys Lys Arg Thr Lys Asp Met Gly Glu Ile Asn Arg Glu Leu Ser Ser
35 40 45
His Val Gly Arg Val Val Lys Asp Gly Lys Ile Thr Gly Glu Glu Val
50 55 60
Leu Ala Leu Phe Glu Arg Asp Leu Ser Val Leu Lys Arg Ser Ile Met
65 70 75 80
Ser Leu Pro Asn Leu Ala Asp Val Tyr Val Ser Leu Asp Met Val Arg
85 90 95
Gln Thr Ala Ile Glu Asn Met Val Phe Gln Met Gly Ala Val Gly Val
100 105 110
Ser Lys Phe Pro Gly Met Leu Arg Cys Leu Lys Ala Lys Asp Trp Asp
115 120 125
Gly Ala Tyr Arg Asn Ala Leu Asp Ser Ala Trp Ala Arg Gln Thr Pro
130 135 140
Asn Arg Ala Lys Arg Val Ala Ser Val Leu Lys Leu Gly Ser Tyr Ala
145 150 155 160
Pro Tyr Gly Phe
<210> 34
<211> 164
<212> PRT
<213> 人工序列
<220>
<223> 来源于无N末端甲硫氨酸和具有C122S的气单胞菌噬菌体Ah1的内溶素
<400> 34
Leu Ala Gln Met Leu Lys Gln Asp Glu Gly Tyr Lys Glu Thr Val Tyr
1 5 10 15
Trp Asp Thr Glu Gly Tyr Pro Thr Ile Gly Ile Gly His Leu Ile Leu
20 25 30
Lys Lys Arg Thr Lys Asp Met Gly Glu Ile Asn Arg Glu Leu Ser Ser
35 40 45
His Val Gly Arg Val Val Lys Asp Gly Lys Ile Thr Gly Glu Glu Val
50 55 60
Leu Ala Leu Phe Glu Arg Asp Leu Ser Val Leu Lys Arg Ser Ile Met
65 70 75 80
Ser Leu Pro Asn Leu Ala Asp Val Tyr Val Ser Leu Asp Met Val Arg
85 90 95
Gln Thr Ala Ile Glu Asn Met Val Phe Gln Met Gly Ala Val Gly Val
100 105 110
Ser Lys Phe Pro Gly Met Leu Arg Ser Leu Lys Ala Lys Asp Trp Asp
115 120 125
Gly Ala Tyr Arg Asn Ala Leu Asp Ser Ala Trp Ala Arg Gln Thr Pro
130 135 140
Asn Arg Ala Lys Arg Val Ala Ser Val Leu Lys Leu Gly Ser Tyr Ala
145 150 155 160
Pro Tyr Gly Phe
<210> 35
<211> 161
<212> PRT
<213> 沙雷氏菌噬菌体PS2
<400> 35
Thr Ile Phe Glu Met Leu Ala Phe Asp Glu Gly Leu Lys Leu Thr Val
1 5 10 15
Tyr Leu Asp Thr Glu Gly Phe Trp Thr Val Gly Ile Gly His Leu Leu
20 25 30
Thr Lys Asn Pro Ser Lys Ala Val Ala Ile Ala Glu Leu Asp Lys Leu
35 40 45
Val Gly Arg Ser Thr Gly Gly Thr Ile Thr Lys Ala Glu Ala Glu Arg
50 55 60
Ile Phe Ala Gln Asp Val Ala Lys Ser Glu Lys Gly Ile Gln Gly Asn
65 70 75 80
Ala Val Leu Gly Pro Val Tyr Ala Gly Leu Asp Ala Thr Arg Lys Met
85 90 95
Ala Leu Val Asn Met Thr Phe Gln Leu Gly Val Ala Gly Ala Ala Gly
100 105 110
Phe Thr Asn Ser Met Lys Leu Leu Ala Ala Lys Gln Trp Lys Glu Ala
115 120 125
Ala Ile Asn Leu Ala Lys Ser Lys Trp Tyr Asn Gln Thr Pro Asn Arg
130 135 140
Ala Lys Arg Val Ile Ser Val Phe Glu Thr Gly Thr Leu Ala Ala Tyr
145 150 155 160
Lys
<210> 36
<211> 165
<212> PRT
<213> 气单胞菌噬菌体As-szw
<400> 36
Leu Glu Lys Met Leu Lys Phe Asp Glu Gly Ser Lys Leu Ser Val Tyr
1 5 10 15
Trp Asp Thr Glu Gly Tyr Pro Thr Ile Gly Ile Gly His Leu Ile Lys
20 25 30
Arg Leu Arg Thr Lys Asp Met Gly Glu Ile Asn Arg Glu Leu Ser Ser
35 40 45
His Val Gly Arg Val Ile Thr Asp Gly Lys Ile Thr Gln Ser Glu Glu
50 55 60
Ser Gln Leu Phe Ala Lys Asp Leu Glu Val Val Arg Asn Ser Met Lys
65 70 75 80
Gly Tyr Val Asp Leu Trp Ser Thr Tyr Val Gly Leu Asp Glu Val Arg
85 90 95
Lys Thr Ala Leu Glu Asn Met Val Phe Gln Met Gly Ala Lys Gly Val
100 105 110
Asn Gly Phe Pro Ser Met Leu Arg Ala Met Arg Ser Lys Asn Trp Val
115 120 125
Glu Ala Lys Lys His Gly Leu Ala Ser Ala Trp Ser Arg Gln Thr Pro
130 135 140
Asn Arg Ala Lys Arg Val Thr Asp Val Ile Glu Thr Gly Thr Tyr Lys
145 150 155 160
Gly Tyr Pro Phe Ala
165
<210> 37
<211> 6
<212> PRT
<213> 人工的
<220>
<223> 合成序列
<400> 37
Lys Arg Lys Lys Arg Lys
1 5
<210> 38
<211> 5
<212> PRT
<213> 人工的
<220>
<223> 合成序列
<220>
<221> misc_feature
<222> (3)..(3)
<223> Xaa可以是任何天然存在的氨基酸
<400> 38
Lys Arg Xaa Lys Arg
1 5
<210> 39
<211> 5
<212> PRT
<213> 人工的
<220>
<223> 合成序列
<400> 39
Lys Arg Ser Lys Arg
1 5
<210> 40
<211> 5
<212> PRT
<213> 人工的
<220>
<223> 合成序列
<400> 40
Lys Arg Gly Ser Gly
1 5
<210> 41
<211> 9
<212> PRT
<213> 人工的
<220>
<223> 合成序列
<400> 41
Lys Arg Lys Lys Arg Lys Lys Arg Lys
1 5
<210> 42
<211> 9
<212> PRT
<213> 人工的
<220>
<223> 合成序列
<400> 42
Arg Arg Arg Arg Arg Arg Arg Arg Arg
1 5
<210> 43
<211> 8
<212> PRT
<213> 人工的
<220>
<223> 合成序列
<400> 43
Lys Lys Lys Lys Lys Lys Lys Lys
1 5
<210> 44
<211> 10
<212> PRT
<213> 人工的
<220>
<223> 合成序列
<400> 44
Lys Arg Lys Lys Arg Lys Lys Arg Lys Lys
1 5 10
<210> 45
<211> 12
<212> PRT
<213> 人工的
<220>
<223> 合成序列
<400> 45
Lys Arg Lys Lys Arg Lys Lys Arg Lys Lys Arg Lys
1 5 10
<210> 46
<211> 14
<212> PRT
<213> 人工的
<220>
<223> 合成序列
<400> 46
Lys Arg Lys Lys Arg Lys Lys Arg Lys Lys Arg Lys Lys Arg
1 5 10
<210> 47
<211> 16
<212> PRT
<213> 人工的
<220>
<223> 合成序列
<400> 47
Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys
1 5 10 15
<210> 48
<211> 18
<212> PRT
<213> 人工的
<220>
<223> 合成序列
<400> 48
Lys Arg Lys Lys Arg Lys Lys Arg Lys Lys Arg Lys Lys Arg Lys Lys
1 5 10 15
Arg Lys
<210> 49
<211> 19
<212> PRT
<213> 人工的
<220>
<223> 合成序列
<400> 49
Lys Arg Lys Lys Arg Lys Lys Arg Lys Lys Arg Lys Lys Arg Lys Lys
1 5 10 15
Arg Lys Lys
<210> 50
<211> 19
<212> PRT
<213> 人工的
<220>
<223> 合成序列
<400> 50
Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg
1 5 10 15
Arg Arg Arg
<210> 51
<211> 19
<212> PRT
<213> 人工的
<220>
<223> 合成序列
<400> 51
Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys
1 5 10 15
Lys Lys Lys
<210> 52
<211> 20
<212> PRT
<213> 人工的
<220>
<223> 合成序列
<400> 52
Lys Arg Lys Lys Arg Lys Lys Arg Lys Arg Ser Lys Arg Lys Lys Arg
1 5 10 15
Lys Lys Arg Lys
20
<210> 53
<211> 21
<212> PRT
<213> 人工的
<220>
<223> 合成序列
<400> 53
Lys Arg Lys Lys Arg Lys Lys Arg Lys Arg Ser Lys Arg Lys Lys Arg
1 5 10 15
Lys Lys Arg Lys Lys
20
<210> 54
<211> 21
<212> PRT
<213> 人工的
<220>
<223> 合成序列
<400> 54
Lys Arg Lys Lys Arg Lys Lys Arg Lys Lys Arg Lys Lys Arg Lys Lys
1 5 10 15
Arg Lys Lys Arg Lys
20
<210> 55
<211> 22
<212> PRT
<213> 人工的
<220>
<223> 合成序列
<400> 55
Lys Arg Lys Lys Arg Lys Lys Arg Lys Arg Gly Ser Gly Lys Arg Lys
1 5 10 15
Lys Arg Lys Lys Arg Lys
20
<210> 56
<211> 24
<212> PRT
<213> 人工的
<220>
<223> 合成序列
<400> 56
Lys Arg Lys Lys Arg Lys Lys Arg Lys Arg Gly Ser Gly Ser Gly Lys
1 5 10 15
Arg Lys Lys Arg Lys Lys Arg Lys
20
<210> 57
<211> 25
<212> PRT
<213> 人工的
<220>
<223> 合成序列
<400> 57
Lys Arg Lys Lys Arg Lys Lys Arg Lys Lys Arg Lys Lys Arg Lys Lys
1 5 10 15
Arg Lys Lys Arg Lys Lys Arg Lys Lys
20 25
<210> 58
<211> 31
<212> PRT
<213> 人工的
<220>
<223> 合成序列
<400> 58
Lys Arg Lys Lys Arg Lys Lys Arg Lys Arg Ser Lys Arg Lys Lys Arg
1 5 10 15
Lys Lys Arg Lys Arg Ser Lys Arg Lys Lys Arg Lys Lys Arg Lys
20 25 30
<210> 59
<211> 38
<212> PRT
<213> 人工的
<220>
<223> 合成序列
<400> 59
Lys Arg Lys Lys Arg Lys Lys Arg Lys Arg Gly Ser Gly Ser Gly Lys
1 5 10 15
Arg Lys Lys Arg Lys Lys Arg Lys Gly Ser Gly Ser Gly Lys Arg Lys
20 25 30
Lys Arg Lys Lys Arg Lys
35
<210> 60
<211> 39
<212> PRT
<213> 人工的
<220>
<223> 合成序列
<400> 60
Lys Arg Lys Lys Arg Lys Lys Arg Lys Lys Arg Lys Lys Arg Lys Lys
1 5 10 15
Arg Lys Lys Arg Lys Lys Arg Lys Lys Arg Lys Lys Arg Lys Lys Arg
20 25 30
Lys Lys Arg Lys Lys Arg Lys
35
<210> 61
<211> 42
<212> PRT
<213> 人工的
<220>
<223> 合成序列
<400> 61
Lys Arg Lys Lys Arg Lys Lys Arg Lys Arg Ser Lys Arg Lys Lys Arg
1 5 10 15
Lys Lys Arg Lys Arg Ser Lys Arg Lys Lys Arg Lys Lys Arg Lys Arg
20 25 30
Ser Lys Arg Lys Lys Arg Lys Lys Arg Lys
35 40
<210> 62
<211> 37
<212> PRT
<213> 智人
<400> 62
Leu Leu Gly Asp Phe Phe Arg Lys Ser Lys Glu Lys Ile Gly Lys Glu
1 5 10 15
Phe Lys Arg Ile Val Gln Arg Ile Lys Asp Phe Leu Arg Asn Leu Val
20 25 30
Pro Arg Thr Glu Ser
35
<210> 63
<211> 29
<212> PRT
<213> 未知
<220>
<223> 绵羊SMAP-29
<400> 63
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> 64
<211> 13
<212> PRT
<213> 未知
<220>
<223> 牛吲哚菌肽
<400> 64
Ile Leu Pro Trp Lys Trp Pro Trp Trp Pro Trp Arg Arg
1 5 10
<210> 65
<211> 18
<212> PRT
<213> 未知
<220>
<223> 猪Protegrin
<400> 65
Arg Gly Gly Arg Leu Cys Tyr Cys Arg Arg Arg Phe Cys Val Cys Val
1 5 10 15
Gly Arg
<210> 66
<211> 31
<212> PRT
<213> 未知
<220>
<223> 哺乳动物(猪)杀菌肽P1
<400> 66
Ser Trp Leu Ser Lys Thr Ala Lys Lys Leu Glu Asn Ser Ala Lys Lys
1 5 10 15
Arg Ile Ser Glu Gly Ile Ala Ile Ala Ile Gln Gly Gly Pro Arg
20 25 30
<210> 67
<211> 23
<212> PRT
<213> 未知
<220>
<223> 蛙滑抓蟾素
<400> 67
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> 68
<211> 25
<212> PRT
<213> 未知
<220>
<223> 鱼Pleurocidin
<400> 68
Gly Trp Gly Ser Phe Phe Lys Lys Ala Ala His Val Gly Lys His Val
1 5 10 15
Gly Lys Ala Ala Leu Thr His Tyr Leu
20 25
<210> 69
<211> 36
<212> PRT
<213> 埃及伊蚊
<400> 69
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> 70
<211> 40
<212> PRT
<213> 黑腹果蝇
<400> 70
Gly Trp Leu Lys Lys Ile Gly Lys Lys Ile Glu Arg Val Gly Gln His
1 5 10 15
Thr Arg Asp Ala Thr Ile Gln Gly Leu Gly Ile Pro Gln Gln Ala Ala
20 25 30
Asn Val Ala Ala Thr Ala Arg Gly
35 40
<210> 71
<211> 21
<212> PRT
<213> 未知
<220>
<223> 脊椎动物Buforin II
<400> 71
Thr Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val His
1 5 10 15
Arg Leu Leu Arg Lys
20
<210> 72
<211> 39
<212> PRT
<213> 未知
<220>
<223> 蝇杀菌肽IA
<400> 72
Gly Trp Leu Lys Lys Ile Gly Lys Lys Ile Glu Arg Val Gly Gln His
1 5 10 15
Thr Arg Asp Ala Thr Ile Gln Gly Leu Gly Ile Ala Gln Gln Ala Ala
20 25 30
Asn Val Ala Ala Thr Ala Arg
35
<210> 73
<211> 17
<212> PRT
<213> 意大利蜜蜂
<400> 73
Ala Asn Arg Pro Val Tyr Ile Pro Pro Pro Arg Pro Pro His Pro Arg
1 5 10 15
Leu
<210> 74
<211> 24
<212> PRT
<213> 未知
<220>
<223> 蛙Ascaphine 5
<400> 74
Gly Ile Lys Asp Trp Ile Lys Gly Ala Ala Lys Lys Leu Ile Lys Thr
1 5 10 15
Val Ala Ser His Ile Ala Asn Gln
20
<210> 75
<211> 22
<212> PRT
<213> 未知
<220>
<223> 蛙Nigrocine 2
<400> 75
Gly Leu Leu Ser Lys Val Leu Gly Val Gly Lys Lys Val Leu Cys Gly
1 5 10 15
Val Ser Gly Leu Val Cys
20
<210> 76
<211> 24
<212> PRT
<213> 未知
<220>
<223> 蛙Pseudin 1
<400> 76
Gly Leu Asn Thr Leu Lys Lys Val Phe Gln Gly Leu His Glu Ala Ile
1 5 10 15
Lys Leu Ile Asn Asn His Val Gln
20
<210> 77
<211> 18
<212> PRT
<213> 未知
<220>
<223> 蛙牛蛙防御肽
<400> 77
Phe Leu Gly Gly Leu Ile Val Pro Ala Met Ile Cys Ala Val Thr Lys
1 5 10 15
Lys Cys
<210> 78
<211> 26
<212> PRT
<213> 未知
<220>
<223> 蜂毒肽
<400> 78
Gly Ile Gly Ala Val Leu Lys Val Leu Thr Thr Gly Leu Pro Ala Leu
1 5 10 15
Ile Ser Trp Ile Lys Arg Lys Arg Gln Gln
20 25
<210> 79
<211> 25
<212> PRT
<213> 未知
<220>
<223> 蜘蛛Lycotoxin 1
<400> 79
Ile Trp Leu Thr Ala Leu Lys Phe Leu Gly Lys His Ala Ala Lys Lys
1 5 10 15
Leu Ala Lys Gln Gln Leu Ser Lys Leu
20 25
<210> 80
<211> 19
<212> PRT
<213> 未知
<220>
<223> 鱼Parasin 1
<400> 80
Lys Gly Arg Gly Lys Gln Gly Gly Lys Val Arg Ala Lys Ala Lys Thr
1 5 10 15
Arg Ser Ser
<210> 81
<211> 39
<212> PRT
<213> 未知
<220>
<223> 蟾蜍Buforin I
<400> 81
Ala Gly Arg Gly Lys Gln Gly Gly Lys Val Arg Ala Lys Ala Lys Thr
1 5 10 15
Arg Ser Ser Arg Ala Gly Leu Gln Phe Pro Val Gly Arg Val His Arg
20 25 30
Leu Leu Arg Lys Gly Asn Tyr
35
<210> 82
<211> 34
<212> PRT
<213> 未知
<220>
<223> 蛙皮抑菌肽
<400> 82
Ala Leu Trp Lys Thr Met Leu Lys Lys Leu Gly Thr Met Ala Leu His
1 5 10 15
Ala Gly Lys Ala Ala Leu Gly Ala Ala Ala Asp Thr Ile Ser Gln Gly
20 25 30
Thr Gln
<210> 83
<211> 12
<212> PRT
<213> 未知
<220>
<223> 奶牛Bactenecin 1
<400> 83
Arg Leu Cys Arg Ile Val Val Ile Arg Val Cys Arg
1 5 10
<210> 84
<211> 21
<212> PRT
<213> 未知
<220>
<223> 昆虫Thanatin
<400> 84
Gly Ser Lys Lys Pro Val Pro Ile Ile Tyr Cys Asn Arg Arg Thr Gly
1 5 10 15
Lys Cys Gln Arg Met
20
<210> 85
<211> 19
<212> PRT
<213> 未知
<220>
<223> 蛙Brevinin 1T
<400> 85
Val Asn Pro Ile Ile Leu Gly Val Leu Pro Lys Val Cys Leu Ile Thr
1 5 10 15
Lys Lys Cys
<210> 86
<211> 26
<212> PRT
<213> 未知
<220>
<223> 蛙Ranateurin 1
<400> 86
Ser Met Leu Ser Val Leu Lys Asn Leu Gly Lys Val Gly Leu Gly Phe
1 5 10 15
Val Ala Cys Lys Ile Asn Ile Lys Gln Cys
20 25
<210> 87
<211> 46
<212> PRT
<213> 未知
<220>
<223> 蛙Esculentin 1
<400> 87
Gly Ile Phe Ser Lys Leu Gly Arg Lys Lys Ile Lys Asn Leu Leu Ile
1 5 10 15
Ser Gly Leu Lys Asn Val Gly Lys Glu Val Gly Met Asp Val Val Arg
20 25 30
Thr Gly Ile Lys Ile Ala Gly Cys Lys Ile Lys Gly Glu Cys
35 40 45
<210> 88
<211> 17
<212> PRT
<213> 美洲鲎
<400> 88
Arg Trp Cys Phe Arg Val Cys Tyr Arg Gly Ile Cys Tyr Arg Lys Cys
1 5 10 15
Arg
<210> 89
<211> 25
<212> PRT
<213> 未知
<220>
<223> 蝎Androctonin
<400> 89
Arg Ser Val Cys Arg Gln Ile Lys Ile Cys Arg Arg Arg Gly Gly Cys
1 5 10 15
Tyr Tyr Lys Cys Thr Asn Arg Pro Tyr
20 25
<210> 90
<211> 30
<212> PRT
<213> 智人
<400> 90
Asp Cys Tyr Cys Arg Ile Pro Ala Cys Ile Ala Gly Glu Arg Arg Tyr
1 5 10 15
Gly Thr Cys Ile Tyr Gln Gly Arg Leu Trp Ala Phe Cys Cys
20 25 30
<210> 91
<211> 38
<212> PRT
<213> 未知
<220>
<223> 奶牛β-防御素
<400> 91
Asn Pro Val Ser Cys Val Arg Asn Lys Gly Ile Cys Val Pro Ile Arg
1 5 10 15
Cys Pro Gly Ser Met Lys Gln Ile Gly Thr Cys Val Gly Arg Ala Val
20 25 30
Lys Cys Cys Arg Lys Lys
35
<210> 92
<211> 18
<212> PRT
<213> 未知
<220>
<223> 猴θ-防御素
<400> 92
Gly Phe Cys Arg Cys Leu Cys Arg Arg Gly Val Cys Arg Cys Ile Cys
1 5 10 15
Thr Arg
<210> 93
<211> 40
<212> PRT
<213> 未知
<220>
<223> 昆虫防御素(sapecin A)
<400> 93
Ala Thr Cys Asp Leu Leu Ser Gly Thr Gly Ile Asn His Ser Ala Cys
1 5 10 15
Ala Ala His Cys Leu Leu Arg Gly Asn Arg Gly Gly Tyr Cys Asn Gly
20 25 30
Lys Ala Val Cys Val Cys Arg Asn
35 40
<210> 94
<211> 46
<212> PRT
<213> 未知
<220>
<223> 植物Thionin (crambin)
<400> 94
Thr Thr Cys Cys Pro Ser Ile Val Ala Arg Ser Asn Phe Asn Val Cys
1 5 10 15
Arg Ile Pro Gly Thr Pro Glu Ala Ile Cys Ala Thr Tyr Thr Gly Cys
20 25 30
Ile Ile Ile Pro Gly Ala Thr Cys Pro Gly Asp Tyr Ala Asn
35 40 45
<210> 95
<211> 50
<212> PRT
<213> 未知
<220>
<223> 来自萝卜的防御素
<400> 95
Gln Lys Leu Cys Gln Arg Pro Ser Gly Thr Trp Ser Gly Val Cys Gly
1 5 10 15
Asn Asn Asn Ala Cys Lys Asn Gln Cys Ile Arg Leu Glu Lys Ala Arg
20 25 30
His Gly Ser Cys Asn Tyr Val Phe Pro Ala His Cys Ile Cys Tyr Phe
35 40 45
Pro Cys
50
<210> 96
<211> 30
<212> PRT
<213> 金环蛇
<400> 96
Lys Phe Phe Arg Lys Leu Lys Lys Ser Val Lys Lys Arg Ala Lys Glu
1 5 10 15
Phe Phe Lys Lys Pro Arg Val Ile Gly Val Ser Ile Pro Phe
20 25 30
<210> 97
<211> 44
<212> PRT
<213> 黑腹果蝇
<400> 97
Asp Cys Leu Ser Gly Arg Tyr Lys Gly Pro Cys Ala Val Trp Asp Asn
1 5 10 15
Glu Thr Cys Arg Arg Val Cys Lys Glu Glu Gly Arg Ser Ser Gly His
20 25 30
Cys Ser Pro Ser Leu Lys Cys Trp Cys Glu Gly Cys
35 40
<210> 98
<211> 25
<212> PRT
<213> 智人
<400> 98
Asp Thr His Phe Pro Ile Cys Ile Phe Cys Cys Gly Cys Cys His Arg
1 5 10 15
Ser Lys Cys Gly Met Cys Cys Lys Thr
20 25
<210> 99
<211> 44
<212> PRT
<213> 未知
<220>
<223> 奶牛Bac 5
<400> 99
Arg Phe Arg Pro Pro Ile Arg Arg Pro Pro Ile Arg Pro Pro Phe Tyr
1 5 10 15
Pro Pro Phe Arg Pro Pro Ile Arg Pro Pro Ile Phe Pro Pro Ile Arg
20 25 30
Pro Pro Phe Arg Pro Pro Leu Gly Arg Pro Phe Pro
35 40
<210> 100
<211> 39
<212> PRT
<213> 未知
<220>
<223> 猪PR-39
<400> 100
Arg Arg Arg Pro Arg Pro Pro Tyr Leu Pro Arg Pro Arg Pro Pro Pro
1 5 10 15
Phe Phe Pro Pro Arg Leu Pro Pro Arg Ile Pro Pro Gly Phe Pro Pro
20 25 30
Arg Phe Pro Pro Arg Phe Pro
35
<210> 101
<211> 20
<212> PRT
<213> 未知
<220>
<223> 昆虫吡咯菌素
<400> 101
Val Asp Lys Gly Ser Tyr Leu Pro Arg Pro Thr Pro Pro Arg Pro Ile
1 5 10 15
Tyr Asn Arg Asn
20
<210> 102
<211> 24
<212> PRT
<213> 智人
<400> 102
Asp Ser His Ala Lys Arg His His Gly Tyr Lys Arg Lys Phe His Glu
1 5 10 15
Lys His His Ser His Arg Gly Tyr
20
<210> 103
<211> 19
<212> PRT
<213> 未知
<220>
<223> ECP19
<400> 103
Arg Pro Pro Gln Phe Thr Arg Ala Gln Trp Phe Ala Ile Gln His Ile
1 5 10 15
Ser Leu Asn
<210> 104
<211> 23
<212> PRT
<213> 未知
<220>
<223> MSI-594
<400> 104
Gly Ile Gly Lys Phe Leu Lys Lys Ala Lys Lys Gly Ile Gly Ala Val
1 5 10 15
Leu Lys Val Leu Thr Thr Gly
20
<210> 105
<211> 35
<212> PRT
<213> 未知
<220>
<223> TL-ColM
<400> 105
Met Glu Thr Leu Thr Val His Ala Pro Ser Pro Ser Thr Asn Leu Pro
1 5 10 15
Ser Tyr Gly Asn Gly Ala Phe Ser Leu Ser Ala Pro His Val Pro Gly
20 25 30
Ala Gly Pro
35
<210> 106
<211> 18
<212> PRT
<213> 未知
<220>
<223> SBO
<400> 106
Lys Leu Lys Lys Ile Ala Gln Lys Ile Lys Asn Phe Phe Ala Lys Leu
1 5 10 15
Val Ala
<210> 107
<211> 30
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 107
Lys Phe Phe Arg Lys Leu Lys Lys Ser Val Lys Lys Arg Ala Lys Arg
1 5 10 15
Phe Phe Lys Lys Pro Arg Val Ile Gly Val Ser Ile Pro Phe
20 25 30
<210> 108
<211> 34
<212> PRT
<213> 美洲鲎
<400> 108
Gly Phe Lys Leu Lys Gly Met Ala Arg Ile Ser Cys Leu Pro Asn Gly
1 5 10 15
Gln Trp Ser Asn Phe Pro Pro Lys Cys Ile Arg Glu Cys Ala Met Val
20 25 30
Ser Ser
<210> 109
<211> 27
<212> PRT
<213> 人工的
<220>
<223> 合成序列
<400> 109
Lys Arg Trp Val Lys Arg Val Lys Arg Val Lys Arg Trp Val Lys Arg
1 5 10 15
Val Val Arg Val Val Lys Arg Trp Val Lys Arg
20 25
<210> 110
<211> 25
<212> PRT
<213> 人工序列
<220>
<223> 合成序列; MW2
<400> 110
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> 111
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 序列,其不是序列基序的一部分,如果序列基序含有至少3个不相邻的组氨酸残基
<400> 111
Ala Ala Leu Thr His
1 5
<210> 112
<211> 4
<212> PRT
<213> 人工序列
<220>
<223> 第一组氨基酸的序列内成对氨基酸嵌段的实例
<400> 112
Leu Lys Arg Glu
1
<210> 113
<211> 5
<212> PRT
<213> 人工序列
<220>
<223> 第一组氨基酸的序列内三联体氨基酸嵌段的实例
<400> 113
Leu Lys Arg Lys Glu
1 5
<210> 114
<211> 4
<212> PRT
<213> 人工序列
<220>
<223> 第一组氨基酸的N末端三联体嵌段的实例
<400> 114
Lys Arg Lys Glu
1
<210> 115
<211> 4
<212> PRT
<213> 人工序列
<220>
<223> 第一组氨基酸的C末端三联体嵌段的实例
<400> 115
Leu Lys Arg Lys
1
<210> 116
<211> 7
<212> PRT
<213> 人工序列
<220>
<223> 从序列基序排除序列的实例,如果存在第一组氨基酸的三联体
<400> 116
Arg Arg Arg Gly Leu Arg His
1 5
<210> 117
<211> 4
<212> PRT
<213> 人工序列
<220>
<223> 序列中不允许的序列的实例
<400> 117
Lys Arg Lys Lys
1
<210> 118
<211> 4
<212> PRT
<213> 人工序列
<220>
<223> 序列中不允许的序列的实例
<400> 118
Arg Arg Arg Arg
1
<210> 119
<211> 18
<212> PRT
<213> 未知
<220>
<223> SMAP-29绵羊;aa1-18
<400> 119
Arg Gly Leu Arg Arg Leu Gly Arg Lys Ile Ala His Gly Val Lys Lys
1 5 10 15
Tyr Gly
<210> 120
<211> 25
<212> PRT
<213> 人工序列
<220>
<223> 来源于天蚕素(埃及伊蚊)的突变的肽
<400> 120
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> 121
<211> 28
<212> PRT
<213> 人工序列
<220>
<223> 来源于BMAP-28的突变的肽
<400> 121
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> 122
<211> 17
<212> PRT
<213> 人工序列
<220>
<223> 来源于MSI-78 (4-20)肽的突变的肽
<400> 122
Arg Phe Leu Arg Arg Ala Arg Arg Phe Gly Arg Ala Phe Val Arg Ile
1 5 10 15
Leu
<210> 123
<211> 26
<212> PRT
<213> 人工序列
<220>
<223> 来源于magainin的突变的肽
<400> 123
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> 124
<211> 20
<212> PRT
<213> 人工序列
<220>
<223> 来源于HPA-NT3肽的突变的肽
<400> 124
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> 125
<211> 29
<212> PRT
<213> 人工序列
<220>
<223> 来源于肉毒素α亚基的氨基酸298-326的突变的肽
<400> 125
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> 126
<211> 27
<212> PRT
<213> 人工序列
<220>
<223> 来源于CagL蛋白的氨基酸26-48的突变的肽
<400> 126
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> 127
<211> 22
<212> PRT
<213> 人工序列
<220>
<223> 来源于CagL蛋白的氨基酸26-48的突变的肽
<400> 127
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> 128
<211> 19
<212> PRT
<213> 人工序列
<220>
<223> 来源于IE1蛋白的氨基酸178-196的突变的肽
<400> 128
Tyr Lys Arg Ala Phe Lys Lys Val Leu Lys Arg Ile Arg Arg Tyr Ala
1 5 10 15
Lys Arg Ser
<210> 129
<211> 24
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 129
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> 130
<211> 20
<212> PRT
<213> 未知
<220>
<223> CAP18AA
<400> 130
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> 131
<211> 20
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 131
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> 132
<211> 29
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 132
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> 133
<211> 29
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 133
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 His His His His His His
20 25
<210> 134
<211> 29
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 134
Arg Gly Ile Arg Lys Val Leu Lys Phe Ala Lys Arg Leu Phe Arg Lys
1 5 10 15
Ile Gly Arg Lys Pro Lys Gly Leu Ile Arg Val Gly Ala
20 25
<210> 135
<211> 25
<212> PRT
<213> 人工序列
<220>
<223> 合成序列
<400> 135
Gly Arg Leu Phe Lys Arg Leu Ala Lys Lys Val Ala Lys Thr Val Arg
1 5 10 15
Lys Phe Gly Arg Lys Ile Gly Ala Leu
20 25
<210> 136
<211> 29
<212> PRT
<213> 藏羚羊
<400> 136
Arg Gly Leu Arg Arg Leu Gly Arg Lys Ile Leu His Gly Leu Lys Thr
1 5 10 15
Tyr Gly Pro Ile Val Ile Pro Leu Ile Arg Leu Gly Gly
20 25
<210> 137
<211> 4
<212> PRT
<213> 人工的
<220>
<223> 接头
<400> 137
Gly Ala Gly Ala
1
<210> 138
<211> 8
<212> PRT
<213> 人工的
<220>
<223> 接头
<400> 138
Gly Ala Gly Ala Gly Ala Gly Ala
1 5
<210> 139
<211> 12
<212> PRT
<213> 人工的
<220>
<223> 接头
<400> 139
Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala Gly Ala
1 5 10
<210> 140
<211> 195
<212> PRT
<213> 人工序列
<220>
<223> SMAP-29肽和克氏柠檬酸杆菌噬菌体CkP1内溶素的融合物,后者具有C54S突变的另外的技术修饰
<400> 140
Ala Arg Gly Leu Arg Arg Leu Gly Arg Lys Ile Ala His Gly Val Lys
1 5 10 15
Lys Tyr Gly Pro Thr Val Leu Arg Ile Ile Arg Ile Ala Gly Gly Ser
20 25 30
Asn Ile Phe Lys Met Leu Arg Ile Asp Glu Gly Tyr Asp Ser Lys Ile
35 40 45
Tyr Lys Asp Thr Glu Gly Phe Trp Thr Ile Gly Ile Gly His Leu Leu
50 55 60
Thr Arg Asp Pro Ser Leu Glu Val Ala Lys Arg Glu Leu Asp Lys Leu
65 70 75 80
Val Gly Arg Lys Ser Asn Gly Gln Ile Thr Gln Ser Glu Ala Glu Lys
85 90 95
Ile Phe Ala Asp Asp Val Asp Lys Ala Ile Asn Gly Ile Lys Lys Asn
100 105 110
Ala Ser Leu Lys Pro Val Tyr Asp Ser Leu Asp Gly Asp Asp Pro Arg
115 120 125
Gln Ala Ala Leu Ile Asn Met Val Phe Gln Met Gly Val Ala Gly Val
130 135 140
Ala Gly Phe Thr Asn Ser Met Arg Met Val Lys Glu Lys Arg Trp Ala
145 150 155 160
Asp Ala Ala Val Asn Leu Ala Gln Ser Lys Trp Tyr Arg Gln Thr Pro
165 170 175
Asn Arg Ala Lys Arg Val Ile Glu Thr Phe Arg Thr Gly Thr Trp Asn
180 185 190
Ala Tyr Lys
195
<210> 141
<211> 195
<212> PRT
<213> 人工序列
<220>
<223> 包含针对肽组分的优选序列基序的肽与肠细菌噬菌体CC31内溶素的融合物,后者具有C54S突变的另外的技术修饰
<400> 141
Ala Arg Gly Leu Arg Arg Leu Gly Arg Lys Ile Ala Arg Gly Val Lys
1 5 10 15
Lys Tyr Gly Pro Thr Val Leu Arg Ile Ile Arg Ile Ala Gly Gly Ser
20 25 30
Asp Ile Phe Gly Met Leu Arg Ile Asp Glu Gly Tyr Asp Ser Lys Ile
35 40 45
Tyr Lys Asp Thr Glu Gly Phe Trp Thr Ile Gly Ile Gly His Leu Leu
50 55 60
Thr Arg Asp Pro Ser Leu Asp Val Ala Lys Arg Glu Leu Asp Lys Leu
65 70 75 80
Val Gly Arg Pro Ser Asn Gly Gln Ile Thr Lys Ala Glu Ala Glu Ala
85 90 95
Ile Phe Ala Lys Asp Val Asp Lys Ala Thr Arg Gly Ile Leu Gly Asn
100 105 110
Ala Val Leu Lys Pro Val Tyr Asp Val Leu Asp Gly Val Arg Arg Ala
115 120 125
Ala Leu Ile Asn Met Val Phe Gln Met Gly Val Ala Gly Val Ala Ser
130 135 140
Phe Pro Ala Ser Met Arg Leu Leu Lys Ser Lys Gln Trp Glu Ala Ala
145 150 155 160
Ala Lys Glu Leu Ala Asn Ser Lys Trp Tyr Arg Gln Thr Pro Asn Arg
165 170 175
Ala Lys Arg Val Ile Ala Thr Phe Lys Thr Gly Thr Trp Lys Ala Tyr
180 185 190
Glu Asn Leu
195
<210> 142
<211> 195
<212> PRT
<213> 人工序列
<220>
<223> 包含针对肽组分的优选序列基序的肽与克氏柠檬酸杆菌噬菌体CkP1内溶素的融合物,后者具有C54S突变的另外的技术修饰
<400> 142
Met Arg Gly Leu Arg Arg Leu Gly Arg Lys Ile Ala His Gly Val Lys
1 5 10 15
Lys Tyr Gly Pro Thr Val Leu Arg His His His His His His Gly Ser
20 25 30
Asn Ile Phe Lys Met Leu Arg Ile Asp Glu Gly Tyr Asp Ser Lys Ile
35 40 45
Tyr Lys Asp Thr Glu Gly Phe Trp Thr Ile Gly Ile Gly His Leu Leu
50 55 60
Thr Arg Asp Pro Ser Leu Glu Val Ala Lys Arg Glu Leu Asp Lys Leu
65 70 75 80
Val Gly Arg Lys Ser Asn Gly Gln Ile Thr Gln Ser Glu Ala Glu Lys
85 90 95
Ile Phe Ala Asp Asp Val Asp Lys Ala Ile Asn Gly Ile Lys Lys Asn
100 105 110
Ala Ser Leu Lys Pro Val Tyr Asp Ser Leu Asp Gly Asp Asp Pro Arg
115 120 125
Gln Ala Ala Leu Ile Asn Met Val Phe Gln Met Gly Val Ala Gly Val
130 135 140
Ala Gly Phe Thr Asn Ser Met Arg Met Val Lys Glu Lys Arg Trp Ala
145 150 155 160
Asp Ala Ala Val Asn Leu Ala Gln Ser Lys Trp Tyr Arg Gln Thr Pro
165 170 175
Asn Arg Ala Lys Arg Val Ile Glu Thr Phe Arg Thr Gly Thr Trp Asn
180 185 190
Ala Tyr Lys
195
<210> 143
<211> 195
<212> PRT
<213> 人工序列
<220>
<223> 包含针对肽组分优选序列的肽与肠细菌噬菌体CC31内溶素的融合物,后者具有C54S突变的另外的技术修饰
<400> 143
Ala Arg Gly Ile Arg Lys Val Leu Lys Phe Ala Lys Arg Leu Phe Arg
1 5 10 15
Lys Ile Gly Arg Lys Pro Lys Gly Leu Ile Arg Val Gly Ala Gly Ser
20 25 30
Asp Ile Phe Gly Met Leu Arg Ile Asp Glu Gly Tyr Asp Ser Lys Ile
35 40 45
Tyr Lys Asp Thr Glu Gly Phe Trp Thr Ile Gly Ile Gly His Leu Leu
50 55 60
Thr Arg Asp Pro Ser Leu Asp Val Ala Lys Arg Glu Leu Asp Lys Leu
65 70 75 80
Val Gly Arg Pro Ser Asn Gly Gln Ile Thr Lys Ala Glu Ala Glu Ala
85 90 95
Ile Phe Ala Lys Asp Val Asp Lys Ala Thr Arg Gly Ile Leu Gly Asn
100 105 110
Ala Val Leu Lys Pro Val Tyr Asp Val Leu Asp Gly Val Arg Arg Ala
115 120 125
Ala Leu Ile Asn Met Val Phe Gln Met Gly Val Ala Gly Val Ala Ser
130 135 140
Phe Pro Ala Ser Met Arg Leu Leu Lys Ser Lys Gln Trp Glu Ala Ala
145 150 155 160
Ala Lys Glu Leu Ala Asn Ser Lys Trp Tyr Arg Gln Thr Pro Asn Arg
165 170 175
Ala Lys Arg Val Ile Ala Thr Phe Lys Thr Gly Thr Trp Lys Ala Tyr
180 185 190
Glu Asn Leu
195
<210> 144
<211> 193
<212> PRT
<213> 人工序列
<220>
<223> 包含针对肽组分的优选序列的肽与沙雷氏菌噬菌体CHI14内溶素的融合物,后者具有C54S突变的另外的技术修饰
<400> 144
Ala Lys Phe Phe Arg Lys Leu Lys Lys Ser Val Lys Lys Arg Ala Lys
1 5 10 15
Arg Phe Phe Lys Lys Pro Arg Val Ile Gly Val Ser Ile Pro Phe Gly
20 25 30
Ser Asp Ile Phe Gly Met Leu Arg Ile Asp Glu Gly Tyr Asp Ser Lys
35 40 45
Ile Tyr Lys Asp Thr Glu Gly Tyr Trp Thr Ile Gly Ile Gly His Leu
50 55 60
Leu Thr Lys Asn Pro Ser Leu Ser Val Ala Lys Ala Glu Leu Asp Lys
65 70 75 80
Leu Val Gly Arg Ser Ser Asn Gly Gln Ile Thr Gln Asp Glu Ala Glu
85 90 95
Ser Ile Phe Ala Lys Asp Val Glu Lys Ala Val Lys Gly Ile Gln Gly
100 105 110
Asn Ser Val Leu Lys Pro Val Tyr Asp Ser Leu Asp Glu Ile Arg Arg
115 120 125
Ala Ala Leu Ile Asn Met Val Phe Gln Met Gly Val Ala Gly Val Ala
130 135 140
Gly Phe Thr Asn Ser Met Arg Met Leu Lys Glu Lys Arg Trp Asp Glu
145 150 155 160
Ala Ala Val Asn Leu Ala Lys Ser Arg Trp Tyr Asn Gln Thr Thr Asn
165 170 175
Arg Ala Lys Arg Val Ile Ser Thr Phe Lys Thr Gly Thr Trp Gly Ala
180 185 190
Tyr
<210> 145
<211> 197
<212> PRT
<213> 人工序列
<220>
<223> 包含针对肽组分的优选序列基序的肽与气单胞菌噬菌体Ah1的内溶素的融合物,后者具有C122S突变的另外的技术修饰
<400> 145
Ala Arg Gly Leu Arg Arg Leu Gly Arg Lys Ile Ala Arg Gly Val Lys
1 5 10 15
Lys Tyr Gly Pro Thr Val Leu Arg Ile Ile Arg Ile Ala Gly Gly Ser
20 25 30
Ala Leu Ala Gln Met Leu Lys Gln Asp Glu Gly Tyr Lys Glu Thr Val
35 40 45
Tyr Trp Asp Thr Glu Gly Tyr Pro Thr Ile Gly Ile Gly His Leu Ile
50 55 60
Leu Lys Lys Arg Thr Lys Asp Met Gly Glu Ile Asn Arg Glu Leu Ser
65 70 75 80
Ser His Val Gly Arg Val Val Lys Asp Gly Lys Ile Thr Gly Glu Glu
85 90 95
Val Leu Ala Leu Phe Glu Arg Asp Leu Ser Val Leu Lys Arg Ser Ile
100 105 110
Met Ser Leu Pro Asn Leu Ala Asp Val Tyr Val Ser Leu Asp Met Val
115 120 125
Arg Gln Thr Ala Ile Glu Asn Met Val Phe Gln Met Gly Ala Val Gly
130 135 140
Val Ser Lys Phe Pro Gly Met Leu Arg Ser Leu Lys Ala Lys Asp Trp
145 150 155 160
Asp Gly Ala Tyr Arg Asn Ala Leu Asp Ser Ala Trp Ala Arg Gln Thr
165 170 175
Pro Asn Arg Ala Lys Arg Val Ala Ser Val Leu Lys Leu Gly Ser Tyr
180 185 190
Ala Pro Tyr Gly Phe
195
<210> 146
<211> 198
<212> PRT
<213> 人工序列
<220>
<223> 包含根据SEQ ID NO 107的肽与气单胞菌噬菌体Ah1的内溶素的融合物,后者具有C122S突变的另外的技术修饰
<400> 146
Ala Lys Phe Phe Arg Lys Leu Lys Lys Ser Val Lys Lys Arg Ala Lys
1 5 10 15
Arg Phe Phe Lys Lys Pro Arg Val Ile Gly Val Ser Ile Pro Phe Gly
20 25 30
Ser Ala Leu Ala Gln Met Leu Lys Gln Asp Glu Gly Tyr Lys Glu Thr
35 40 45
Val Tyr Trp Asp Thr Glu Gly Tyr Pro Thr Ile Gly Ile Gly His Leu
50 55 60
Ile Leu Lys Lys Arg Thr Lys Asp Met Gly Glu Ile Asn Arg Glu Leu
65 70 75 80
Ser Ser His Val Gly Arg Val Val Lys Asp Gly Lys Ile Thr Gly Glu
85 90 95
Glu Val Leu Ala Leu Phe Glu Arg Asp Leu Ser Val Leu Lys Arg Ser
100 105 110
Ile Met Ser Leu Pro Asn Leu Ala Asp Val Tyr Val Ser Leu Asp Met
115 120 125
Val Arg Gln Thr Ala Ile Glu Asn Met Val Phe Gln Met Gly Ala Val
130 135 140
Gly Val Ser Lys Phe Pro Gly Met Leu Arg Ser Leu Lys Ala Lys Asp
145 150 155 160
Trp Asp Gly Ala Tyr Arg Asn Ala Leu Asp Ser Ala Trp Ala Arg Gln
165 170 175
Thr Pro Asn Arg Ala Lys Arg Val Ala Ser Val Leu Lys Leu Gly Ser
180 185 190
Tyr Ala Pro Tyr Gly Phe
195
<210> 147
<211> 194
<212> PRT
<213> 人工序列
<220>
<223> 包含针对肽组分的优选序列基序的肽和沙雷氏菌噬菌体PS2内溶素的融合物
<400> 147
Ala Arg Gly Leu Arg Arg Leu Gly Arg Lys Ile Ala Arg Gly Val Lys
1 5 10 15
Lys Tyr Gly Pro Thr Val Leu Arg Ile Ile Arg Ile Ala Gly Gly Ser
20 25 30
Ala Thr Ile Phe Glu Met Leu Ala Phe Asp Glu Gly Leu Lys Leu Thr
35 40 45
Val Tyr Leu Asp Thr Glu Gly Phe Trp Thr Val Gly Ile Gly His Leu
50 55 60
Leu Thr Lys Asn Pro Ser Lys Ala Val Ala Ile Ala Glu Leu Asp Lys
65 70 75 80
Leu Val Gly Arg Ser Thr Gly Gly Thr Ile Thr Lys Ala Glu Ala Glu
85 90 95
Arg Ile Phe Ala Gln Asp Val Ala Lys Ser Glu Lys Gly Ile Gln Gly
100 105 110
Asn Ala Val Leu Gly Pro Val Tyr Ala Gly Leu Asp Ala Thr Arg Lys
115 120 125
Met Ala Leu Val Asn Met Thr Phe Gln Leu Gly Val Ala Gly Ala Ala
130 135 140
Gly Phe Thr Asn Ser Met Lys Leu Leu Ala Ala Lys Gln Trp Lys Glu
145 150 155 160
Ala Ala Ile Asn Leu Ala Lys Ser Lys Trp Tyr Asn Gln Thr Pro Asn
165 170 175
Arg Ala Lys Arg Val Ile Ser Val Phe Glu Thr Gly Thr Leu Ala Ala
180 185 190
Tyr Lys
<210> 148
<211> 195
<212> PRT
<213> 人工序列
<220>
<223> 肽Cathelecidin-BF和沙雷氏菌噬菌体PS2的融合物
<400> 148
Ala Lys Phe Phe Arg Lys Leu Lys Lys Ser Val Lys Lys Arg Ala Lys
1 5 10 15
Glu Phe Phe Lys Lys Pro Arg Val Ile Gly Val Ser Ile Pro Phe Gly
20 25 30
Ser Ala Thr Ile Phe Glu Met Leu Ala Phe Asp Glu Gly Leu Lys Leu
35 40 45
Thr Val Tyr Leu Asp Thr Glu Gly Phe Trp Thr Val Gly Ile Gly His
50 55 60
Leu Leu Thr Lys Asn Pro Ser Lys Ala Val Ala Ile Ala Glu Leu Asp
65 70 75 80
Lys Leu Val Gly Arg Ser Thr Gly Gly Thr Ile Thr Lys Ala Glu Ala
85 90 95
Glu Arg Ile Phe Ala Gln Asp Val Ala Lys Ser Glu Lys Gly Ile Gln
100 105 110
Gly Asn Ala Val Leu Gly Pro Val Tyr Ala Gly Leu Asp Ala Thr Arg
115 120 125
Lys Met Ala Leu Val Asn Met Thr Phe Gln Leu Gly Val Ala Gly Ala
130 135 140
Ala Gly Phe Thr Asn Ser Met Lys Leu Leu Ala Ala Lys Gln Trp Lys
145 150 155 160
Glu Ala Ala Ile Asn Leu Ala Lys Ser Lys Trp Tyr Asn Gln Thr Pro
165 170 175
Asn Arg Ala Lys Arg Val Ile Ser Val Phe Glu Thr Gly Thr Leu Ala
180 185 190
Ala Tyr Lys
195
<210> 149
<211> 190
<212> PRT
<213> 人工序列
<220>
<223> 包含针对肽组分的优选序列基序的肽与克氏柠檬酸杆菌噬菌体CkP1的内溶素的融合物,后者具有C54S突变的另外的技术修饰
<400> 149
Gly Arg Leu Phe Lys Arg Leu Ala Lys Lys Val Ala Lys Thr Val Arg
1 5 10 15
Lys Phe Gly Arg Lys Ile Gly Ala Leu Gly Ser Asn Ile Phe Lys Met
20 25 30
Leu Arg Ile Asp Glu Gly Tyr Asp Ser Lys Ile Tyr Lys Asp Thr Glu
35 40 45
Gly Phe Trp Thr Ile Gly Ile Gly His Leu Leu Thr Arg Asp Pro Ser
50 55 60
Leu Glu Val Ala Lys Arg Glu Leu Asp Lys Leu Val Gly Arg Lys Ser
65 70 75 80
Asn Gly Gln Ile Thr Gln Ser Glu Ala Glu Lys Ile Phe Ala Asp Asp
85 90 95
Val Asp Lys Ala Ile Asn Gly Ile Lys Lys Asn Ala Ser Leu Lys Pro
100 105 110
Val Tyr Asp Ser Leu Asp Gly Asp Asp Pro Arg Gln Ala Ala Leu Ile
115 120 125
Asn Met Val Phe Gln Met Gly Val Ala Gly Val Ala Gly Phe Thr Asn
130 135 140
Ser Met Arg Met Val Lys Glu Lys Arg Trp Ala Asp Ala Ala Val Asn
145 150 155 160
Leu Ala Gln Ser Lys Trp Tyr Arg Gln Thr Pro Asn Arg Ala Lys Arg
165 170 175
Val Ile Glu Thr Phe Arg Thr Gly Thr Trp Asn Ala Tyr Lys
180 185 190
<210> 150
<211> 195
<212> PRT
<213> 人工序列
<220>
<223> 包含针对肽组分的优选序列基序的肽与克氏柠檬酸杆菌噬菌体CkP1的内溶素的融合物,后者具有C54S突变的另外的技术修饰
<400> 150
Ala Arg Gly Leu Arg Arg Leu Gly Arg Lys Ile Leu His Gly Leu Lys
1 5 10 15
Thr Tyr Gly Pro Ile Val Ile Pro Leu Ile Arg Leu Gly Gly Gly Ser
20 25 30
Asn Ile Phe Lys Met Leu Arg Ile Asp Glu Gly Tyr Asp Ser Lys Ile
35 40 45
Tyr Lys Asp Thr Glu Gly Phe Trp Thr Ile Gly Ile Gly His Leu Leu
50 55 60
Thr Arg Asp Pro Ser Leu Glu Val Ala Lys Arg Glu Leu Asp Lys Leu
65 70 75 80
Val Gly Arg Lys Ser Asn Gly Gln Ile Thr Gln Ser Glu Ala Glu Lys
85 90 95
Ile Phe Ala Asp Asp Val Asp Lys Ala Ile Asn Gly Ile Lys Lys Asn
100 105 110
Ala Ser Leu Lys Pro Val Tyr Asp Ser Leu Asp Gly Asp Asp Pro Arg
115 120 125
Gln Ala Ala Leu Ile Asn Met Val Phe Gln Met Gly Val Ala Gly Val
130 135 140
Ala Gly Phe Thr Asn Ser Met Arg Met Val Lys Glu Lys Arg Trp Ala
145 150 155 160
Asp Ala Ala Val Asn Leu Ala Gln Ser Lys Trp Tyr Arg Gln Thr Pro
165 170 175
Asn Arg Ala Lys Arg Val Ile Glu Thr Phe Arg Thr Gly Thr Trp Asn
180 185 190
Ala Tyr Lys
195
<210> 151
<211> 198
<212> PRT
<213> 人工序列
<220>
<223> 包含针对肽组分的优选序列基序的肽与气单胞菌噬菌体
As-szw内溶素的融合物
<400> 151
Ala Arg Gly Leu Arg Arg Leu Gly Arg Lys Ile Ala Arg Gly Val Lys
1 5 10 15
Lys Tyr Gly Pro Thr Val Leu Arg Ile Ile Arg Ile Ala Gly Gly Ser
20 25 30
Ala Leu Glu Lys Met Leu Lys Phe Asp Glu Gly Ser Lys Leu Ser Val
35 40 45
Tyr Trp Asp Thr Glu Gly Tyr Pro Thr Ile Gly Ile Gly His Leu Ile
50 55 60
Lys Arg Leu Arg Thr Lys Asp Met Gly Glu Ile Asn Arg Glu Leu Ser
65 70 75 80
Ser His Val Gly Arg Val Ile Thr Asp Gly Lys Ile Thr Gln Ser Glu
85 90 95
Glu Ser Gln Leu Phe Ala Lys Asp Leu Glu Val Val Arg Asn Ser Met
100 105 110
Lys Gly Tyr Val Asp Leu Trp Ser Thr Tyr Val Gly Leu Asp Glu Val
115 120 125
Arg Lys Thr Ala Leu Glu Asn Met Val Phe Gln Met Gly Ala Lys Gly
130 135 140
Val Asn Gly Phe Pro Ser Met Leu Arg Ala Met Arg Ser Lys Asn Trp
145 150 155 160
Val Glu Ala Lys Lys His Gly Leu Ala Ser Ala Trp Ser Arg Gln Thr
165 170 175
Pro Asn Arg Ala Lys Arg Val Thr Asp Val Ile Glu Thr Gly Thr Tyr
180 185 190
Lys Gly Tyr Pro Phe Ala
195
<210> 152
<211> 192
<212> PRT
<213> 人工序列
<220>
<223> 包含针对肽组分的优选序列基序的肽与沙雷氏菌噬菌体CHI14内溶素的融合物,后者具有C54S突变的另外的技术修饰
<400> 152
Ala Arg Gly Leu Arg Arg Leu Gly Arg Lys Ile Ala Arg Gly Val Lys
1 5 10 15
Lys Tyr Gly Pro Thr Val Leu Arg Ile Ile Arg Ile Ala Gly Gly Ser
20 25 30
Asp Ile Phe Gly Met Leu Arg Ile Asp Glu Gly Tyr Asp Ser Lys Ile
35 40 45
Tyr Lys Asp Thr Glu Gly Tyr Trp Thr Ile Gly Ile Gly His Leu Leu
50 55 60
Thr Lys Asn Pro Ser Leu Ser Val Ala Lys Ala Glu Leu Asp Lys Leu
65 70 75 80
Val Gly Arg Ser Ser Asn Gly Gln Ile Thr Gln Asp Glu Ala Glu Ser
85 90 95
Ile Phe Ala Lys Asp Val Glu Lys Ala Val Lys Gly Ile Gln Gly Asn
100 105 110
Ser Val Leu Lys Pro Val Tyr Asp Ser Leu Asp Glu Ile Arg Arg Ala
115 120 125
Ala Leu Ile Asn Met Val Phe Gln Met Gly Val Ala Gly Val Ala Gly
130 135 140
Phe Thr Asn Ser Met Arg Met Leu Lys Glu Lys Arg Trp Asp Glu Ala
145 150 155 160
Ala Val Asn Leu Ala Lys Ser Arg Trp Tyr Asn Gln Thr Thr Asn Arg
165 170 175
Ala Lys Arg Val Ile Ser Thr Phe Lys Thr Gly Thr Trp Gly Ala Tyr
180 185 190
<210> 153
<211> 6
<212> PRT
<213> 人工序列
<220>
<223> His-Tag (6x)
<400> 153
His His His His His His
1 5
<210> 154
<211> 196
<212> PRT
<213> 人工序列
<220>
<223> 天蚕素(埃及伊文)肽与弧菌噬菌体VvAW1(YP_007518361.1)的融合物
<400> 154
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 Gly Gly Ser Gly Gly Gly Ser Gly Ser Met
35 40 45
Gly Phe Lys Phe Ser Glu Arg Ser Lys Ser Arg Met Ala Gly Val His
50 55 60
Pro Glu Leu Val Leu Val Phe His Glu Ala Leu Ala Val Ser Pro Ile
65 70 75 80
Asp Phe Gly Ile Pro Glu His Gly Gly Leu Arg Ser Ala Glu Glu Gln
85 90 95
Tyr Ser Leu Phe Leu Asp Asn Lys Ser Lys Ala Asp Gly Tyr Asn Lys
100 105 110
Leu Ser Asn His Gln Ser Gly Asn Ala Leu Asp Phe Tyr Ala Tyr Leu
115 120 125
Asn Gly Ala Ala Ser Trp Asp Lys Val His Leu Ala Met Val Ala Ala
130 135 140
Thr Ile Leu Ser Thr Ala Ala Arg Leu Lys Glu Gln Gly Lys Ile Ser
145 150 155 160
Ile Ser Ile Arg Trp Gly Gly Thr Phe Gly Asn Lys Gly Arg Ser Phe
165 170 175
His Gly Trp Asp Tyr Pro His Met Glu Val Ile Ser Leu Glu His His
180 185 190
His His His His
195
<210> 155
<211> 295
<212> PRT
<213> 人工序列
<220>
<223> 天蚕素(埃及伊文)肽与模块化KZ144内溶素和lys68内溶素的突变的细胞壁结合结构域的融合物
<400> 155
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 Lys
35 40 45
Val Leu Arg Lys Gly Asp Arg Gly Asp Glu Val Ser Gln Leu Gln Thr
50 55 60
Leu Leu Asn Leu Ser Gly Tyr Asp Val Gly Lys Pro Asp Gly Ile Phe
65 70 75 80
Gly Asn Asn Thr Phe Asn Gln Val Val Lys Phe Gln Lys Asp Asn Ser
85 90 95
Leu Asp Ser Asp Gly Ile Val Gly Lys Asn Thr Trp Ala Glu Leu Phe
100 105 110
Ser Lys Tyr Ser Pro Pro Ile Pro Tyr Lys Thr Ile Pro Met Ser Asn
115 120 125
Arg Asn Ile Ser Asp Asn Gly Ile Lys Phe Thr Ala Ala Phe Glu Gly
130 135 140
Phe Arg Gly Thr Ala Tyr Arg Ala Thr Lys Asn Glu Lys Tyr Leu Thr
145 150 155 160
Ile Gly Tyr Gly His Tyr Gly Ala Asp Val Lys Glu Gly Gln Lys Ile
165 170 175
Thr Glu Gly Gln Gly Leu Leu Leu Leu His Lys Asp Met Val Lys Ala
180 185 190
Val Ala Ala Val Asp Ala Val Ala His Pro Pro Leu Asn Gln Ser Gln
195 200 205
Phe Asp Ala Met Cys Asp Leu Val Tyr Asn Ala Gly Val Gly Val Ile
210 215 220
Ala Ala Ser Thr Gly Thr Gly Gln Ala Leu Arg Lys Gly Asp Val Ala
225 230 235 240
Thr Leu Arg Asn Lys Leu Thr Gln Phe His Tyr Gln Asn Gly Lys Ser
245 250 255
Leu Leu Gly Leu Arg Arg Arg Ala Ala Gly Arg Val Ala Leu Phe Asp
260 265 270
Gly Met Leu Trp Gln Gln Ala Glu Ala Ile Gly Arg Gly Ala Lys Leu
275 280 285
Glu His His His His His His
290 295
<210> 156
<211> 223
<212> PRT
<213> 人工序列
<220>
<223> 经修饰的肽(SEQ ID NO:110)与假单胞菌噬菌体 vB_PsyM_KIL1内溶素的融合物
<400> 156
Met Gly Leu Arg Lys Ala Leu Arg Lys Phe Arg Lys Lys Ile Lys Glu
1 5 10 15
Ala Leu Lys Lys Ile Gly Gly Gly Gly Ser Gly Ser Met Leu Ser Glu
20 25 30
Lys Ser Phe Val Glu Ala Ala Ala Ser Leu Gly Cys Glu Val Ala Ala
35 40 45
Ile Lys Ala Ile Ala Ser Val Glu Thr Lys Gly Ser Ala Trp Ile Thr
50 55 60
Pro Gly Val Pro Gln Ile Leu Tyr Glu Arg His Ile Met Ala Arg Leu
65 70 75 80
Leu Lys Ala Lys Gly Val Pro Ile Ala Gly Leu Pro Ser Asp Leu Val
85 90 95
Asn Thr Thr Pro Gly Gly Tyr Gly Lys Phe Ser Glu Gln His Gly Lys
100 105 110
Leu Asp Arg Ala Val Lys Ile Asp Arg Glu Cys Ala Leu Gln Ser Cys
115 120 125
Ser Trp Gly Met Phe Gln Leu Met Gly Phe Asn Tyr Lys Leu Cys Gly
130 135 140
Tyr Ala Thr Val Gln Ala Phe Val Asn Ala Met Tyr Lys Ser Glu Asp
145 150 155 160
Glu Gln Leu Asn Ala Phe Val Gly Phe Ile Lys Ser Asn Leu Gln Leu
165 170 175
Asn Asp Ala Leu Lys Ser Lys Asp Trp Ala Thr Val Ala Arg Leu Tyr
180 185 190
Asn Gly Ala Asp Tyr Lys Ile Asn Ser Tyr Asp Gln Lys Leu Ala Val
195 200 205
Ala Tyr Glu Ser Asn Lys Arg Leu Glu His His His His His His
210 215 220
Claims (16)
1.一种多肽,其包含革兰氏阴性内溶素和选自以下的肽:抗微生物肽、两亲性肽、阳离子肽、寿司肽或防御素,
其中所述内溶素又是包含根据SEQ ID NO:2的序列的内溶素,
条件是:
a)所述多肽既不包含根据SEQ ID NO:3的序列,也不包含根据SEQ ID NO:4的序列,也不包含根据SEQ ID NO:5的序列,
b)所述内溶素不是肠杆菌噬菌体JS98的EpJS98_gp116,
c)如果所述多肽包含SEQ ID NO:6所示的序列,则肽选自以下:抗微生物肽、两亲性肽、寿司肽或防御素,
d)如果所述内溶素具有选自以下的序列:
和另外仅缺少N-末端甲硫氨酸的相应序列,则所述多肽不包含i)模块化革兰氏阴性内溶素或ii)噬菌体尾/基板蛋白的细胞壁结合结构域,
e)如果所述内溶素具有根据以下的氨基酸2-164的序列,则所述多肽不包含i)模块革兰氏阴性内溶素或ii)噬菌体尾/基板蛋白的细胞壁结合结构域:
f)如果所述内溶素具有根据以下的氨基酸2-161的序列,则所述多肽不包含i)模块革兰氏阴性内溶素或ii)噬菌体尾/基板蛋白的细胞壁结合结构域:
。
2.根据权利要求1所述的多肽,其中所述内溶素是包含根据SEQ ID NO:7的序列的内溶素。
3.根据权利要求1或权利要求2所述的多肽,其中所述内溶素选自以下:SEQ ID NO:6、SEQ ID NO:28、SEQ ID NO:29、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:34、SEQ ID NO:35、SEQ ID NO:36以及与SEQ ID NO:6、SEQ ID NO:28、SEQID NO:29、SEQ ID NO:30、SEQ ID NO:31、SEQ ID NO:32、SEQ ID NO:33、SEQ ID NO:34、SEQID NO:35和/或SEQ ID NO:36具有至少80%序列同一性的序列。
4.根据权利要求2或权利要求3所述的多肽,其中所述内溶素包含选自以下的序列:SEQID NO:8、SEQ ID NO:9、SEQ ID NO:10和SEQ ID NO:11。
5.根据前述权利要求中任一项所述的多肽,其中所述肽是抗微生物肽或两亲性肽。
6.根据前述权利要求中任一项所述的多肽,其中所述肽包含选自以下的序列:KRK和SEQ ID NO:37-136,特别地其中所述肽包含根据SEQ ID NO:63或根据SEQ ID NO:132的序列。
7.根据前述权利要求中任一项所述的多肽,其中所述多肽包含SEQ ID NO:140或SEQID NO:141所示的氨基酸序列,或者与SEQ ID NO:140和/或SEQ ID NO:141具有至少80%序列同一性的序列。
8.一种多肽,其包含选自以下的肽的序列:SEQ ID NO:107、SEQ ID NO:133、SEQ IDNO:134、SEQ ID NO:135和SEQ ID NO:136,以及胞壁质水解酶的序列。
9.根据前述权利要求中任一项所述的多肽,其中所述多肽降解至少一种革兰氏阴性细菌物种的肽聚糖,特别是其中所述多肽降解大肠杆菌细菌和/或铜绿假单胞菌细菌的肽聚糖。
10.根据权利要求9所述的多肽,其中在不存在其他外膜透化物质的情况下所述多肽降解至少一种革兰氏阴性细菌物种的肽聚糖,特别是其中在不存在外膜透化物质的情况下所述多肽降解大肠杆菌细菌和/或铜绿假单胞菌细菌的肽聚糖。
11.根据权利要求9所述的多肽,其中在不存在外膜透化物质的情况下所述多肽对大肠杆菌菌株RKI 06-08410的最小抑制浓度(MIC)为20μg/ml或更低。
12.编码权利要求1至11中任一项所述的多肽的核酸。
13.包含根据权利要求12所述的核酸的载体。
14.宿主细胞,其包含根据权利要求1至11中任一项所述的多肽,根据权利要求12所述的核酸,和/或根据权利要求13所述的载体。
15.根据权利要求1至11中任一项所述的多肽,用于通过手术或疗法治疗人体或动物体的方法中的用途或者用于在人体或动物体上实践的诊断方法中的用途,其中在不添加其他外膜透化物质的情况下施用所述多肽。
16.根据权利要求1至11中任一项所述的多肽作为非治疗性消毒剂的用途,其中在不添加其他外膜透化物质的情况下施用所述多肽。
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CN113801864A (zh) * | 2021-08-13 | 2021-12-17 | 青岛农业大学 | 一种用于编码溶酶菌lysin6的基因及其应用 |
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