CN109963578A - 针对金黄色葡萄球菌的新抗微生物剂 - Google Patents

针对金黄色葡萄球菌的新抗微生物剂 Download PDF

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CN109963578A
CN109963578A CN201780071621.0A CN201780071621A CN109963578A CN 109963578 A CN109963578 A CN 109963578A CN 201780071621 A CN201780071621 A CN 201780071621A CN 109963578 A CN109963578 A CN 109963578A
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M.比布尔
M.格列斯尔
K.纽曼
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Lysando AG
Lysando Holding AG
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Abstract

本发明涉及对金黄色葡萄球菌细菌有活性的抗微生物剂领域。特别地，本发明涉及包含LysK内溶素的CHAP域、溶葡萄球菌酶的M23内肽酶域、ALE‑1的细胞壁结合域(CBD)和选自下组的进一步的肽的多肽：抗微生物肽、两亲性肽、阳离子肽、疏水性肽、寿司肽和防御素。此外，本发明涉及编码此类多肽的核酸、包含此类核酸的载体、以及相应的宿主细胞、组合物和装置。最后，本发明涉及本发明多肽的应用，特别是在制药领域中的应用。

Description

针对金黄色葡萄球菌的新抗微生物剂
本发明涉及对金黄色葡萄球菌细菌有活性的抗微生物剂领域。特别地，本发明涉及包含LysK内溶素的CHAP域、溶葡萄球菌酶的M23内肽酶域、ALE-1的细胞壁结合域(CBD)和选自下组的进一步的肽的多肽：抗微生物肽、两亲性肽、阳离子肽、疏水性肽、寿司肽和防御素。此外，本发明涉及编码此类多肽的核酸、包含此类核酸的载体、以及相应的宿主细胞、组合物和装置。最后，本发明涉及本发明多肽的应用，特别是在制药领域中的应用。
细菌病原体对人类健康构成重大威胁。尽管本领域已知各种类型的具有杀细菌或抑细菌活性的药剂(例如抗生素)，但对这些药剂，特别是抗生素的微生物抗性正在稳步增加。代表健康问题的病原体之一是金黄色葡萄球菌(Stafylococcus aureus)细菌。金黄色葡萄球菌是菌血症，感染性心内膜炎以及骨和关节感染的最常见原因之一。此外，它可以导致皮肤和软组织感染以及食物中毒。金黄色葡萄球菌迅速产生对抗生素的抗性，如多药物抗性金黄色葡萄球菌(MDR)、甲氧西林抗性金黄色葡萄球菌(MRSA)和对万古霉素的易感性降低(万古霉素中间体菌株)所证明。由于抗性增加降低常规抗生素的效用，一直需要新的抗微生物剂来控制金黄色葡萄球菌的数量，例如在医源(医院)环境中。
对金黄色葡萄球菌细菌具有高活性的药剂是溶葡萄球菌酶。溶葡萄球菌酶是一种肽聚糖水解酶，其能够降解金黄色葡萄球菌细菌的细胞壁肽聚糖。它是由模仿葡萄球菌溶葡萄球菌生物变种(Staphylococcus simulans biovar staphylolyticus)产生的。溶葡萄球菌酶具有两个域：N端催化性M23内肽酶域和C端细胞壁结合域(CBD)。然而，金黄色葡萄球菌也迅速产生对溶葡萄球菌酶的抗性。
最近，Becker et al.(Sci Rep.2016 Apr 28；6:25063；通过引用并入本文)报道了融合蛋白的产生，其中组合了来自溶葡萄球菌肽和LysK内溶素的抗微生物活性。得到的融合蛋白显著降低抗性菌株形成的发生。
然而，仍然不断需要对金黄色葡萄球菌细菌有活性的新抗细菌剂。优选地，所述药剂显示降低的抗性菌株形成的发生，而平行地表现出高的抗细菌活性，如低的最小抑制浓度(MIC)反映的。
因此，本发明要解决的问题是提供针对金黄色葡萄球菌细菌的新抗细菌剂，其显示出抗性菌株形成的降低的发生，而平行地表现出高的抗细菌活性。
此目的通过权利要求书中定义的和下面阐述的主题来解决。
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盐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，从而产生缀合的多肽，其也包括在如本文所用的术语“多肽”中。如本文使用的该术语还意图包括蛋白质。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，所述多肽表现出本领域通常使用的任选修饰，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。从下面的描述中可明显看出，根据本发明的多肽是非天然存在的多肽。如本文所用，术语“多肽”不限于氨基酸聚合物链的特定长度，但通常多肽将表现出超过250个氨基酸的长度。通常但非必要地，本发明的典型多肽的长度不超过约750个氨基酸，优选长度不超过约450个氨基酸。
如本文所用，术语“变体序列”是指与相应的参考序列相比表现出一种或多种添加，缺失，插入和/或取代及其组合的氨基酸序列。这包括例如缺失/插入，插入/缺失，缺失/添加，添加/缺失，插入/添加，添加/插入等的组合。然而，本领域技术人员将理解与参考序列中相应相同位置处存在的氨基酸残基不同的变体序列的某些位置处氨基酸残基的存在不是例如缺失和随后在相同位置处插入的组合，而是如本文所定义的取代。相反，如果在本文中提及添加、缺失、插入和取代中的一个或多个的组合，则序列中不同位置处的变化的组合是意图的，例如，N末端的添加和序列内缺失。此类衍生序列将与相应的参考序列，例如给定的SEQ ID NO表现出一定水平的序列同一性，其优选至少80％，至少85％，至少90％，至少95％，至少96％，至少97％，至少98％或至少99％。优选的变体序列是亲本分子，例如给定的SEQ ID NO的片段，保留了亲本分子的活性，即在一般水平上表现出与相应亲本分子相同的活性。然而，所述活性可以与相应的亲本分子相同，更高或更低。还优选的变体序列是由亲本序列，例如给定的SEQ ID NO内的保守氨基酸取代产生，再次在一般水平上保留亲本分子活性的那些。
如本文所用，术语“％序列同一性”必须理解如下：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(所谓的局部长度)(其更适合于不同长度的序列)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替换或缺失主题序列中多达5％(100个中的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公知的。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 et al.(1993),PNAS USA,90:5873-5877的算法。此类算法集成在BLAST系列程序中，例如，BLAST或NBLAST程序(还参见Altschul etal.,1990,J.Mol.Biol.215,403-410或Altschul et al.(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 and Lipman(1988),Proc.Natl.Acad.Sci.U.S.A85,2444-2448)。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 Sequence Analysis Package,第9.1版(Devereux etal,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 and 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。如果在本文中提及与参考序列共享特定程度的序列同一性的氨基酸序列，则所述序列差异优选由于保守氨基酸取代所致。优选地，此类序列保留参考序列的活性，例如，尽管可能速度较慢。另外，如果在本文中提及在特定百分比的序列同一性下共享“至少”的序列，则优选不包括100％序列同一性。
如本文所用，术语“进一步的肽”是指本发明多肽的氨基酸序列内的氨基酸子序列。所述序列可以是阳离子肽、聚阳离子肽、两亲性肽、疏水性肽、寿司肽和/或抗微生物肽的序列。该术语不涉及传统标签，如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(cystein)标签、FLAG标签或本领域已知的其他标签、硫氧还蛋白或麦芽糖结合蛋白(MBP)。优选地，进一步的肽的序列具有至少约3至至多约50，优选至多约39个氨基酸残基的长度。进一步的肽序列本身不提供任何以下酶活性：内肽酶、壳多糖酶、T4如胞壁质酶、lambda样胞壁质酶、N-乙酰基-胞壁酰-L-丙氨酸-酰胺酶(酰胺酶)、胞壁酰-L-丙氨酸-酰胺酶、胞壁质酶、裂解性转糖基酶(lytictransglycosylase)(C)、裂解性转糖基酶(M)、N-乙酰基-胞壁质酶(溶菌酶)、N-乙酰基-氨基葡萄糖苷酶或转糖基酶。通常，进一步的肽序列根本不提供任何酶活性。
如本文所用，术语“阳离子肽”指具有带正电荷的氨基酸残基的肽。优选地，阳离子肽的pKa值为9.0或更高。通常，阳离子肽的至少四个氨基酸残基可带正电荷，例如赖氨酸或精氨酸。“带正电荷”指在约生理条件下具有净正电荷的氨基酸残基的侧链。如本文所用，术语“阳离子肽”还指聚阳离子肽，但也包括其中包含例如小于20％、优选小于10％的带正电荷的氨基酸残基的阳离子肽。
如本文所用，术语“聚阳离子肽”指主要由带正电荷的氨基酸残基组成的肽，特别是赖氨酸和/或精氨酸残基。如果至少约20、30、40、50、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，特别是赖氨酸和/或精氨酸残基，则肽为主要由带正电荷的氨基酸残基组成。作为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，作为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“抗微生物肽”(AMP)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天然存在的肽。因此，如本文所用的术语“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、抗传染、抗感染和/或杀菌、杀藻、杀阿米巴、杀微生物、杀细菌、杀真菌、杀寄生物、杀原生动物、杀原生生物特性的任何肽。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、导管素(cathelicidin)、颗粒溶素(granulysin)、富组蛋白(histatin)、牛皮癣素(psoriasin)、表皮素(dermicidine)或肝杀菌肽(hepcidin)。抗微生物肽可以天然存在于昆虫、鱼、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小萝卜、蚕蛾、狼蛛、蛙中，优选在非洲爪蟾(Xenopuslaevis)、林蛙(Rana frogs)中，更优选在牛蛙(Rana catesbeiana)、蟾蜍、优选亚洲蟾蜍中华大蟾蜍(Bufobufogargarizans)中，蝇，优选在果蝇(Drosophila)中，更优选在黑腹果蝇(Drosophila melanogaster)中，在埃及伊蚊(Aedesa egypti)中，在蜜蜂、大黄蜂(bumblebee)，优选在熊蜂(Bombus pascuorum)中，麻蝇，优选在棕尾别麻蝇(Sarcophaga peregrine)中，蝎子、马蹄蟹、鲶鱼(catfish)，优选在鲶鱼(Parasilurus asotus)中，牛、猪、绵羊、猪、牛、猴和人。如本文所用，“抗微生物肽”(AMP)可以特别地是并非阳离子肽、聚阳离子肽、两亲性肽、寿司肽、防御素和疏水肽，但仍然表现出抗微生物活性的肽。
如本文所用，术语“寿司肽”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到蛋白质-蛋白质相互作用域的作用。含有寿司域的肽已经显示具有抗微生物活性。优选地，寿司肽是天然存在的肽。
如本文所用，术语“防御素”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作用，破坏外来物质例如传染性细菌和/或传染性病毒和/或真菌。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、indolicidin和magainin。如本文所用，术语“防御素”指来自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“两亲性肽”是指具有亲水和疏水官能团的肽。优选地，如本文所用的术语“两亲性肽”是指具有限定的亲水和疏水基团排列的肽，例如，两亲性肽可以是例如α螺旋，沿螺旋的一侧主要具有非极性侧链，沿其表面的其余部分具有极性残基。
如本文所用，术语“疏水基团”优选指基本上不溶于水，但可溶于油相的化学基团，例如氨基酸侧链，在油相中的溶解度高于在水中或在水相中的溶解度。在水中，具有疏水侧链的氨基酸残基彼此相互作用以产生非水环境。具有疏水侧链的氨基酸残基的实例是缬氨酸，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，酪氨酸和脯氨酸残基。
如本文所用，术语“疏水性肽”是指疏水性肽，其优选主要由具有疏水基团的氨基酸残基组成。此类肽优选由通常疏水性的氨基酸残基组成，即至少约20,30,40,50,60,70,75,80,85,90,95或至少约100％的氨基酸残基是疏水性的氨基酸残基。非疏水性的氨基酸残基优选是中性的，优选不是亲水性的。
如本文所用，术语“标签”是指氨基酸序列，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个氨基酸序列中，用于a)改善总氨基酸序列或多肽的表达，b)促进纯化总氨基酸序列或多肽，c)促进总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。标签的例子是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(cystein)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加标签的多肽适用于例如不同的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“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”意味着可选地可以存在另外的物质。术语“包含”包括特别设想的实施方案，其落入其范围“由......组成”(即排除存在另外的其他物质)和“包括但不由......组成”(即，需要存在另外的其他物质)，前者更优选。
令人惊讶地，本发明的发明人发现了新的多肽试剂，它们表现出高的抗细菌活性，并且平行地表现出降低的抗性菌株形成的发生。在这两方面，本发明的多肽代表Becker etal.(Sci Rep.2016 Apr 28；6:25063)的最佳融合蛋白L-K构建体的改进。
因此，本发明的第一方面涉及多肽，其包含：
i)LysK内溶素的CHAP域(或其与LysK内溶素的CHAP域表现出至少80％的序列同一性的变体序列)；
ii)溶葡萄球菌酶的M23内肽酶域(或其与溶葡萄球菌酶的M23内肽酶域表现出至少80％的序列同一性的变体序列)；
iii)ALE-1的细胞壁结合域(CBD)(或其与ALE-1的细胞壁结合域(CBD)表现出至少90％序列同一性的变体)，和
iv)选自抗微生物肽，两亲性肽，阳离子肽，疏水性肽，寿司肽和防御素的进一步的肽。
本发明的多肽包含至少四个序列元件。第一个元件是LysK内溶素的CHAP域(半胱氨酸-组氨酸依赖性酰胺水解酶/肽酶域)(参见SEQ ID NO:1)或其变体序列。除LysK的CHAP域外，本发明的多肽也可以包含LysK的较长序列元件和相应的变体序列。例如，多肽可以包含SEQ ID NO:2的序列，其反映除N末端甲硫氨酸外的LysK内溶素的N端部分(包括CHAP域)。第二个元件是溶葡萄球菌酶的M23内肽酶域(参见SEQ ID NO:3)或其变体序列。该域是细菌素溶葡萄球菌酶的催化域。除了溶葡萄球菌酶的M23内肽酶域外，本发明的多肽也可以包含较长的序列元件(和相应的变体序列)，例如SEQ ID NO:4的序列，其反映溶葡萄球菌酶的N端部分(氨基酸4-152；包括M23内肽酶域)。第三个元件是ALE-1内溶素的细胞壁结合域(CBD)(参见SEQ ID NO:5)或其变体序列。这是一个非催化域。除了ALE-1内溶素的CBD之外，本发明的多肽也可以包含ALE-1内溶素的较长序列元件(和相应的变体序列)，例如SEQ IDNO:6的序列，其反映ALE-1内溶素的C端部分(氨基酸234-327；包括CBD域)。两个催化域(CHAP，M23)和CBD域(ALE-1的CBD域)原则上可以以任何顺序发生。然而，优选地，不同元件(无论它们是天然存在的还是变体序列)如下排列(从N末端到C末端)：CHAP域-M23内肽酶-ALE-1的CBD。也可能存在有其他的例如居间序列元件。优选地，居间序列是短接头序列，在每种情况下不超过10个氨基酸，更优选不超过5个氨基酸的长度。最优选地，它们的长度仅为一个或两个氨基酸。接头序列优选为包含一个或多个甘氨酸残基的柔性序列。此类接头的实例是甘氨酸-丝氨酸接头或序列GGGGS(SEQ ID NO:7)。优选地，本发明多肽的第四个元件，进一步的肽(见下文)位于由两个催化域(CHAP，M23)和CBD域(ALE-1的CBD域)形成的单元的N-或C-末端，C末端位置是更优选的。如果两个催化域(CHAP，M23)和CBD域形成单元(即肽位于所述单元的C或N端)，则所述单元的长度优选小于500个氨基酸，优选小于450个氨基酸。优选的是，根据本发明第一方面的多肽至少包含SEQ ID NO:3(M23)和/或SEQ ID NO:5(ALE-1)的氨基酸序列。最优选地，根据本发明的多肽在任何情况下包含根据SEQ ID NO:5的氨基酸序列。最后这两个元件(M23，ALE-1)的优选排列反映在SEQ ID NO:8中。关于CHAP域，本发明提供了许多可能的变体序列，即绝对不强制明确使用SEQ ID NO:1或SEQ ID NO:2的氨基酸序列。可能的突变(相对于SEQ ID NO:2鉴定)是例如K17E，K28N，H51Q，T86A，G154S或G154C，E172G，T173N或A174R。最优选的是SEQ ID NO:2的变体序列，其包含突变K17E和H51Q。最优选地，所述变体序列包含突变K17E，K28N，H51Q，T86A和G154S。已显示这些突变改善了根据本发明的多肽的抗细菌活性并增加热稳定性。如果使用天然存在的SEQ IDNO:2的序列，则本发明的前三个元件的优选排列反映在SEQ ID NO:9(无N-末端甲硫氨酸)和SEQ ID NO:10中。考虑用于本发明的还有其变体序列，例如，与SEQ ID NO:8，SEQ ID NO:9或SEQ ID NO:10包含至少80％的序列同一性的变体序列。
除前三种元件(CHAP域-M23内肽酶-ALE-1的CBD)外，本发明的多肽包括第四种元件，即选自抗微生物肽，两亲性肽，阳离子肽，疏水肽，寿司肽和防御素的肽。优选地，所述进一步的肽序列与LysK内溶素的CHAP域，溶葡萄球菌酶的M23内肽酶域和ALE-1的细胞壁结合域(CBD)异源，即进一步的肽序列不存在于LysK内溶素序列，溶葡萄球菌酶序列和/或ALE-1内溶素序列中。优选地，本发明多肽的第四种元件位于由两个催化域(CHAP，M23)和CBD域(ALE-1的CBD域)形成的单元的N或C末端，C末端位置更优选。因此，合适的排列是：a)CHAP域-M23内肽酶-ALE-1肽的CBD-肽，和b)肽-CHAP域-M23内肽酶-ALE-1的CBD。肽可以直接或通过居间序列，例如接头序列与酶单元(CHAP域-M23内肽酶-AML-1的CBD)连接。
可用作进一步的肽的阳离子/聚阳离子氨基酸序列的实例列于下表中。
表1：
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2：
进一步的还可以是由Ding JL,Li P,Ho BCell Mol Life Sci.2008 Apr；65(7-8):1202-19.The Sushi peptides:structural characterization and mode of actionagainst Gram-negative bacteria描述的寿司肽。特别优选的是根据SEQ ID NO:95的寿司1肽。其他优选的寿司肽是寿司肽S1和S3及其倍数(Tan et al,FASEB J.2000Sep；14(12):1801-13)。
优选的疏水性肽是具有根据SEQ ID NO:96的氨基酸序列的Walmagh1和具有氨基酸序列Phe-Phe-Val-Ala-Pro(SEQ ID NO:97)的疏水性肽。
优选的两亲性肽是根据SEQ ID NO:98的T4溶菌酶的α4-螺旋和具有根据SEQ IDNO:99的氨基酸序列的WLBU2-变体和根据SEQ ID NO:100的Walmagh 2。
更优选地，进一步的肽的序列选自SEQ ID NO:36，SEQ ID NO:37，SEQ ID NO:38，SEQ ID NO:39和SEQ ID NO:94。最优选地，进一步的肽的序列选自SEQ ID NO:38和SEQ IDNO:94。
特别是在本发明多肽由宿主细胞重组表达的情况下，优选本发明多肽在N-末端包含甲硫氨酸残基。
本发明的多肽可另外包含一种或多种标签序列。此类标签序列可以例如位于本发明多肽的N-或C-末端，或位于肽序列和酶单元(CHAP域-M23内肽酶-AML-1的CBD)之间。在优选的实施方案中，一种或多种标签序列位于酶单元(CHAP域-M23内肽酶-AML-1的CBD)的C端侧。一种或多种标签序列可以例如直接或通过短接头与酶单元连接(参见上文)。标签的许多例子在本领域中是已知的，其中一些已经在上面提到过。在本发明的上下文中，特别优选的标签序列是His标签，优选根据SEQ ID NO:101的His标签。包含酶单元(CHAP域-M23内肽酶-ALE-1的CBD)和标签的优选序列是SEQ ID NO:102和SEQ ID NO:103(在N末端都具有任选的甲硫氨酸)。肽序列优选位于C端(例如SEQ ID NO:102或SEQ ID NO:103的C端)，直接或通过接头序列与其连接。
根据本发明的多肽的特别优选的实例是包含SEQ ID NO:104(具有N-末端甲硫氨酸的SEQ ID NO:105)或SEQ ID NO:106(具有N-末端甲硫氨酸的SEQ ID NO:107)的序列的多肽。其他实例是SEQ ID NO:108(具有N-末端甲硫氨酸的SEQ ID NO:109)和SEQ ID NO:110(具有N-末端甲硫氨酸的SEQ ID NO:111)。本发明还考虑利用包含这8个序列中任一个的变体序列的多肽，特别是与这8个序列中的任何一个序列至少表现出至少80％，至少85％，至少90％，至少95％，至少96％，至少97％，至少98％或至少99％的序列同一性的变体序列。优选地，所述多肽比各自的参考序列更加热稳定和/或表现出更高的活性。优选地，所述变体序列仍包含SEQ ID NO:38或SEQ ID NO:94的序列，和/或仍包含SEQ ID NO:5或SEQID NO:6的序列。变异位点特别是本发明鉴定出合适突变的位点。变体序列也可以缺乏N-末端甲硫氨酸。
在其他方面，本发明涉及包含改进的LysK CHAP域的多肽。发明人已经鉴定了改善抗细菌活性和/或热稳定性的突变。由于蛋白质域能够独立于其他域折叠，因此这些性质将得到保留，并且当用其他域，例如其他内溶素的其他域改组此域时可以利用这些性质。
因此，本发明在本发明的第二方面涉及进一步的多肽，所述多肽包含SEQ ID NO:1的变体序列，其中所述变体序列包含与SEQ ID NO:1的氨基酸序列的至少80％的序列同一性，并且其中所述变体序列与SEQ ID NO:1的氨基酸序列相比表现出突变H8N和/或T43A。由于所述多肽需要表现出这两种突变中的至少一种，所述多肽不包含与SEQ ID NO:1的氨基酸序列的100％序列同一性。优选地，本发明第二方面的多肽至少表现出H8N突变，并且最优选表现出这两种突变。
在第三方面，本发明涉及包含SEQ ID NO:2的变体序列的进一步的多肽，其中所述变体序列包含与SEQ ID NO:2的氨基酸序列的至少80％的序列同一性，并且其中与SEQ IDNO:2的氨基酸序列相比，所述变体序列表现出一种或多种选自K16E，K27N，H50Q，T85A，G154C，G154S的突变。优选地，根据本发明第三方面的多肽包含与SEQ ID NO:2的氨基酸序列相比至少表现出突变K16E和H50Q的变体序列。甚至更优选地，根据本发明第三方面的多肽包含与SEQ ID NO:2的氨基酸序列相比至少表现出突变K16E，K27N，H50Q，T85A和G154S的变体序列。
本发明第二或第三方面的多肽可以包含进一步的序列元件，特别是进一步的域，例如为融合蛋白提供(例如针对葡萄球菌，例如金黄色葡萄球菌)抗细菌活性的域。例如，本发明第二或第三方面的多肽可包含肽聚糖水解的至少一个(例如，一个，两个或超过两个)催化域。如果本发明第二或第三方面的多肽包含超过一个另外的域，则所述域可以衍生自不同的来源，即彼此可以是异源的。
根据本发明第二或第三方面的多肽的实例可以是多肽，其中所述变体序列与SEQID NO:105，SEQ ID NO:107，SEQ ID NO:109，或SEQ ID NO:111的氨基酸序列表现出至少95％的序列同一性，优选地与SEQ ID NO:105，SEQ ID NO:107，SEQ ID NO:109，或SEQ IDNO:111的氨基酸序列表现出至少96％，至少97％，至少98％或至少99％的序列同一性。
根据本发明任何方面的多肽的长度原则上不受限制，但优选长度不会过大。优选地，根据本发明的多肽的总长度不超过约600个氨基酸，优选不超过约500个氨基酸。
优选地，根据本发明的多肽的特征在于降解金黄色葡萄球菌细菌的肽聚糖的能力。如果酶是活性的，则肽聚糖层的降解将导致浊度下降，这可以通过光度法测量(参见例如Briers et al.,J.Biochem.Biophys Methods 70:531-533,(2007)。
本发明还涉及编码一种或多种本发明的发明性多肽的核酸。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或其杂合体。它们可以是单链或双链的。可具有小转录物或整个基因组，例如噬菌体基因组的大小。如本文所用，编码本发明的一种或多种发明性多肽的核酸可以是反映有义链的核酸。同样，也包括反义链。核酸可以包括用于表达本发明多肽的异源启动子。
另一方面，本发明涉及载体，其包含根据本发明的核酸。此类载体可以是例如允许表达本发明多肽的表达载体。所述表达可以是组成性或诱导型的。载体也可以是克隆载体，其包含用于克隆目的的本发明多肽的核酸序列。
另一方面，本发明涉及宿主细胞，其包含根据本发明的多肽，根据本发明的核酸，和/或根据本发明的载体。宿主细胞尤其可选自细菌细胞和酵母细胞。特别优选的宿主细胞是大肠杆菌细胞。
在另一方面，本发明涉及组合物，其包含根据本发明的多肽，根据本发明的核酸，根据本发明的载体和/或根据本发明的宿主细胞，并且还包含合适的稀释剂，赋形剂或载体。优选的组合物包含根据本发明的多肽。优选地，根据本发明的组合物包含药学上可接受的稀释剂，赋形剂或载体。此类组合物可以是药物组合物。此外，根据本发明的组合物可以是水溶液(优选缓冲液或生理溶液)、粉末、栓剂、乳液、悬浮液、凝胶、洗剂、乳膏、油膏剂(salve)、软膏剂、可注射溶液、糖浆剂、喷雾剂、吸入剂或任何其他医学合理盖伦派医学(galenic)组合物或配制剂、涂层组合物，优选植入物涂层组合物、支架涂层组合物或导管涂层组合物、或生物材料，优选骨水泥，所述组合物包含根据本发明的多肽，根据本发明的核酸，根据本发明的载体和/或根据本发明的宿主细胞。
在另一方面，本发明涉及装置，特别是医学装置，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，根据本发明的宿主细胞和/或根据本发明的组合物。此类装置(或其至少一部分)可以例如用根据本发明的多肽，根据本发明的核酸，根据本发明的载体，根据本发明的宿主细胞，和/或根据本发明的组合物涂覆或浸渍。特别优选用根据本发明的多肽或根据本发明的组合物涂覆或浸渍。此类装置可以例如是植入物，支架或导管。这些装置可以在例如心脏或整形外科手术中得到应用。本发明的其他合适的装置可以是包含根据本发明的多肽(或根据本发明的核酸，根据本发明的载体，根据本发明的宿主细胞，和/或根据本发明的组合物)的石膏，敷布(compress)或敷料(dressing)。这些可以在例如伤口护理中得到应用。
在另一方面，本发明涉及根据本发明的多肽，根据本发明的核酸，根据本发明的载体，根据本发明的宿主细胞，和/或根据本发明的组合物，其用于通过手术或疗法治疗人或动物身体的方法或对人或动物身体实施的诊断方法。
对人或动物身体实施的诊断方法可以涉及从受试者取样，建立细菌培养物和分析向培养物添加例如本发明的多肽是否抑制细菌生长。如果如此，则可能存在金黄色葡萄球菌。
如果在本文中使用本发明的核酸或本发明的载体，则优选地，所述核酸或载体提供从细胞分泌本发明的多肽。如果使用本发明的宿主细胞，则优选相应的宿主细胞分泌本发明的多肽。
本发明还涉及根据本发明的多肽(和同样地根据本发明的核酸，根据本发明的载体，根据本发明的宿主细胞，和/或根据本发明的组合物)，用于治疗或预防感染的方法，特别是用于治疗或预防涉及金黄色葡萄球菌细菌的感染的方法。在这方面，本发明还涉及根据本发明的多肽(和同样地根据本发明的核酸，根据本发明的载体，根据本发明的宿主细胞和/或根据本发明的组合物)，用于治疗，改善或预防皮炎(如特应性皮炎)或耳炎的方法。金黄色葡萄球菌细菌经常参与这些疾病状态，例如，作为继发感染。
本发明还涉及根据本发明的多肽(和同样地根据本发明的核酸，根据本发明的载体，根据本发明的宿主细胞，和/或根据本发明的组合物)，用于治疗受试者伤口的方法，特别是急性伤口如医源性伤口或慢性伤口。受试者可以是例如动物或人。
本发明还涉及治疗或预防受试者中由金黄色葡萄球菌引起的感染的方法，该方法包括使所述受试者与根据本发明的多肽(或同样地，根据本发明的核酸，根据本发明的载体，根据本发明的宿主细胞和/或根据本发明的组合物)接触。特别地，本发明涉及治疗或预防受试者中涉及金黄色葡萄球菌细菌的感染的方法，该方法包括使所述受试者与根据本发明的多肽接触。
在另一方面，本发明涉及根据本发明的多肽(或根据本发明的核酸，根据本发明的载体，根据本发明的宿主细胞，和/或根据本发明的组合物)用于对无生命的表面、组合物和/或物体进行消毒的用途，特别是在医院环境或医生诊室中。
另一方面，本发明涉及根据本发明的多肽(或根据本发明的核酸，根据本发明的载体，根据本发明的宿主细胞和/或根据本发明的组合物)用于防止无生命表面，组合物和/或物体被细菌污染，特别是用于防止金黄色葡萄球菌细菌的污染的用途。
在另一方面，本发明涉及对无生命表面，组合物和/或物体进行消毒的方法，特别是在医院环境或医生诊室中，其中该方法包括使无生命的表面，组合物和/或物体接触根据本发明的多肽(或根据本发明的核酸，根据本发明的载体，根据本发明的宿主细胞，和/或根据本发明的组合物)。
在另一方面，本发明涉及用于防止无生命表面，组合物和/或物体被细菌污染的方法，特别是用于防止金黄色葡萄球菌污染，其中该方法包括使无生命表面，组合物和/或物体接触根据本发明的多肽(或根据本发明的核酸，根据本发明的载体，根据本发明的宿主细胞和/或根据本发明的组合物)。
实施例
在下文中，呈现了说明本发明的实施方案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案的范围。实际上，除了本文所述的那些方案之外，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：本发明的两种融合蛋白对金黄色葡萄球菌的抗细菌活性
Becker et al.(Sci Rep.2016 Apr 28；6:25063)报道了具有降低的抗性金黄色葡萄球菌菌株形成发生的融合蛋白L-K的构建。为了提供针对金黄色葡萄球菌的其他改进的融合蛋白，本发明人产生了两种融合蛋白，每种融合蛋白包含LysK内溶素的CHAP域，溶葡萄球菌酶的M23内肽酶域；和ALE-1的细胞壁结合域(CBD)。另外，融合蛋白之一包含组织蛋白酶G(77-83)肽(SEQ ID NO:94)，另一种包含阳离子肽KNK(SEQ ID NO:38)。测定所得融合蛋白(SEQ ID NO:105和SEQ ID NO:107)的抗细菌活性和抗性形成。为此目的，进行MIC(最小抑制浓度)测定法(见下文)达几个培养周期。
MIC测定法
将金黄色葡萄球菌Sp10在(Luria-Bertani)培养基中培养，并在Mueller-Hinton培养基中以1:10稀释。在光密度OD600为约0.6时，将细菌以1:10在相同培养基中稀释，然后以1:500稀释。使用不同浓度的蛋白质/庆大霉素和20μl的终体积，将蛋白质缓冲液(20mMHEPES，500mM NaCl，pH7.4)和蛋白质或庆大霉素移液到96孔板中。使用的蛋白质是根据SEQID NO:105和SEQ ID NO:107的融合蛋白。将180μl细菌细胞或培养基(Mueller-Hinton)对照给予96孔板并混合。将板在37℃温育18-22小时，测量孔的OD600值测定细菌生长。将具有与培养基对照显示相同的OD600值的最低蛋白质/庆大霉素浓度的孔作为MIC。对于下一个周期，使用来自亚MIC孔的细菌溶液。亚MIC孔是比测试的MIC浓度次低的浓度的孔，即具有最高浓度的蛋白质/庆大霉素但仍然高于培养基对照的OD600的孔。对于抗性测定的进一步周期，取出来自先前周期的此亚MIC孔的细菌用于接着的过夜培养。
结果列于下表3a和b中。
表3a:
下表3b以相对于初始MIC(周期1)的倍数变化显示了表3a的所述结果。
表3b：
如从上表3b中明显，这两种本发明的融合蛋白随着时间比庆大霉素更不易于形成抗性菌株。
此外，与对现有技术的融合蛋白L-K(参见Becker et al.(Sci Rep.2016 Apr 28；6:25063))报道的结果相比，这两种本发明的融合蛋白均显示令人惊讶地降低了更多周期后抗性菌株形成的发生(12个周期后这两种本发明的融合蛋白的1.5倍MIC变化相对于10个周期后Becker等人的L-K融合蛋白的2倍MIC变化(参见Becker等人的图1B)，而平行地表现出更高的抗细菌活性(本发明的融合蛋白的初始MIC为4μg/ml相对于Becker等人的L-K融合蛋白的初始MIC为7.8μg/ml)。
实施例2：根据本发明的多肽的变体
本发明的发明人还创建了上述多肽的变体。为此目的，在SEQ ID NO:SEQ ID NO:105的序列中引入突变。
表4：
下表5显示了这些突变对SEQ ID NO:105的融合蛋白的活性的结果。如本文中使用，菌株DSM 346指金黄色葡萄球菌菌株DSM 346(Leibniz-Institut DSMZ-德国微生物菌种保藏中心(Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH),Braunschweig,Germany)。S64和S69是从Rob Lavigne教授(KatholiekeUniversiteitLeuven,Belgium)获得的金黄色葡萄球菌菌株S64和S69。
表5:
所有突变体保留了对金黄色葡萄球菌的抗细菌活性。
也针对SEQ ID NO:107的序列验证了上文提及的突变中的一些。另外，将上述突变中的一些组合。另外使用突变G154S替换G154C。下表6显示了对SEQ ID NO:107的融合蛋白测试的突变
表6：
克隆 Mut1 Mut2 Mut3 Mut4 Mut 5 Mut 6
11 K17E H51Q
12 K17E H51Q E172G T173N A174R
13 K28N T86A G154S
14 K28N T86A G154S E172G T173N A174R
15 K17E H51Q K28N T86A G154S
下表7显示了这些突变对SEQ ID NO:107的融合蛋白的活性的结果。
表7：
所有突变体保留对金黄色葡萄球菌的抗细菌活性。
另外，与未修饰的融合蛋白相比对所述突变体的两种评估了热稳定性。用菌株金黄色葡萄球菌DSM 346进行了热稳定性测定法。将蛋白质稀释到0,3mg/ml的浓度，接着于不同的温度温育20分钟(参见下表8)。在此温育时间后进行标准MIC测定法。蛋白质仍然显示(高)活性的温度越高，蛋白质的热稳定性越好。
表8：
因此，克隆11和15与未修饰的融合蛋白相比显示增加的热稳定性。
序列表
<110> 莱桑多公司
<120> 针对金黄色葡萄球菌的新抗微生物剂
<130> LYS-043 PCT
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<151> 2016-11-18
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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> 35
<211> 42
<212> PRT
<213> 人工
<220>
<223> 合成序列
<400> 35
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> 36
<211> 3
<212> PRT
<213> 人工序列
<220>
<223> 阳离子肽KNA
<400> 36
Lys Asn Ala
1
<210> 37
<211> 6
<212> PRT
<213> 人工序列
<220>
<223> 阳离子肽GGSKNA
<400> 37
Gly Gly Ser Lys Asn Ala
1 5
<210> 38
<211> 3
<212> PRT
<213> 人工序列
<220>
<223> 阳离子肽KNK
<400> 38
Lys Asn Lys
1
<210> 39
<211> 6
<212> PRT
<213> 人工序列
<220>
<223> 阳离子肽GGSKNK
<400> 39
Gly Gly Ser Lys Asn Lys
1 5
<210> 40
<211> 37
<212> PRT
<213> 智人
<400> 40
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> 41
<211> 29
<212> PRT
<213> 未知
<220>
<223> SMAP-29绵羊
<400> 41
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> 42
<211> 13
<212> PRT
<213> 未知
<220>
<223> Indolicidine牛
<400> 42
Ile Leu Pro Trp Lys Trp Pro Trp Trp Pro Trp Arg Arg
1 5 10
<210> 43
<211> 18
<212> PRT
<213> 未知
<220>
<223> Protegrin猪
<400> 43
Arg Gly Gly Arg Leu Cys Tyr Cys Arg Arg Arg Phe Cys Val Cys Val
1 5 10 15
Gly Arg
<210> 44
<211> 31
<212> PRT
<213> 未知
<220>
<223> 杀菌肽P1 哺乳动物(猪)
<400> 44
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> 45
<211> 23
<212> PRT
<213> 未知
<220>
<223> Magainin蛙
<400> 45
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> 46
<211> 25
<212> PRT
<213> 未知
<220>
<223> Pleurocidin 鱼
<400> 46
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> 47
<211> 36
<212> PRT
<213> 埃及伊蚊
<400> 47
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> 48
<211> 40
<212> PRT
<213> 黑腹果蝇
<400> 48
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> 49
<211> 21
<212> PRT
<213> 未知
<220>
<223> Buforin II 脊椎动物
<400> 49
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> 50
<211> 39
<212> PRT
<213> 未知
<220>
<223> 麻蝇杀菌肽IA 蝇
<400> 50
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> 51
<211> 17
<212> PRT
<213>蜜蜂
<400> 51
Ala Asn Arg Pro Val Tyr Ile Pro Pro Pro Arg Pro Pro His Pro Arg
1 5 10 15
Leu
<210> 52
<211> 24
<212> PRT
<213> 未知
<220>
<223> Ascaphine 5 蛙
<400> 52
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> 53
<211> 22
<212> PRT
<213> 未知
<220>
<223> Nigrocine 2蛙
<400> 53
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> 54
<211> 24
<212> PRT
<213> 未知
<220>
<223> Pseudin 1林蛙
<400> 54
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> 55
<211> 18
<212> PRT
<213> 未知
<220>
<223> Ranalexin 蛙
<400> 55
Phe Leu Gly Gly Leu Ile Val Pro Ala Met Ile Cys Ala Val Thr Lys
1 5 10 15
Lys Cys
<210> 56
<211> 26
<212> PRT
<213> 未知
<220>
<223> 蜂毒肽蜜蜂
<400> 56
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> 57
<211> 25
<212> PRT
<213> 未知
<220>
<223> Lycotoxin 1蜘蛛
<400> 57
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> 58
<211> 19
<212> PRT
<213> 未知
<220>
<223> Parasin 1 鱼
<400> 58
Lys Gly Arg Gly Lys Gln Gly Gly Lys Val Arg Ala Lys Ala Lys Thr
1 5 10 15
Arg Ser Ser
<210> 59
<211> 39
<212> PRT
<213> 未知
<220>
<223> Buforin I蟾蜍
<400> 59
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> 60
<211> 34
<212> PRT
<213> 未知
<220>
<223> Dermaseptin 1 蛙
<400> 60
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> 61
<211> 12
<212> PRT
<213> 未知
<220>
<223> Bactenecin 1 牛
<400> 61
Arg Leu Cys Arg Ile Val Val Ile Arg Val Cys Arg
1 5 10
<210> 62
<211> 21
<212> PRT
<213> 未知
<220>
<223> Thanatin 昆虫
<400> 62
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> 63
<211> 19
<212> PRT
<213> 未知
<220>
<223> Brevinin 1T 林蛙
<400> 63
Val Asn Pro Ile Ile Leu Gly Val Leu Pro Lys Val Cys Leu Ile Thr
1 5 10 15
Lys Lys Cys
<210> 64
<211> 26
<212> PRT
<213> 未知
<220>
<223> Ranateurin 1林蛙
<400> 64
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> 65
<211> 46
<212> PRT
<213> 未知
<220>
<223> Esculentin 1林蛙
<400> 65
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> 66
<211> 17
<212> PRT
<213> 美洲鲎
<400> 66
Arg Trp Cys Phe Arg Val Cys Tyr Arg Gly Ile Cys Tyr Arg Lys Cys
1 5 10 15
Arg
<210> 67
<211> 25
<212> PRT
<213> 未知
<220>
<223> Androctonin蝎子
<400> 67
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> 68
<211> 30
<212> PRT
<213> 智人
<400> 68
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> 69
<211> 38
<212> PRT
<213> 未知
<220>
<223> beta-防御素牛
<400> 69
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> 70
<211> 18
<212> PRT
<213> 未知
<220>
<223> theta-防御素猴
<400> 70
Gly Phe Cys Arg Cys Leu Cys Arg Arg Gly Val Cys Arg Cys Ile Cys
1 5 10 15
Thr Arg
<210> 71
<211> 40
<212> PRT
<213> 未知
<220>
<223> 防御素(sapecin A) 昆虫
<400> 71
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> 72
<211> 46
<212> PRT
<213> 未知
<220>
<223> Thionin (花菜蛋白) 植物
<400> 72
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> 73
<211> 50
<212> PRT
<213> 未知
<220>
<223> 来自萝卜的防御素
<400> 73
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> 74
<211> 44
<212> PRT
<213>黑腹果蝇
<400> 74
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> 75
<211> 25
<212> PRT
<213> 智人
<400> 75
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> 76
<211> 44
<212> PRT
<213> 未知
<220>
<223> Bac 5牛
<400> 76
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> 77
<211> 39
<212> PRT
<213> 未知
<220>
<223> PR-39猪
<400> 77
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> 78
<211> 20
<212> PRT
<213> 未知
<220>
<223> Pyrrhocoricin 昆虫
<400> 78
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> 79
<211> 24
<212> PRT
<213> 智人
<400> 79
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> 80
<211> 19
<212> PRT
<213> 未知
<220>
<223> ECP19
<400> 80
Arg Pro Pro Gln Phe Thr Arg Ala Gln Trp Phe Ala Ile Gln His Ile
1 5 10 15
Ser Leu Asn
<210> 81
<211> 23
<212> PRT
<213> 未知
<220>
<223> MSI-594
<400> 81
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> 82
<211> 35
<212> PRT
<213> 未知
<220>
<223> TL-ColM
<400> 82
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> 83
<211> 18
<212> PRT
<213> 未知
<220>
<223> SBO
<400> 83
Lys Leu Lys Lys Ile Ala Gln Lys Ile Lys Asn Phe Phe Ala Lys Leu
1 5 10 15
Val Ala
<210> 84
<211> 26
<212> PRT
<213> 未知
<220>
<223> Macedocin
<400> 84
Gly Lys Asn Gly Val Phe Lys Thr Ile Ser His Glu Cys His Leu Asn
1 5 10 15
Thr Trp Ala Phe Leu Ala Thr Cys Cys Ser
20 25
<210> 85
<211> 22
<212> PRT
<213> 未知
<220>
<223> Macedocin (Trunc)
<400> 85
Gly Lys Asn Gly Val Phe Lys Thr Ile Ser His Glu Cys His Leu Asn
1 5 10 15
Thr Trp Ala Phe Leu Ala
20
<210> 86
<211> 28
<212> PRT
<213> 未知
<220>
<223> D16
<400> 86
Ala Cys Lys Leu Lys Ser Leu Leu Lys Thr Leu Ser Lys Ala Lys Lys
1 5 10 15
Lys Lys Leu Lys Thr Leu Leu Lys Ala Leu Ser Lys
20 25
<210> 87
<211> 17
<212> PRT
<213> 未知
<220>
<223> CPF-C1
<400> 87
Gly Phe Gly Ser Leu Leu Gly Lys Ala Leu Arg Leu Gly Ala Asn Val
1 5 10 15
Leu
<210> 88
<211> 34
<212> PRT
<213> 未知
<220>
<223> TL-ColM
<400> 88
Glu Thr Leu Thr Val His Ala Pro Ser Pro Ser Thr Asn Leu Pro Ser
1 5 10 15
Tyr Gly Asn Gly Ala Phe Ser Leu Ser Ala Pro His Val Pro Gly Ala
20 25 30
Gly Pro
<210> 89
<211> 26
<212> PRT
<213> 未知
<220>
<223> TM-174E
<400> 89
Leu Ile Ser Lys Gly Trp Pro Tyr Leu Leu Val Val Val Leu Gly Ala
1 5 10 15
Thr Ile Tyr Phe Trp Gly Asn Ser Asn Gly
20 25
<210> 90
<211> 45
<212> PRT
<213> 未知
<220>
<223> ECP45
<400> 90
Arg Pro Pro Gln Phe Thr Arg Ala Gln Trp Phe Ala Ile Gln His Ile
1 5 10 15
Ser Leu Asn Pro Pro Arg Cys Thr Ile Ala Met Arg Ala Ile Asn Asn
20 25 30
Tyr Arg Trp Arg Cys Lys Asn Gln Asn Thr Phe Leu Arg
35 40 45
<210> 91
<211> 50
<212> PRT
<213> 未知
<220>
<223> 大肠菌素E3_1-51 (S37F)
<400> 91
Ser Gly Gly Asp Gly Arg Gly His Asn Thr Gly Ala His Ser Thr Ser
1 5 10 15
Gly Asn Ile Asn Gly Gly Pro Thr Gly Leu Gly Val Gly Gly Gly Ala
20 25 30
Ser Asp Gly Phe Gly Trp Ser Ser Glu Asn Asn Pro Trp Gly Gly Gly
35 40 45
Ser Gly
50
<210> 92
<211> 68
<212> PRT
<213> 未知
<220>
<223> 大肠菌素E3_1-69 (S37F)
<400> 92
Ser Gly Gly Asp Gly Arg Gly His Asn Thr Gly Ala His Ser Thr Ser
1 5 10 15
Gly Asn Ile Asn Gly Gly Pro Thr Gly Leu Gly Val Gly Gly Gly Ala
20 25 30
Ser Asp Gly Phe Gly Trp Ser Ser Glu Asn Asn Pro Trp Gly Gly Gly
35 40 45
Ser Gly Ser Gly Ile His Trp Gly Gly Gly Ser Gly His Gly Asn Gly
50 55 60
Gly Gly Asn Gly
65
<210> 93
<211> 52
<212> PRT
<213> 未知
<220>
<223> 大肠菌素D_1-53
<400> 93
Ser Asp Tyr Glu Gly Ser Gly Pro Thr Glu Gly Ile Asp Tyr Gly His
1 5 10 15
Ser Met Val Val Trp Pro Ser Thr Gly Leu Ile Ser Gly Gly Asp Val
20 25 30
Lys Pro Gly Gly Ser Ser Gly Ile Ala Pro Ser Met Pro Pro Gly Trp
35 40 45
Gly Asp Tyr Ser
50
<210> 94
<211> 7
<212> PRT
<213> 智人
<400> 94
His Pro Gln Tyr Asn Gln Arg
1 5
<210> 95
<211> 34
<212> PRT
<213> 美洲鲎
<400> 95
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> 96
<211> 18
<212> PRT
<213> 人工
<220>
<223> 合成序列
<400> 96
Gly Phe Phe Ile Pro Ala Val Ile Leu Pro Ser Ile Ala Phe Leu Ile
1 5 10 15
Val Pro
<210> 97
<211> 5
<212> PRT
<213> 人工
<220>
<223> 合成序列
<400> 97
Phe Phe Val Ala Pro
1 5
<210> 98
<211> 13
<212> PRT
<213> 未知
<220>
<223> T4 溶菌酶的alpha4螺旋
<400> 98
Pro Asn Arg Ala Lys Arg Val Ile Thr Thr Phe Arg Thr
1 5 10
<210> 99
<211> 27
<212> PRT
<213> 人工
<220>
<223> 合成序列
<400> 99
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> 100
<211> 25
<212> PRT
<213> 人工序列
<220>
<223> 合成序列; MW2
<400> 100
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> 101
<211> 6
<212> PRT
<213> 人工序列
<220>
<223> His-标签(6x)
<400> 101
His His His His His His
1 5
<210> 102
<211> 430
<212> PRT
<213> 人工序列
<220>
<223> 融合蛋白，包含 LysK內溶素的CHAP域 , 溶葡萄球菌酶的M23
内肽酶域，ALE-1细胞壁结合域和 His6-标签，不含N末端甲硫氨酸
<400> 102
Ala Lys Thr Gln Ala Glu Ile Asn Lys Arg Leu Asp Ala Tyr Ala Lys
1 5 10 15
Gly Thr Val Asp Ser Pro Tyr Arg Val Lys Lys Ala Thr Ser Tyr Asp
20 25 30
Pro Ser Phe Gly Val Met Glu Ala Gly Ala Ile Asp Ala Asp Gly Tyr
35 40 45
Tyr His Ala Gln Cys Gln Asp Leu Ile Thr Asp Tyr Val Leu Trp Leu
50 55 60
Thr Asp Asn Lys Val Arg Thr Trp Gly Asn Ala Lys Asp Gln Ile Lys
65 70 75 80
Gln Ser Tyr Gly Thr Gly Phe Lys Ile His Glu Asn Lys Pro Ser Thr
85 90 95
Val Pro Lys Lys Gly Trp Ile Ala Val Phe Thr Ser Gly Ser Tyr Glu
100 105 110
Gln Trp Gly His Ile Gly Ile Val Tyr Asp Gly Gly Asn Thr Ser Thr
115 120 125
Phe Thr Ile Leu Glu Gln Asn Trp Asn Gly Tyr Ala Asn Lys Lys Pro
130 135 140
Thr Lys Arg Val Asp Asn Tyr Tyr Gly Leu Thr His Phe Ile Glu Ile
145 150 155 160
Pro Val Lys Ala Gly Thr Thr Val Lys Lys Glu Thr Ala Lys Lys Ser
165 170 175
Ala Thr Ser His Glu His Ser Ala Gln Trp Leu Asn Asn Tyr Lys Lys
180 185 190
Gly Tyr Gly Tyr Gly Pro Tyr Pro Leu Gly Ile Asn Gly Gly Met His
195 200 205
Tyr Gly Val Asp Phe Phe Met Asn Ile Gly Thr Pro Val Lys Ala Ile
210 215 220
Ser Ser Gly Lys Ile Val Glu Ala Gly Trp Ser Asn Tyr Gly Gly Gly
225 230 235 240
Asn Gln Ile Gly Leu Ile Glu Asn Asp Gly Val His Arg Gln Trp Tyr
245 250 255
Met His Leu Ser Lys Tyr Asn Val Lys Val Gly Asp Tyr Val Lys Ala
260 265 270
Gly Gln Ile Ile Gly Trp Ser Gly Ser Thr Gly Tyr Ser Thr Ala Pro
275 280 285
His Leu His Phe Gln Arg Met Val Asn Ser Phe Ser Asn Ser Thr Ala
290 295 300
Gln Asp Pro Met Pro Phe Leu Lys Ser Ala Gly Tyr Gly Lys Ala Gly
305 310 315 320
Gly Thr Val Thr Pro Thr Pro Asn Pro Gly Asn Gly Tyr Lys Thr Asn
325 330 335
Lys Tyr Gly Thr Leu Tyr Lys Ser Glu Ser Ala Ser Phe Thr Ala Asn
340 345 350
Thr Asp Ile Ile Thr Arg Leu Thr Gly Pro Phe Arg Ser Met Pro Gln
355 360 365
Ser Gly Val Leu Arg Lys Gly Leu Thr Ile Lys Tyr Asp Glu Val Met
370 375 380
Lys Gln Asp Gly His Val Trp Val Gly Tyr Asn Thr Asn Ser Gly Lys
385 390 395 400
Arg Val Tyr Leu Pro Val Arg Thr Trp Asn Glu Ser Thr Gly Glu Leu
405 410 415
Gly Pro Leu Trp Gly Thr Ile Lys His His His His His His
420 425 430
<210> 103
<211> 432
<212> PRT
<213> 人工序列
<220>
<223> 融合蛋白，包含 LysK內溶素的CHAP域 , 溶葡萄球菌酶的M23
内肽酶域，ALE-1的细胞壁结合域和 His6-标签，经由GS接头；不含N末端甲硫氨酸
<400> 103
Ala Lys Thr Gln Ala Glu Ile Asn Lys Arg Leu Asp Ala Tyr Ala Lys
1 5 10 15
Gly Thr Val Asp Ser Pro Tyr Arg Val Lys Lys Ala Thr Ser Tyr Asp
20 25 30
Pro Ser Phe Gly Val Met Glu Ala Gly Ala Ile Asp Ala Asp Gly Tyr
35 40 45
Tyr His Ala Gln Cys Gln Asp Leu Ile Thr Asp Tyr Val Leu Trp Leu
50 55 60
Thr Asp Asn Lys Val Arg Thr Trp Gly Asn Ala Lys Asp Gln Ile Lys
65 70 75 80
Gln Ser Tyr Gly Thr Gly Phe Lys Ile His Glu Asn Lys Pro Ser Thr
85 90 95
Val Pro Lys Lys Gly Trp Ile Ala Val Phe Thr Ser Gly Ser Tyr Glu
100 105 110
Gln Trp Gly His Ile Gly Ile Val Tyr Asp Gly Gly Asn Thr Ser Thr
115 120 125
Phe Thr Ile Leu Glu Gln Asn Trp Asn Gly Tyr Ala Asn Lys Lys Pro
130 135 140
Thr Lys Arg Val Asp Asn Tyr Tyr Gly Leu Thr His Phe Ile Glu Ile
145 150 155 160
Pro Val Lys Ala Gly Thr Thr Val Lys Lys Glu Thr Ala Lys Lys Ser
165 170 175
Ala Thr Ser His Glu His Ser Ala Gln Trp Leu Asn Asn Tyr Lys Lys
180 185 190
Gly Tyr Gly Tyr Gly Pro Tyr Pro Leu Gly Ile Asn Gly Gly Met His
195 200 205
Tyr Gly Val Asp Phe Phe Met Asn Ile Gly Thr Pro Val Lys Ala Ile
210 215 220
Ser Ser Gly Lys Ile Val Glu Ala Gly Trp Ser Asn Tyr Gly Gly Gly
225 230 235 240
Asn Gln Ile Gly Leu Ile Glu Asn Asp Gly Val His Arg Gln Trp Tyr
245 250 255
Met His Leu Ser Lys Tyr Asn Val Lys Val Gly Asp Tyr Val Lys Ala
260 265 270
Gly Gln Ile Ile Gly Trp Ser Gly Ser Thr Gly Tyr Ser Thr Ala Pro
275 280 285
His Leu His Phe Gln Arg Met Val Asn Ser Phe Ser Asn Ser Thr Ala
290 295 300
Gln Asp Pro Met Pro Phe Leu Lys Ser Ala Gly Tyr Gly Lys Ala Gly
305 310 315 320
Gly Thr Val Thr Pro Thr Pro Asn Pro Gly Asn Gly Tyr Lys Thr Asn
325 330 335
Lys Tyr Gly Thr Leu Tyr Lys Ser Glu Ser Ala Ser Phe Thr Ala Asn
340 345 350
Thr Asp Ile Ile Thr Arg Leu Thr Gly Pro Phe Arg Ser Met Pro Gln
355 360 365
Ser Gly Val Leu Arg Lys Gly Leu Thr Ile Lys Tyr Asp Glu Val Met
370 375 380
Lys Gln Asp Gly His Val Trp Val Gly Tyr Asn Thr Asn Ser Gly Lys
385 390 395 400
Arg Val Tyr Leu Pro Val Arg Thr Trp Asn Glu Ser Thr Gly Glu Leu
405 410 415
Gly Pro Leu Trp Gly Thr Ile Lys Gly Ser His His His His His His
420 425 430
<210> 104
<211> 439
<212> PRT
<213> 人工序列
<220>
<223> 融合蛋白，包含 LysK內溶素的CHAP域 , 溶葡萄球菌酶的M23
内肽酶域 ，ALE-1的细胞壁结合域，His6标签和合成肽HPQYNQR；不含N端甲硫氨酸
<400> 104
Ala Lys Thr Gln Ala Glu Ile Asn Lys Arg Leu Asp Ala Tyr Ala Lys
1 5 10 15
Gly Thr Val Asp Ser Pro Tyr Arg Val Lys Lys Ala Thr Ser Tyr Asp
20 25 30
Pro Ser Phe Gly Val Met Glu Ala Gly Ala Ile Asp Ala Asp Gly Tyr
35 40 45
Tyr His Ala Gln Cys Gln Asp Leu Ile Thr Asp Tyr Val Leu Trp Leu
50 55 60
Thr Asp Asn Lys Val Arg Thr Trp Gly Asn Ala Lys Asp Gln Ile Lys
65 70 75 80
Gln Ser Tyr Gly Thr Gly Phe Lys Ile His Glu Asn Lys Pro Ser Thr
85 90 95
Val Pro Lys Lys Gly Trp Ile Ala Val Phe Thr Ser Gly Ser Tyr Glu
100 105 110
Gln Trp Gly His Ile Gly Ile Val Tyr Asp Gly Gly Asn Thr Ser Thr
115 120 125
Phe Thr Ile Leu Glu Gln Asn Trp Asn Gly Tyr Ala Asn Lys Lys Pro
130 135 140
Thr Lys Arg Val Asp Asn Tyr Tyr Gly Leu Thr His Phe Ile Glu Ile
145 150 155 160
Pro Val Lys Ala Gly Thr Thr Val Lys Lys Glu Thr Ala Lys Lys Ser
165 170 175
Ala Thr Ser His Glu His Ser Ala Gln Trp Leu Asn Asn Tyr Lys Lys
180 185 190
Gly Tyr Gly Tyr Gly Pro Tyr Pro Leu Gly Ile Asn Gly Gly Met His
195 200 205
Tyr Gly Val Asp Phe Phe Met Asn Ile Gly Thr Pro Val Lys Ala Ile
210 215 220
Ser Ser Gly Lys Ile Val Glu Ala Gly Trp Ser Asn Tyr Gly Gly Gly
225 230 235 240
Asn Gln Ile Gly Leu Ile Glu Asn Asp Gly Val His Arg Gln Trp Tyr
245 250 255
Met His Leu Ser Lys Tyr Asn Val Lys Val Gly Asp Tyr Val Lys Ala
260 265 270
Gly Gln Ile Ile Gly Trp Ser Gly Ser Thr Gly Tyr Ser Thr Ala Pro
275 280 285
His Leu His Phe Gln Arg Met Val Asn Ser Phe Ser Asn Ser Thr Ala
290 295 300
Gln Asp Pro Met Pro Phe Leu Lys Ser Ala Gly Tyr Gly Lys Ala Gly
305 310 315 320
Gly Thr Val Thr Pro Thr Pro Asn Pro Gly Asn Gly Tyr Lys Thr Asn
325 330 335
Lys Tyr Gly Thr Leu Tyr Lys Ser Glu Ser Ala Ser Phe Thr Ala Asn
340 345 350
Thr Asp Ile Ile Thr Arg Leu Thr Gly Pro Phe Arg Ser Met Pro Gln
355 360 365
Ser Gly Val Leu Arg Lys Gly Leu Thr Ile Lys Tyr Asp Glu Val Met
370 375 380
Lys Gln Asp Gly His Val Trp Val Gly Tyr Asn Thr Asn Ser Gly Lys
385 390 395 400
Arg Val Tyr Leu Pro Val Arg Thr Trp Asn Glu Ser Thr Gly Glu Leu
405 410 415
Gly Pro Leu Trp Gly Thr Ile Lys His His His His His His Leu Lys
420 425 430
His Pro Gln Tyr Asn Gln Arg
435
<210> 105
<211> 440
<212> PRT
<213> 人工序列
<220>
<223> 融合蛋白，包含 LysK內溶素的CHAP域 , 溶葡萄球菌酶的M23
内肽酶域，ALE-1的细胞壁结合域 ，His6标签和合成肽HPQYNQR
<400> 105
Met Ala Lys Thr Gln Ala Glu Ile Asn Lys Arg Leu Asp Ala Tyr Ala
1 5 10 15
Lys Gly Thr Val Asp Ser Pro Tyr Arg Val Lys Lys Ala Thr Ser Tyr
20 25 30
Asp Pro Ser Phe Gly Val Met Glu Ala Gly Ala Ile Asp Ala Asp Gly
35 40 45
Tyr Tyr His Ala Gln Cys Gln Asp Leu Ile Thr Asp Tyr Val Leu Trp
50 55 60
Leu Thr Asp Asn Lys Val Arg Thr Trp Gly Asn Ala Lys Asp Gln Ile
65 70 75 80
Lys Gln Ser Tyr Gly Thr Gly Phe Lys Ile His Glu Asn Lys Pro Ser
85 90 95
Thr Val Pro Lys Lys Gly Trp Ile Ala Val Phe Thr Ser Gly Ser Tyr
100 105 110
Glu Gln Trp Gly His Ile Gly Ile Val Tyr Asp Gly Gly Asn Thr Ser
115 120 125
Thr Phe Thr Ile Leu Glu Gln Asn Trp Asn Gly Tyr Ala Asn Lys Lys
130 135 140
Pro Thr Lys Arg Val Asp Asn Tyr Tyr Gly Leu Thr His Phe Ile Glu
145 150 155 160
Ile Pro Val Lys Ala Gly Thr Thr Val Lys Lys Glu Thr Ala Lys Lys
165 170 175
Ser Ala Thr Ser His Glu His Ser Ala Gln Trp Leu Asn Asn Tyr Lys
180 185 190
Lys Gly Tyr Gly Tyr Gly Pro Tyr Pro Leu Gly Ile Asn Gly Gly Met
195 200 205
His Tyr Gly Val Asp Phe Phe Met Asn Ile Gly Thr Pro Val Lys Ala
210 215 220
Ile Ser Ser Gly Lys Ile Val Glu Ala Gly Trp Ser Asn Tyr Gly Gly
225 230 235 240
Gly Asn Gln Ile Gly Leu Ile Glu Asn Asp Gly Val His Arg Gln Trp
245 250 255
Tyr Met His Leu Ser Lys Tyr Asn Val Lys Val Gly Asp Tyr Val Lys
260 265 270
Ala Gly Gln Ile Ile Gly Trp Ser Gly Ser Thr Gly Tyr Ser Thr Ala
275 280 285
Pro His Leu His Phe Gln Arg Met Val Asn Ser Phe Ser Asn Ser Thr
290 295 300
Ala Gln Asp Pro Met Pro Phe Leu Lys Ser Ala Gly Tyr Gly Lys Ala
305 310 315 320
Gly Gly Thr Val Thr Pro Thr Pro Asn Pro Gly Asn Gly Tyr Lys Thr
325 330 335
Asn Lys Tyr Gly Thr Leu Tyr Lys Ser Glu Ser Ala Ser Phe Thr Ala
340 345 350
Asn Thr Asp Ile Ile Thr Arg Leu Thr Gly Pro Phe Arg Ser Met Pro
355 360 365
Gln Ser Gly Val Leu Arg Lys Gly Leu Thr Ile Lys Tyr Asp Glu Val
370 375 380
Met Lys Gln Asp Gly His Val Trp Val Gly Tyr Asn Thr Asn Ser Gly
385 390 395 400
Lys Arg Val Tyr Leu Pro Val Arg Thr Trp Asn Glu Ser Thr Gly Glu
405 410 415
Leu Gly Pro Leu Trp Gly Thr Ile Lys His His His His His His Leu
420 425 430
Lys His Pro Gln Tyr Asn Gln Arg
435 440
<210> 106
<211> 438
<212> PRT
<213> 人工序列
<220>
<223> 融合蛋白，包含 LysK內溶素的CHAP域 , 溶葡萄球菌酶的M23
内肽酶域 ，ALE-1的细胞壁结合域，His6标签和合成肽KNK；不含N端甲硫氨酸
<400> 106
Ala Lys Thr Gln Ala Glu Ile Asn Lys Arg Leu Asp Ala Tyr Ala Lys
1 5 10 15
Gly Thr Val Asp Ser Pro Tyr Arg Val Lys Lys Ala Thr Ser Tyr Asp
20 25 30
Pro Ser Phe Gly Val Met Glu Ala Gly Ala Ile Asp Ala Asp Gly Tyr
35 40 45
Tyr His Ala Gln Cys Gln Asp Leu Ile Thr Asp Tyr Val Leu Trp Leu
50 55 60
Thr Asp Asn Lys Val Arg Thr Trp Gly Asn Ala Lys Asp Gln Ile Lys
65 70 75 80
Gln Ser Tyr Gly Thr Gly Phe Lys Ile His Glu Asn Lys Pro Ser Thr
85 90 95
Val Pro Lys Lys Gly Trp Ile Ala Val Phe Thr Ser Gly Ser Tyr Glu
100 105 110
Gln Trp Gly His Ile Gly Ile Val Tyr Asp Gly Gly Asn Thr Ser Thr
115 120 125
Phe Thr Ile Leu Glu Gln Asn Trp Asn Gly Tyr Ala Asn Lys Lys Pro
130 135 140
Thr Lys Arg Val Asp Asn Tyr Tyr Gly Leu Thr His Phe Ile Glu Ile
145 150 155 160
Pro Val Lys Ala Gly Thr Thr Val Lys Lys Glu Thr Ala Lys Lys Ser
165 170 175
Ala Thr Ser His Glu His Ser Ala Gln Trp Leu Asn Asn Tyr Lys Lys
180 185 190
Gly Tyr Gly Tyr Gly Pro Tyr Pro Leu Gly Ile Asn Gly Gly Met His
195 200 205
Tyr Gly Val Asp Phe Phe Met Asn Ile Gly Thr Pro Val Lys Ala Ile
210 215 220
Ser Ser Gly Lys Ile Val Glu Ala Gly Trp Ser Asn Tyr Gly Gly Gly
225 230 235 240
Asn Gln Ile Gly Leu Ile Glu Asn Asp Gly Val His Arg Gln Trp Tyr
245 250 255
Met His Leu Ser Lys Tyr Asn Val Lys Val Gly Asp Tyr Val Lys Ala
260 265 270
Gly Gln Ile Ile Gly Trp Ser Gly Ser Thr Gly Tyr Ser Thr Ala Pro
275 280 285
His Leu His Phe Gln Arg Met Val Asn Ser Phe Ser Asn Ser Thr Ala
290 295 300
Gln Asp Pro Met Pro Phe Leu Lys Ser Ala Gly Tyr Gly Lys Ala Gly
305 310 315 320
Gly Thr Val Thr Pro Thr Pro Asn Pro Gly Asn Gly Tyr Lys Thr Asn
325 330 335
Lys Tyr Gly Thr Leu Tyr Lys Ser Glu Ser Ala Ser Phe Thr Ala Asn
340 345 350
Thr Asp Ile Ile Thr Arg Leu Thr Gly Pro Phe Arg Ser Met Pro Gln
355 360 365
Ser Gly Val Leu Arg Lys Gly Leu Thr Ile Lys Tyr Asp Glu Val Met
370 375 380
Lys Gln Asp Gly His Val Trp Val Gly Tyr Asn Thr Asn Ser Gly Lys
385 390 395 400
Arg Val Tyr Leu Pro Val Arg Thr Trp Asn Glu Ser Thr Gly Glu Leu
405 410 415
Gly Pro Leu Trp Gly Thr Ile Lys Gly Ser His His His His His His
420 425 430
Gly Gly Ser Lys Asn Lys
435
<210> 107
<211> 439
<212> PRT
<213> 人工序列
<220>
<223> 融合蛋白，包含 LysK內溶素的CHAP域 , 溶葡萄球菌酶的M23
内肽酶域 ，ALE-1的细胞壁结合域，His6标签和合成肽KNK
<400> 107
Met Ala Lys Thr Gln Ala Glu Ile Asn Lys Arg Leu Asp Ala Tyr Ala
1 5 10 15
Lys Gly Thr Val Asp Ser Pro Tyr Arg Val Lys Lys Ala Thr Ser Tyr
20 25 30
Asp Pro Ser Phe Gly Val Met Glu Ala Gly Ala Ile Asp Ala Asp Gly
35 40 45
Tyr Tyr His Ala Gln Cys Gln Asp Leu Ile Thr Asp Tyr Val Leu Trp
50 55 60
Leu Thr Asp Asn Lys Val Arg Thr Trp Gly Asn Ala Lys Asp Gln Ile
65 70 75 80
Lys Gln Ser Tyr Gly Thr Gly Phe Lys Ile His Glu Asn Lys Pro Ser
85 90 95
Thr Val Pro Lys Lys Gly Trp Ile Ala Val Phe Thr Ser Gly Ser Tyr
100 105 110
Glu Gln Trp Gly His Ile Gly Ile Val Tyr Asp Gly Gly Asn Thr Ser
115 120 125
Thr Phe Thr Ile Leu Glu Gln Asn Trp Asn Gly Tyr Ala Asn Lys Lys
130 135 140
Pro Thr Lys Arg Val Asp Asn Tyr Tyr Gly Leu Thr His Phe Ile Glu
145 150 155 160
Ile Pro Val Lys Ala Gly Thr Thr Val Lys Lys Glu Thr Ala Lys Lys
165 170 175
Ser Ala Thr Ser His Glu His Ser Ala Gln Trp Leu Asn Asn Tyr Lys
180 185 190
Lys Gly Tyr Gly Tyr Gly Pro Tyr Pro Leu Gly Ile Asn Gly Gly Met
195 200 205
His Tyr Gly Val Asp Phe Phe Met Asn Ile Gly Thr Pro Val Lys Ala
210 215 220
Ile Ser Ser Gly Lys Ile Val Glu Ala Gly Trp Ser Asn Tyr Gly Gly
225 230 235 240
Gly Asn Gln Ile Gly Leu Ile Glu Asn Asp Gly Val His Arg Gln Trp
245 250 255
Tyr Met His Leu Ser Lys Tyr Asn Val Lys Val Gly Asp Tyr Val Lys
260 265 270
Ala Gly Gln Ile Ile Gly Trp Ser Gly Ser Thr Gly Tyr Ser Thr Ala
275 280 285
Pro His Leu His Phe Gln Arg Met Val Asn Ser Phe Ser Asn Ser Thr
290 295 300
Ala Gln Asp Pro Met Pro Phe Leu Lys Ser Ala Gly Tyr Gly Lys Ala
305 310 315 320
Gly Gly Thr Val Thr Pro Thr Pro Asn Pro Gly Asn Gly Tyr Lys Thr
325 330 335
Asn Lys Tyr Gly Thr Leu Tyr Lys Ser Glu Ser Ala Ser Phe Thr Ala
340 345 350
Asn Thr Asp Ile Ile Thr Arg Leu Thr Gly Pro Phe Arg Ser Met Pro
355 360 365
Gln Ser Gly Val Leu Arg Lys Gly Leu Thr Ile Lys Tyr Asp Glu Val
370 375 380
Met Lys Gln Asp Gly His Val Trp Val Gly Tyr Asn Thr Asn Ser Gly
385 390 395 400
Lys Arg Val Tyr Leu Pro Val Arg Thr Trp Asn Glu Ser Thr Gly Glu
405 410 415
Leu Gly Pro Leu Trp Gly Thr Ile Lys Gly Ser His His His His His
420 425 430
His Gly Gly Ser Lys Asn Lys
435
<210> 108
<211> 438
<212> PRT
<213> 人工序列
<220>
<223> 融合蛋白，包含 LysK內溶素的CHAP域 , 溶葡萄球菌酶的M23
内肽酶域 ，ALE-1的细胞壁结合域，His6标签和合成肽KNK；包括突变K17E
和H51Q；不含N末端甲硫氨酸
<400> 108
Ala Lys Thr Gln Ala Glu Ile Asn Lys Arg Leu Asp Ala Tyr Ala Glu
1 5 10 15
Gly Thr Val Asp Ser Pro Tyr Arg Val Lys Lys Ala Thr Ser Tyr Asp
20 25 30
Pro Ser Phe Gly Val Met Glu Ala Gly Ala Ile Asp Ala Asp Gly Tyr
35 40 45
Tyr Gln Ala Gln Cys Gln Asp Leu Ile Thr Asp Tyr Val Leu Trp Leu
50 55 60
Thr Asp Asn Lys Val Arg Thr Trp Gly Asn Ala Lys Asp Gln Ile Lys
65 70 75 80
Gln Ser Tyr Gly Thr Gly Phe Lys Ile His Glu Asn Lys Pro Ser Thr
85 90 95
Val Pro Lys Lys Gly Trp Ile Ala Val Phe Thr Ser Gly Ser Tyr Glu
100 105 110
Gln Trp Gly His Ile Gly Ile Val Tyr Asp Gly Gly Asn Thr Ser Thr
115 120 125
Phe Thr Ile Leu Glu Gln Asn Trp Asn Gly Tyr Ala Asn Lys Lys Pro
130 135 140
Thr Lys Arg Val Asp Asn Tyr Tyr Gly Leu Thr His Phe Ile Glu Ile
145 150 155 160
Pro Val Lys Ala Gly Thr Thr Val Lys Lys Glu Thr Ala Lys Lys Ser
165 170 175
Ala Thr Ser His Glu His Ser Ala Gln Trp Leu Asn Asn Tyr Lys Lys
180 185 190
Gly Tyr Gly Tyr Gly Pro Tyr Pro Leu Gly Ile Asn Gly Gly Met His
195 200 205
Tyr Gly Val Asp Phe Phe Met Asn Ile Gly Thr Pro Val Lys Ala Ile
210 215 220
Ser Ser Gly Lys Ile Val Glu Ala Gly Trp Ser Asn Tyr Gly Gly Gly
225 230 235 240
Asn Gln Ile Gly Leu Ile Glu Asn Asp Gly Val His Arg Gln Trp Tyr
245 250 255
Met His Leu Ser Lys Tyr Asn Val Lys Val Gly Asp Tyr Val Lys Ala
260 265 270
Gly Gln Ile Ile Gly Trp Ser Gly Ser Thr Gly Tyr Ser Thr Ala Pro
275 280 285
His Leu His Phe Gln Arg Met Val Asn Ser Phe Ser Asn Ser Thr Ala
290 295 300
Gln Asp Pro Met Pro Phe Leu Lys Ser Ala Gly Tyr Gly Lys Ala Gly
305 310 315 320
Gly Thr Val Thr Pro Thr Pro Asn Pro Gly Asn Gly Tyr Lys Thr Asn
325 330 335
Lys Tyr Gly Thr Leu Tyr Lys Ser Glu Ser Ala Ser Phe Thr Ala Asn
340 345 350
Thr Asp Ile Ile Thr Arg Leu Thr Gly Pro Phe Arg Ser Met Pro Gln
355 360 365
Ser Gly Val Leu Arg Lys Gly Leu Thr Ile Lys Tyr Asp Glu Val Met
370 375 380
Lys Gln Asp Gly His Val Trp Val Gly Tyr Asn Thr Asn Ser Gly Lys
385 390 395 400
Arg Val Tyr Leu Pro Val Arg Thr Trp Asn Glu Ser Thr Gly Glu Leu
405 410 415
Gly Pro Leu Trp Gly Thr Ile Lys Gly Ser His His His His His His
420 425 430
Gly Gly Ser Lys Asn Lys
435
<210> 109
<211> 439
<212> PRT
<213> 人工序列
<220>
<223> 融合蛋白，包含 LysK內溶素的CHAP域 , 溶葡萄球菌酶的M23
内肽酶域 ，ALE-1的细胞壁结合域，His6标签和合成肽KNK；包括突变K17E
和H51Q
<400> 109
Met Ala Lys Thr Gln Ala Glu Ile Asn Lys Arg Leu Asp Ala Tyr Ala
1 5 10 15
Glu Gly Thr Val Asp Ser Pro Tyr Arg Val Lys Lys Ala Thr Ser Tyr
20 25 30
Asp Pro Ser Phe Gly Val Met Glu Ala Gly Ala Ile Asp Ala Asp Gly
35 40 45
Tyr Tyr Gln Ala Gln Cys Gln Asp Leu Ile Thr Asp Tyr Val Leu Trp
50 55 60
Leu Thr Asp Asn Lys Val Arg Thr Trp Gly Asn Ala Lys Asp Gln Ile
65 70 75 80
Lys Gln Ser Tyr Gly Thr Gly Phe Lys Ile His Glu Asn Lys Pro Ser
85 90 95
Thr Val Pro Lys Lys Gly Trp Ile Ala Val Phe Thr Ser Gly Ser Tyr
100 105 110
Glu Gln Trp Gly His Ile Gly Ile Val Tyr Asp Gly Gly Asn Thr Ser
115 120 125
Thr Phe Thr Ile Leu Glu Gln Asn Trp Asn Gly Tyr Ala Asn Lys Lys
130 135 140
Pro Thr Lys Arg Val Asp Asn Tyr Tyr Gly Leu Thr His Phe Ile Glu
145 150 155 160
Ile Pro Val Lys Ala Gly Thr Thr Val Lys Lys Glu Thr Ala Lys Lys
165 170 175
Ser Ala Thr Ser His Glu His Ser Ala Gln Trp Leu Asn Asn Tyr Lys
180 185 190
Lys Gly Tyr Gly Tyr Gly Pro Tyr Pro Leu Gly Ile Asn Gly Gly Met
195 200 205
His Tyr Gly Val Asp Phe Phe Met Asn Ile Gly Thr Pro Val Lys Ala
210 215 220
Ile Ser Ser Gly Lys Ile Val Glu Ala Gly Trp Ser Asn Tyr Gly Gly
225 230 235 240
Gly Asn Gln Ile Gly Leu Ile Glu Asn Asp Gly Val His Arg Gln Trp
245 250 255
Tyr Met His Leu Ser Lys Tyr Asn Val Lys Val Gly Asp Tyr Val Lys
260 265 270
Ala Gly Gln Ile Ile Gly Trp Ser Gly Ser Thr Gly Tyr Ser Thr Ala
275 280 285
Pro His Leu His Phe Gln Arg Met Val Asn Ser Phe Ser Asn Ser Thr
290 295 300
Ala Gln Asp Pro Met Pro Phe Leu Lys Ser Ala Gly Tyr Gly Lys Ala
305 310 315 320
Gly Gly Thr Val Thr Pro Thr Pro Asn Pro Gly Asn Gly Tyr Lys Thr
325 330 335
Asn Lys Tyr Gly Thr Leu Tyr Lys Ser Glu Ser Ala Ser Phe Thr Ala
340 345 350
Asn Thr Asp Ile Ile Thr Arg Leu Thr Gly Pro Phe Arg Ser Met Pro
355 360 365
Gln Ser Gly Val Leu Arg Lys Gly Leu Thr Ile Lys Tyr Asp Glu Val
370 375 380
Met Lys Gln Asp Gly His Val Trp Val Gly Tyr Asn Thr Asn Ser Gly
385 390 395 400
Lys Arg Val Tyr Leu Pro Val Arg Thr Trp Asn Glu Ser Thr Gly Glu
405 410 415
Leu Gly Pro Leu Trp Gly Thr Ile Lys Gly Ser His His His His His
420 425 430
His Gly Gly Ser Lys Asn Lys
435
<210> 110
<211> 438
<212> PRT
<213> 人工序列
<220>
<223> 融合蛋白，包含 LysK內溶素的CHAP域 , 溶葡萄球菌酶的M23
内肽酶域，ALE-1的细胞壁结合域， His6标签和合成肽KNK；包括突变
K17E,H51Q, K28N, T86A和G154S；不含N末端甲硫氨酸
<400> 110
Ala Lys Thr Gln Ala Glu Ile Asn Lys Arg Leu Asp Ala Tyr Ala Glu
1 5 10 15
Gly Thr Val Asp Ser Pro Tyr Arg Val Lys Asn Ala Thr Ser Tyr Asp
20 25 30
Pro Ser Phe Gly Val Met Glu Ala Gly Ala Ile Asp Ala Asp Gly Tyr
35 40 45
Tyr Gln Ala Gln Cys Gln Asp Leu Ile Thr Asp Tyr Val Leu Trp Leu
50 55 60
Thr Asp Asn Lys Val Arg Thr Trp Gly Asn Ala Lys Asp Gln Ile Lys
65 70 75 80
Gln Ser Tyr Gly Ala Gly Phe Lys Ile His Glu Asn Lys Pro Ser Thr
85 90 95
Val Pro Lys Lys Gly Trp Ile Ala Val Phe Thr Ser Gly Ser Tyr Glu
100 105 110
Gln Trp Gly His Ile Gly Ile Val Tyr Asp Gly Gly Asn Thr Ser Thr
115 120 125
Phe Thr Ile Leu Glu Gln Asn Trp Asn Gly Tyr Ala Asn Lys Lys Pro
130 135 140
Thr Lys Arg Val Asp Asn Tyr Tyr Ser Leu Thr His Phe Ile Glu Ile
145 150 155 160
Pro Val Lys Ala Gly Thr Thr Val Lys Lys Glu Thr Ala Lys Lys Ser
165 170 175
Ala Thr Ser His Glu His Ser Ala Gln Trp Leu Asn Asn Tyr Lys Lys
180 185 190
Gly Tyr Gly Tyr Gly Pro Tyr Pro Leu Gly Ile Asn Gly Gly Met His
195 200 205
Tyr Gly Val Asp Phe Phe Met Asn Ile Gly Thr Pro Val Lys Ala Ile
210 215 220
Ser Ser Gly Lys Ile Val Glu Ala Gly Trp Ser Asn Tyr Gly Gly Gly
225 230 235 240
Asn Gln Ile Gly Leu Ile Glu Asn Asp Gly Val His Arg Gln Trp Tyr
245 250 255
Met His Leu Ser Lys Tyr Asn Val Lys Val Gly Asp Tyr Val Lys Ala
260 265 270
Gly Gln Ile Ile Gly Trp Ser Gly Ser Thr Gly Tyr Ser Thr Ala Pro
275 280 285
His Leu His Phe Gln Arg Met Val Asn Ser Phe Ser Asn Ser Thr Ala
290 295 300
Gln Asp Pro Met Pro Phe Leu Lys Ser Ala Gly Tyr Gly Lys Ala Gly
305 310 315 320
Gly Thr Val Thr Pro Thr Pro Asn Pro Gly Asn Gly Tyr Lys Thr Asn
325 330 335
Lys Tyr Gly Thr Leu Tyr Lys Ser Glu Ser Ala Ser Phe Thr Ala Asn
340 345 350
Thr Asp Ile Ile Thr Arg Leu Thr Gly Pro Phe Arg Ser Met Pro Gln
355 360 365
Ser Gly Val Leu Arg Lys Gly Leu Thr Ile Lys Tyr Asp Glu Val Met
370 375 380
Lys Gln Asp Gly His Val Trp Val Gly Tyr Asn Thr Asn Ser Gly Lys
385 390 395 400
Arg Val Tyr Leu Pro Val Arg Thr Trp Asn Glu Ser Thr Gly Glu Leu
405 410 415
Gly Pro Leu Trp Gly Thr Ile Lys Gly Ser His His His His His His
420 425 430
Gly Gly Ser Lys Asn Lys
435
<210> 111
<211> 439
<212> PRT
<213> 人工序列
<220>
<223> 融合蛋白，包含 LysK內溶素的CHAP域 , 溶葡萄球菌酶的M23
内肽酶域，ALE-1的细胞壁结合域，His6标签和合成肽KNK；包括突变
K17E,H51Q, K28N, T86A和G154S
<400> 111
Met Ala Lys Thr Gln Ala Glu Ile Asn Lys Arg Leu Asp Ala Tyr Ala
1 5 10 15
Glu Gly Thr Val Asp Ser Pro Tyr Arg Val Lys Asn Ala Thr Ser Tyr
20 25 30
Asp Pro Ser Phe Gly Val Met Glu Ala Gly Ala Ile Asp Ala Asp Gly
35 40 45
Tyr Tyr Gln Ala Gln Cys Gln Asp Leu Ile Thr Asp Tyr Val Leu Trp
50 55 60
Leu Thr Asp Asn Lys Val Arg Thr Trp Gly Asn Ala Lys Asp Gln Ile
65 70 75 80
Lys Gln Ser Tyr Gly Ala Gly Phe Lys Ile His Glu Asn Lys Pro Ser
85 90 95
Thr Val Pro Lys Lys Gly Trp Ile Ala Val Phe Thr Ser Gly Ser Tyr
100 105 110
Glu Gln Trp Gly His Ile Gly Ile Val Tyr Asp Gly Gly Asn Thr Ser
115 120 125
Thr Phe Thr Ile Leu Glu Gln Asn Trp Asn Gly Tyr Ala Asn Lys Lys
130 135 140
Pro Thr Lys Arg Val Asp Asn Tyr Tyr Ser Leu Thr His Phe Ile Glu
145 150 155 160
Ile Pro Val Lys Ala Gly Thr Thr Val Lys Lys Glu Thr Ala Lys Lys
165 170 175
Ser Ala Thr Ser His Glu His Ser Ala Gln Trp Leu Asn Asn Tyr Lys
180 185 190
Lys Gly Tyr Gly Tyr Gly Pro Tyr Pro Leu Gly Ile Asn Gly Gly Met
195 200 205
His Tyr Gly Val Asp Phe Phe Met Asn Ile Gly Thr Pro Val Lys Ala
210 215 220
Ile Ser Ser Gly Lys Ile Val Glu Ala Gly Trp Ser Asn Tyr Gly Gly
225 230 235 240
Gly Asn Gln Ile Gly Leu Ile Glu Asn Asp Gly Val His Arg Gln Trp
245 250 255
Tyr Met His Leu Ser Lys Tyr Asn Val Lys Val Gly Asp Tyr Val Lys
260 265 270
Ala Gly Gln Ile Ile Gly Trp Ser Gly Ser Thr Gly Tyr Ser Thr Ala
275 280 285
Pro His Leu His Phe Gln Arg Met Val Asn Ser Phe Ser Asn Ser Thr
290 295 300
Ala Gln Asp Pro Met Pro Phe Leu Lys Ser Ala Gly Tyr Gly Lys Ala
305 310 315 320
Gly Gly Thr Val Thr Pro Thr Pro Asn Pro Gly Asn Gly Tyr Lys Thr
325 330 335
Asn Lys Tyr Gly Thr Leu Tyr Lys Ser Glu Ser Ala Ser Phe Thr Ala
340 345 350
Asn Thr Asp Ile Ile Thr Arg Leu Thr Gly Pro Phe Arg Ser Met Pro
355 360 365
Gln Ser Gly Val Leu Arg Lys Gly Leu Thr Ile Lys Tyr Asp Glu Val
370 375 380
Met Lys Gln Asp Gly His Val Trp Val Gly Tyr Asn Thr Asn Ser Gly
385 390 395 400
Lys Arg Val Tyr Leu Pro Val Arg Thr Trp Asn Glu Ser Thr Gly Glu
405 410 415
Leu Gly Pro Leu Trp Gly Thr Ile Lys Gly Ser His His His His His
420 425 430
His Gly Gly Ser Lys Asn Lys
435

Claims (34)

1.多肽，其包含：
i)LysK内溶素的CHAP域或其与LysK内溶素的CHAP域表现出至少80％的序列同一性的变体；
ii)溶葡萄球菌酶的M23内肽酶域或其与溶葡萄球菌酶的M23内肽酶域表现出至少80％的序列同一性的变体；
iii)ALE-1的细胞壁结合域(CBD)或其与ALE-1的细胞壁结合域(CBD)表现出至少90％的序列同一性的变体，和
iv)选自下组的进一步的肽：抗微生物肽、两亲性肽、阳离子肽、疏水性肽、寿司肽和防御素。
2.根据权利要求1所述的多肽，其中所述多肽包含：
i)SEQ ID NO:1的氨基酸序列，或其与SEQ ID NO:1的氨基酸序列表现出至少80％序列同一性的变体，
ii)SEQ ID NO:3的氨基酸序列，或其与SEQ ID NO:3的氨基酸序列表现出至少80％序列同一性的变体，和/或
iii)SEQ ID NO:5的氨基酸序列，或其与SEQ ID NO:5的氨基酸序列表现出至少90％序列同一性的变体。
3.根据权利要求2所述的多肽，其中所述多肽包含SEQ ID NO:1、SEQ ID NO:3和/或SEQID NO:5的氨基酸序列，优选其中所述多肽包含SEQ ID NO:3的氨基酸序列和/或SEQ IDNO:5，甚至更优选其中多肽包含SEQ ID NO:5的氨基酸序列。
4.根据权利要求1所述的多肽，其中所述多肽包含：
i)SEQ ID NO:2的氨基酸序列，或其与SEQ ID NO:2的氨基酸序列表现出至少80％序列同一性的变体，
ii)SEQ ID NO:4的氨基酸序列，或其与SEQ ID NO:4的氨基酸序列表现出至少80％序列同一性的变体，和/或
iii)SEQ ID NO:6的氨基酸序列，或其与SEQ ID NO:6的氨基酸序列表现出至少90％序列同一性的变体。
5.根据权利要求4所述的多肽，其中与SEQ ID NO:2的氨基酸序列相比，SEQ ID NO:2的变体序列表现出选自K16E、K27N、H50Q、T85A、G154C、G154S的一种或多种突变，优选其中与SEQ ID NO:2的氨基酸序列相比，所述变体序列至少表现出突变K16E和H50Q，甚至更优选其中与SEQ ID NO:2的氨基酸序列相比，所述变体序列至少表现出突变K16E、K27N、H50Q、T85A和G154S。
6.根据前述权利要求中任一项的多肽，其中所述多肽包含选自SEQ ID NO:2、SEQ IDNO:4和SEQ ID NO:6的一种或多种序列，优选其中所述多肽包含SEQ ID NO:4和/或SEQ IDNO:6的氨基酸序列，甚至更优选其中所述多肽包含SEQ ID NO:6的氨基酸序列。
7.根据前述权利要求中任一项的多肽，其中所述多肽包含SEQ ID NO:2、SEQ ID NO:4和SEQ ID NO:6的氨基酸序列，优选其中所述多肽包含SEQ ID NO:8或SEQ ID NO:9的氨基酸序列，优选其中多肽包含SEQ ID NO:9的氨基酸序列。
8.根据前述权利要求中任一项的多肽，其中元件或其相应变体的顺序从N末端至C末端是：
a)CHAP域-M23内肽酶-ALE-1的CBD-肽，或
b)肽-CHAP域-M23内肽酶-ALE-1的CBD。
9.根据前述权利要求中任一项的多肽，其中所述多肽包含标签，优选His6标签。
10.根据前述权利要求中任一项的多肽，其中所述进一步的肽是：
i)选自SEQ ID NO:40至94的SEQ ID No的抗微生物肽，
ii)选自SEQ ID NO:98、SEQ ID NO:99和SEQ ID NO:100的两亲性肽，
iii)选自SEQ ID NO:11至39的SEQ ID No的阳离子肽，
iv)根据SEQ ID NO:95的寿司肽，或
iii)选自SEQ ID NO:96和SEQ ID NO:97的疏水性肽。
11.根据权利要求10所述的多肽，其中所述进一步的肽选自SEQ ID NO:38和SEQ IDNO:94。
12.根据前述权利要求中任一项的多肽，其中所述多肽包含选自SEQ ID NO:105、SEQID NO:107、SEQ ID NO:109和SEQ ID NO:111的序列和与这四种序列中的任一种至少90％相同的变体序列。
13.根据前述权利要求中任一项的多肽，其中所述多肽的长度不超过500个氨基酸。
14.根据前述权利要求中任一项的多肽，其中所述多肽能够降解金黄色葡萄球菌细菌的细胞壁。
15.包含SEQ ID NO:1的变体序列的多肽，其中所述变体序列与SEQ ID NO:1的氨基酸序列包含至少80％的序列同一性，并且其中与SEQ ID NO:1的氨基酸序列相比，所述变体序列表现出突变H8N和/或T43A。
16.包含SEQ ID NO:2的变体序列的多肽，其中所述变体序列与SEQ ID NO:2的氨基酸序列包含至少80％的序列同一性，并且其中与SEQ ID NO:2的氨基酸序列相比，所述变体序列表现出选自下组的一种或多种突变：K16E、K27N、H50Q、T85A，、G154C、G154S。
17.根据权利要求16的多肽，其中与SEQ ID NO:2的氨基酸序列相比，所述变体序列至少表现出突变K16E和H50Q。
18.根据权利要求16所述的多肽，其中与SEQ ID NO:2的氨基酸序列相比，所述变体序列至少表现出突变K16E、K27N、H50Q、T85A和G154S。
19.根据权利要求16-18中任一项的多肽，其中所述多肽还包含肽聚糖水解酶的至少一个催化域的序列和/或表现出抗细菌活性，特别是针对金黄色葡萄球菌的抗细菌活性。
20.根据权利要求19所述的多肽，其中所述变体序列与SEQ ID NO:109或SEQ ID NO:111的氨基酸序列表现出至少95％的序列同一性，优选与SEQ ID NO:109或SEQ ID NO:111的氨基酸序列表现出至少96％，至少97％，至少98％或至少99％的序列同一性。
21.根据权利要求19所述的多肽，其中所述多肽包含SEQ ID NO:108或SEQ ID NO:110的序列。
22.编码权利要求1至21中任一项的多肽的核酸。
23.载体，其包含权利要求22的核酸。
24.宿主细胞，其包含根据权利要求1至21中任一项的多肽，根据权利要求22的核酸，或根据权利要求23的载体。
25.组合物，其包含权利要求1至21中任一项的多肽，权利要求22的核酸，权利要求23的载体和/或权利要求24的宿主细胞，并且还包含载体，稀释剂或赋形剂。
26.根据权利要求25所述的组合物，其中所述载体，稀释剂或赋形剂是药学上可接受的。
27.根据权利要求25所述的组合物，其中所述组合物是水溶液(优选缓冲液或生理溶液)、粉末、栓剂、乳液、悬浮液、凝胶、洗剂、乳膏、油膏剂(salve)、软膏剂、可注射溶液、糖浆剂、喷雾剂、吸入剂、涂层组合物，优选植入物涂层组合物、支架涂层组合物或导管涂层组合物、或生物材料，优选骨水泥。
28.装置，其包含根据权利要求1至21中任一项的多肽、根据权利要求22的核酸、根据权利要求23的载体、根据权利要求24的宿主细胞和/或根据权利要求25至27中任一项的组合物。
29.根据权利要求28所述的装置，其中所述装置是医学装置，特别地其中所述装置是植入物、支架、导管、膏药、敷布(compress)或敷料(dressing)。
30.根据权利要求1至21中任一项的多肽、根据权利要求22的核酸、根据权利要求23的载体、根据权利要求24的宿主细胞和/或根据权利要求25至27中任一项的组合物，其用于通过手术或疗法治疗人或动物身体的方法或对人或动物身体实施的诊断方法。
31.根据权利要求30使用的多肽、核酸、载体、宿主细胞或组合物，其中所述多肽、核酸、载体、宿主细胞或组合物用于治疗或预防受试者中的细菌感染，特别是用于治疗或预防金黄色葡萄球菌细菌感染。
32.根据权利要求30使用的多肽、核酸、载体、宿主细胞或组合物，其中所述多肽、核酸、载体、宿主细胞或组合物用于治疗受试者的伤口，特别是急性伤口，例如医源性伤口或慢性伤口，或用于治疗皮炎或耳炎。
33.根据权利要求1至21中任一项的多肽、根据权利要求22的核酸、根据权利要求23的载体、根据权利要求24的宿主细胞和/或根据权利要求25至27中任一项的组合物用于对无生命的表面、组合物和/或物体进行消毒的用途，特别是在医院环境或医生诊室中。
34.根据权利要求1至21中任一项的多肽、根据权利要求22的核酸、根据权利要求23的载体、根据权利要求24的宿主细胞和/或根据权利要求25至27中任一项的组合物用于防止无生命表面，组合物和/或物体被细菌污染，特别是用于防止金黄色葡萄球菌细菌的污染的用途。
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