CN117004590B - 一种抗菌多肽及应用 - Google Patents

一种抗菌多肽及应用 Download PDF

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CN117004590B
CN117004590B CN202310809148.9A CN202310809148A CN117004590B CN 117004590 B CN117004590 B CN 117004590B CN 202310809148 A CN202310809148 A CN 202310809148A CN 117004590 B CN117004590 B CN 117004590B
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polypeptide
antibacterial
resistant
bacteria
cov6
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周磊
林见敏
宋元林
杜春玲
汪小欢
李与凡
王芳
邓燕燕
周霞
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Shanghai Institute Of Major Infectious Diseases And Biosafety
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Abstract

本发明公开了一种抗菌多肽及应用。其来源于新型冠状病毒(SARS‑CoV‑2)的Replicase polyprotein 1a蛋白，其氨基酸序列如SEQ ID No.01或SEQ ID No.03所示。本发明的抗菌多肽对金葡菌等革兰阳性菌和大肠杆菌、肺炎克雷伯菌、鲍曼不动杆菌等常见的临床致病菌具有较强的抗菌活性，对多种多药耐药菌具有较强的抗菌活性，如耐甲氧西林的金葡菌、耐碳青霉烯的大肠杆菌、耐碳青霉烯的肺炎克雷伯菌均对其敏感，特别是耐碳青霉烯的鲍曼不动杆菌对Cov6多肽的MIC低至4μM；本发明抗菌多肽还具有溶血及细胞毒性极低的优点，具备作为药物和食品添加剂开发的条件，具有广泛的应用前景。

Description

一种抗菌多肽及应用
技术领域
本发明涉及一种抗菌多肽及应用，属于生物医药技术领域。
背景技术
抗生素是人类治疗细菌感染性疾病的主要武器。然而，随着抗菌药物的不规范使用，细菌耐药现象越来越严重，甚至出现多种对现有抗菌药物几乎全部耐药的超级细菌。多药耐药菌中最为常见的就是金黄色葡萄球菌(Staphylococcus aureus)，肠杆菌科细菌(Enterobacter species)，铜绿假单胞菌(Pseudomonas aeruginosa)、肺炎克雷伯菌(Klebsiella Pneumoniae)、鲍曼不动杆菌(Acinetobacter baumannii)等多药耐药菌。这几类细菌可通过多种机制产生耐药性。由于现有的抗菌药物作用的机制较为单一，细菌可以通过改变抗菌药物目标蛋白、下调细胞膜通道蛋白表达、产生灭活酶、表达外排泵以及产生生物膜等多种机制产生耐药。尽管人们开发出了诸如利用β内酰胺酶抑制剂等方法来对抗细菌的耐药性，但只能在部分药物和某些细菌中取得了一定的效果，仍然无法治疗大部分的耐药菌感染。因此，开发对多药耐药菌有效的抗菌药物迫在眉睫。
抗微生物多肽(Anti-microbial peptides，AMPs)具有与小分子抗菌物质明显不同的抗菌机制。AMPs多数为具有8-50个氨基酸残基的小分子物质，大多带有正电荷和两亲性，在PH接近中性的水溶液中可形成α螺旋、β折叠等二级结构。AMPs可与细菌细胞膜上的脂质分子层发生非特异性的相互作用，从而破坏细菌胞膜的稳定性，或形成孔状结构使细菌内容物流出导致死亡。部分AMPs还可与细菌胞内的关键酶类或蛋白发生相互作用，从而使菌体生命代谢和/或能量代谢出现障碍导致细菌死亡。因此，AMPs可通过多种机制产生杀菌作用，细菌很难进化出对AMPs的耐药性。尽管AMPs目前还存在者溶血活性强，稳定性差以及生产成本高等缺点，但随着人类对AMPs和细菌生命活动的深入认识，可以采用对多肽进行酰胺化、环化等不同修饰和优化改造等方案来克服以上缺点，为AMPs的实际应用开拓广阔的空间。
目前，已知700多种抗微生物肽序列(www.bbcm.univ.trieste.it/～tossi/search.htm)，包括天蚕抗菌肤(cecropin)、防御素(defensin)、马加宁(magainin)和cathelicidin。
即使目前存在相对大量的可获得的抗微生物肽，仍然存在对可用于抵抗或耐受抗生素和/或其他抗微生物剂的微生物的新的改进的抗微生物肽的增加的需要。此外，溶血及细胞毒性限制了大量抗微生物肽在药物和食品添加剂方面的应用。
发明内容
本发明的目的在于提供一种新型的来源于SARS-CoV-2Replicase polyprotein1a蛋白多肽类抗菌物质。
为了实现上述目的，在本发明的第一方面，提供一种抗菌多肽，所述抗菌多肽具有如SEQ ID No.01或SEQ ID No.03所示的氨基酸序列。
在本发明的第二方面，提供一种编码上述抗菌多肽的核苷酸序列，包括DNA和RNA。
在本发明的第三方面，提供上述抗菌多肽和/或与其功能相同的突变产物在制备防腐产品或抗菌产品中的应用。
优选地，所述抗菌产品包括抗菌生物制品、抗菌添加剂、动物饲料、洗手液、香皂、沐浴液或化妆品。
在本发明的第四方面，提供上述核苷酸序列在制备抗菌产品中的应用。
与现有技术相比，本发明的有益效果在于：
(1)本发明的抗菌多肽Cov6和T5-5对金葡菌等革兰阳性菌和大肠杆菌、鲍曼不动杆菌、肺炎克雷伯菌等常见的临床致病菌具有较强的抗菌活性，对多种多药耐药菌具有较强的抗菌活性，如耐甲氧西林的金葡菌(MRSA)、耐碳青霉烯的大肠杆菌(CRE)、耐碳青霉烯的肺炎克雷伯菌(CRKP)均对其敏感，特别是耐碳青霉烯的鲍曼不动杆菌(CRAB)对Cov6多肽的MIC低至4μM；
(2)本发明的抗菌多肽具有溶血及细胞毒性极低的优点，具备作为药物和食品添加剂开发的条件，具有广泛的应用前景和极高的价值。
附图说明
图1为Cov6多肽的螺旋图(A)和二级结构预测图(B)；
图2为抗菌多肽Cov6对标准菌株金黄色葡萄球菌(A)、大肠杆菌(B)和铜绿假单胞菌(C)的抑菌曲线；
图3为抗菌多肽Cov6和T5-5的溶血活性测试结果；
图4为膜去极化试验检测不同浓度的多肽对大肠杆菌内膜电位的影响。
具体实施方式
为使本发明更明显易懂，兹以优选实施例，并配合附图作详细说明如下。
实施例
1.抗菌多肽Cov6和T5-5的来源：
多肽序列SEQ ID No.01(表1)命名为Cov6，是来源于SARS-CoV-2Replicasepolyprotein 1a蛋白的一段氨基酸序列，该蛋白在UniProKB数据库(https://www.uniprot.org/uniprotkb/)的编号为：P0DTC1·R1A_SARS2。通过对该蛋白的第2754-2775号氨基酸序列(SEQ ID No.02，表1)进行突变和优化改造获得。另外一个多肽序列SEQID No.03(表1)命名为T5-5，同样是来源于SARS-CoV-2Replicase polyprotein 1a蛋白的一段氨基酸序列，是对其进行不同的优化方案获得。另外，按照3个特定核苷酸碱基(密码子)编码1个氨基酸残基的规则，可以查到该多肽对应的核苷酸序列。但由于编码同一个氨基酸的对应密码子可能有2-3个，因此，核苷酸序列不唯一但数量有限。核苷酸序列可用于制作表达多肽的模板。
表1.Cov6和T5-5多肽及其来源多肽序列
SEQ ID No.01 LKKVITKIIKGVNNWLKQLGKV
SEQ ID No.02 TTKIALKGGKIVNNWLKQLIKV
SEQ ID No.03 LKKIVTKIGKIVNNWLKQLGKV
2.抗菌多肽Cov6的理化性质分析：
Cov6多肽含有22个氨基酸残基，相对分子质量为：2521.17；通过在线工具Heliquest计算疏水性(Hydrophobicity，H)值为0.423，疏水力距(Hydrophoticitymoment，μH)值为0.764，含有6个净正电荷(charges，c)(图1A，表2)，通过在线工具ExpasyProtParam预测其等电点为：10.70，脂肪族指数145.91，不稳定指数-9.67(表2)。通过在线工具Pep-fold预测为典型的α螺旋结构(图1B)。以上计算和预测结果显示，该多肽具有合理的疏水性，其疏水性残基分布均匀，含有的正电荷数和典型的α螺旋二级结构均有利于肽和细菌胞膜的磷脂层结合发挥抗菌作用。同时，预测Cov6具有较好的耐热和稳定性，具备抗微生物多肽的一般特性和作为药物开发的基本条件。所用生物信息学工具如下：
物化性质分析网站：
Expasy ProtParam(https://web.expasy.org/protparam/)；
螺旋轮图及疏水性分析网站：
Heliquest(https://heliquest.ipmc.cnrs.fr/)；
二级结构预测网站：
Pep-fold(https://bioserv.rpbs.univ-paris-diderot.fr/services/PEP-FOLD3/)；
表2.Cov6多肽的理化特性
多肽 分子量 H μH c 长度 等电点 脂肪族指数 不稳定指数
Cov6 2521.17 0.423 0.764 +6 22aa 10.70 145.91 -9.67
3.抗菌多肽Cov6和T5-5的抗菌活性检测：
多肽Cov6和T5-5由上海强耀生物技术有限公司通过固相合成法制成冻干粉，纯度在95％以上，并进行脱盐处理。
(1)最低抑菌浓度(Minimum Inhibition Concentration，MIC)检测：
采用临床实验室标准研究所(Clinical and Laboratory Standards Institute，CLSI)推荐的标准肉汤微量稀释法进行MIC检测。待测菌株为：金黄色葡萄球菌(ATCC25923)，大肠杆菌(ATCC25922)和铜绿假单胞菌(ATCC27853)。挑取细菌单克隆，用钙离子调节的MHB2培养基稀释，检测麦氏浊度，将其麦氏浊度调整至0.5McFarland(细菌浊度单位)，此时对应的细菌浓度约为108CFU/ml。稀释100倍后按照每孔50μL接种于96孔板，然后每孔加入50μL浓度梯度稀释的多肽溶液，采用2μM、4μM、8μM、16μM、32μM和64μM浓度梯度，在200rpm摇床37℃共孵育18小时。每小时用酶标仪测定570nm吸光度。标准对照孔加入细菌悬液和MHB2培养基，均为50μL/孔；空白对照孔仅加入MHB2培养基100μL/孔。检测结果显示，Cov6多肽对大肠杆菌(ATCC25922)的MIC为8μM，对金黄色葡萄球菌(ATCC25923)的MIC为8μM，对铜绿假单胞菌(ATCC27853)的MIC>64μM(图2)。T5-5多肽对大肠杆菌(ATCC25922)的MIC为8μM，对金黄色葡萄球菌(ATCC25923)的MIC为64μM，对鲍曼不动杆菌(ATCC19606)的MIC为8μM。
(2)对临床分离的多药耐药菌的抗菌活性检测：
同时检测了该多肽对多种临床分离的多药耐药菌的抗菌活性。这些菌株均来自复旦大学附属中山医院和复旦大学附属中山医院青浦分院微生物室，均为感染性疾病的患者的痰、尿液、血液等标本中分离出的多药耐药菌，对临床上使用的多种抗生素如β-内酰胺类、喹诺酮类、大环内酯类乃至碳青霉烯类抗菌药物具有耐药性。其耐药机制包括产NDM、KPC碳青霉烯酶等。MIC检测结果显示，Cov6多肽对其中多种耐药菌的抗菌效果良好。特别是对多药耐药的鲍曼不动杆菌，其MIC可达4μM(表3)，显示其在对抗耐药菌方面的特殊优势。
表3.Cov6多肽对临床分离的多药耐药菌的抗菌活性
4.抗菌多肽Cov6和T5-5的毒性检测
对Cov6和T5-5多肽进行溶血试验确定其毒性，溶血试验：采集由健康志愿者捐献的1ml肝素抗凝全血，在700×g离心5min，弃去上清。沉淀物用磷酸盐缓冲溶液(PBS)重悬漂洗3次。然后用PBS按照1:40体积稀释。血细胞悬液按照1:1体积比与梯度浓度的多肽(128，64，32，16，8，4，2μM)在37℃条件下共孵育1小时。采用1％Triton-X溶液作为阳性对照，空白PBS溶液作为空白对照，每组设3个复孔。再次700×g离心10min，取各组上清液100μl加入到96孔板，用酶标仪测量OD540nm处的吸光度值。溶血活性计算公式：Hemolysis％＝[A(Sam)–A(Neg)]/[A(Pos)–A(Neg)]×100％；式中A(Sam)代表样品吸光度，A(Neg)为空白对照，A(Pos)为1％Triton-X，完全溶血。溶血试验显示，Cov6和T5-5的溶血活性非常弱，在8倍其大肠杆菌MIC的浓度下溶血性仍低于10％(图3)，说明Cov6和T5-5多肽安全性良好。
5.抗菌多肽Cov6的抗菌机制验证：
膜去极化试验(Membrane depolarization assay)检测细菌内膜电位改变：采用电位敏感性染料DiSC3检测大肠杆菌内膜功能。将大肠杆菌37℃过夜摇菌，转接于新鲜的MHB2培养液中，过夜培养。取菌液在4℃，1000rpm离心10min，用浓度5mM的HEPES缓冲液(含KCl 1M，Disc3荧光染料0.5μM，KCl用于平衡细胞内外的K离子浓度)洗涤重悬。在底部不透明的全黑96孔板每孔加入100μl菌液，采用酶标仪检测检测荧光强度，激发波长为622nm，发射波长为670nm，直至荧光强度基本恒定。然后加入不同浓度的多肽(4×，2×，1×，1/2×，1/4×，1/8×MIC)，按100μl/孔加入到菌液中，37℃避光孵育30分钟，然后采用酶标仪检测荧光强度，激发波长为622nm，发射波长为670nm，直至荧光水平基本不再变化。结果显示随着多肽浓度的升高，荧光强度显著增强，说明多肽对细菌胞膜可产生破坏作用，导致细菌内膜稳定性变差，产生杀菌作用(图4)。
上述实施例仅为本发明的优选实施例，并非对本发明任何形式上和实质上的限制，应当指出，对于本技术领域的普通技术人员，在不脱离本发明的前提下，还将可以做出若干改进和补充，这些改进和补充也应视为本发明的保护范围。

Claims (5)

1. 一种抗菌多肽，其特征在于，所述抗菌多肽的氨基酸序列如SEQ ID No. 01或SEQID No. 03所示。
2.编码权利要求1所述的抗菌多肽的核酸。
3.权利要求1所述的抗菌多肽在制备抗菌产品中的应用。
4.如权利要求3所述的应用，其特征在于，所述抗菌产品包括抗菌添加剂、动物饲料、洗手液、香皂、沐浴液或化妆品。
5.权利要求2所述的核酸在制备抗菌产品中的应用。
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