副溶血性弧菌噬菌体vB_VpP_1裂解酶的生物信息学分析、原核表达及生物活性鉴定  

Bioinformatics Analysis,Prokaryotic Expression and Biological Activity of Lysin from Vibrio parahaemolyticus Phage vB_VpP_1

作　　者：张德福 杨雯静 刘可 刘青青 白梧桐 李凡 吕欣然 柏雪 檀茜倩 李学鹏 励建荣 ZHANG Defu;YANG Wenjing;LIU Ke;LIU Qingqing;BAI Wutong;LI Fan;LÜXinran;BAI Xue;TAN Xiqian;LI Xuepeng;LI Jianrong(Institute of Ocean Research,National and Local Joint Engineering Research Center of Storage,Processing and Safety Control Technology for Fresh Agricultural and Aquatic Products,Food Safety Key Lab of Liaoning Province,College of Food Science and Engineering,Bohai University,Jinzhou 121013,China)

机构地区：[1]渤海大学食品科学与工程学院,渤海大学海洋研究院,生鲜农产品贮藏加工及安全控制技术国家地方联合工程研究中心,辽宁省食品安全重点实验室,辽宁锦州121013

出　　处：《食品科学》2025年第3期83-89,共7页Food Science

基　　金：渤海大学海洋研究院项目(BDHYYJY2024003);渤海大学食品科学与工程学院预研项目。

摘　　要：为了研究副溶血性弧菌噬菌体vB_VpP_1裂解酶重组表达后对副溶血性弧菌的体外裂解作用,本实验根据全基因组测序及功能分析结果初步判断噬菌体vB_VpP_1的gp32基因片段为裂解酶基因,使用Expasy等工具分析了gp32的氨基酸序列组成和结构等;使用Primer 5.0软件设计引物后克隆至pET-28a(+)载体进行原核表达;纯化后的裂解酶作用于宿主菌及乙二胺四乙酸(ethylene diamine tetraacetic acid,EDTA)处理后的宿主菌,测定裂解酶的活性。结果表明,vB_VpP_1裂解酶的三级结构为球形亲水蛋白,预测含有2个催化结构域,符合革兰氏阴性菌噬菌体裂解酶的基本特征,不存在跨膜区域及信号肽。纯化后的噬菌体vB_VpP_1裂解酶活力约为(1 487±182)U/mg,对已被EDTA破坏细胞壁外膜的副溶血性弧菌表现出较强的裂解能力,但不能有效裂解细胞壁完好的副溶血性弧菌。本研究成功构建噬菌体vB_VpP_1裂解酶原核表达载体,表达、纯化后的裂解酶能够裂解细胞壁被破坏的副溶血性弧菌。To study the in vitro cleavage effect of a recombinant lysin from bacteriophage vB_VpP_1 on Vibrio parahaemolyticus,the gp32 gene fragment of vB_VpP_1 was identified as a lysin gene according to the results of whole-genome sequencing(WGS)and functional analysis.The amino acid sequence and structure of gp32 were analyzed by various tools such as Expasy.Primers were designed using Primer 5.0 software and cloned into the pET-28a(+)vector for prokaryotic expression.The lytic activity of the purified lysin against the host bacterium before and after being treated with ethylene diamine tetraacetic acid(EDTA)was measured.Tertiary structure analysis showed that the lysin was a spherical hydrophilic protein,which was predicted to contain two catalytic domains.This was consistent with the basic characteristics of Gram-negative phage lysins.The lysin had no transmembrane region or signal peptide.The purified bacteriophage vB_VpP_1 had a lysin activity of approximately(1487±182)U/mg,which showed a strong capacity to lyse V.parahaemolyticus with damaged cell walls but not V.parahaemolyticus with intact cell walls.The prokaryotic expression vector for bacteriophage vB_VpP_1 lysin was successfully constructed,and the expressed and purified lysin could lyse V.parahaemolyticus with damaged cell walls.

关 键 词：副溶血性弧菌 噬菌体 裂解酶 生物抗菌剂 食品安全 

分 类 号：TS201.3[轻工技术与工程—食品科学]