鮰爱德华菌T6SS eivpP基因缺失突变株的构建  

Construction of eivpP in-frame deletion of pathogenic bacterium Edwardsiella ictaluri

作　　者：郭羿 杨晓宇[1] 倪萍 王一婷 郭思聪 马红丽 赵小然 叶仕根[1] GUO Yi;YANG Xiao-yu;NI Ping;WANG Yi-ting;GUO Si-cong;MA Hong-li;ZHAO Xiao-ran;YE Shi-gen(Key Laboratory of Mariculture & Stock Enhancement in North China s Sea, Ministry of Agriculture and Rural Affairs, Dalian Key Laboratory of Marine Animal Disease Prevention and Control, Dalian Ocean University, Dalian 116023, China)

机构地区：[1]大连海洋大学农业农村部北方海水增养殖重点实验室大连市海珍品疾病防控重点实验室,辽宁大连116023

出　　处：《大连海洋大学学报》2019年第1期27-32,共6页Journal of Dalian Ocean University

基　　金：辽宁省自然科学基金资助项目(2015020802)

摘　　要：为研究鮰爱德华菌Edwardsiella ictaluriⅥ型分泌系统(T6SS)EivpP与其他分泌蛋白之间的相互作用及其在细菌致病过程中的作用机制,在克隆到eivpP基因上、下游同源臂的基础上,采用融合PCR技术制备了eivpP基因缺失用的融合片段,并成功克隆到自杀质粒pDM4内,再将重组自杀质粒转化到大肠杆菌S17-1λ(pir)中,与鮰爱德华菌野生菌株ATCC33202共培养。经同源重组和蔗糖负筛获得鮰爱德华菌eivpP基因缺失突变阳性克隆,经PCR鉴定结果显示,突变株未检测出eivpP基因,即eivpP基因彻底敲除成功。本试验结果可为研究eivpP基因在鮰爱德华菌致病过程中的作用机制提供数据资料,为鮰爱德华菌弱毒疫苗的研发提供理论依据。In order to study the interaction between EivpP and other secreted proteins of Edwardsiella ictaluri type Ⅵ secretion system (T6SS) and its mechanism in the pathogenesis of bacteria, the fusion fragment containing the upstream and downstream homologous arms for the eivpP gene deletion was prepared by fusion PCR and successfully cloned into the suicide plasmid pDM4. The recombinant suicide plasmid was transformed into Eschericha coli S17-1λ( pir ), co-cultured with E.ictaluri wild strain ATCC33202, and E. ictaluri eivpP gene deletion mutation positive clone was obtained by homologous recombination and sucrose reverse screening. The results of PCR identification showed that the eivpP gene was not detected in the mutant strain, that is, the eivpP gene was successfully knocked out. The findings provide data with research of the role of eivpP gene in the pathogenesis of E.ictaluri , and provide a theoretical basis for the development of E.ictaluri vaccine as well.

关 键 词：鮰爱德华菌 eivpP基因 基因敲除 缺失突变 

分 类 号：S943[农业科学—水产养殖]