维氏气单胞菌脂蛋白Lpp基因缺失株的构建及其部分生物学特性分析  

Construction and partial characteristic analysis of lipoprotein Lpp gene mutant in Aeromonas veronii

作　　者：宋格格 盛天鸽 张俊辉[1] 王文东[1] 杨振国[1] 卢强[1] SONG Ge-ge;SHENG Tian-ge;ZHANG Jun-hui;WANG Wen-dong;YANG Zhen-guo;LU Qiang(Institute of Zoonosis,Jilin University,Changchun 130062,China)

机构地区：[1]吉林大学人兽共患病研究所,吉林长春130062

出　　处：《中国兽医科学》2020年第11期1413-1420,共8页Chinese Veterinary Science

基　　金：国家自然科学基金项目(31672686)。

摘　　要：为了探究维氏气单胞菌毒力因子脂蛋白的致病机制,在全基因测序的基础上,通过同源重组缺失脂蛋白基因。首先扩增脂蛋白基因的上下游同源臂,通过重叠PCR连接后,凝胶回收DNA片段;连接克隆载体pMD18-T,转化DH5α感受态细胞,测序成功后提取质粒进行双酶切,连入相同酶切的Pre112自杀性质粒;依次转化DH5α-λpir、WM3064感受态细胞,作为供体菌;和受体菌野生株进行同源重组,并筛选鉴定。结果显示,成功缺失了维氏气单胞菌脂蛋白基因。生物学特性分析结果发现,缺失株和野生株的生长速度相似,生物膜形成能力降低,鞭毛缺失,游动能力减弱,细菌的毒力降低,溶血活性没有明显变化。因此,成功构建了维氏气单胞菌脂蛋白基因缺失株,并分析了其部分生物学特性,为进一步研究脂蛋白基因的功能奠定了一定的基础。In order to investigate the role of lipoprotein in the pathogenic mechanism of Aeromonas veronii,lipoprotein gene was knocked out by homologous recombination on the basis of whole genome sequencing.After the upstream and downstream homologous arms of the lipoprotein gene were amplified by PCR,the gel recovery products were connected by overlapping PCR amplification and then the connected product was recovered by gel.The ligation product was ligated with the cloning vector pMD18-T and transformed into DH5αcompetent cells.After successful sequencing,the plasmid was extracted for double restriction enzyme digestion and connected to the same enzyme digested suicide plasmid Pre112.Subsequently,the product was transformed into DH5α-λpir and WM3064 competent cells successively and as donor bacteria.The donor was homologously recombinanted with wild strains of recipient bacteria and then was screened for identification.The results showed that the lipoprotein gene of A.veronii was successfully deleted.And the biological characteristics analysis showed that the growth rate of the lipoprotein mutant was similar to wild strain,biofilm formation ability was reduced,flagella was absent,motility was weakened,virulence was reduced and hemolytic activity was not changed.This study successfully constructed the lipoprotein gene mutant of A.veronii and analyzed some of its biological characteristics,which laid the foundation for further study of the function of lipoprotein gene.

关 键 词：维氏气单胞菌 脂蛋白基因 基因缺失 生物学特性 

分 类 号：S852.615[农业科学—基础兽医学]