特利波契巴尔通体基因敲除体系的建立

Establishment of in-frame deletion mutant of Bartonella tribocorum

本研究旨在建立基于同源重组技术的特利波契巴尔通体基因敲除方法。研究通过扩增并融合Ⅳ型分泌系统VirB4基因的上下游同源片段,将同源片段与pJM05质粒进行体外拼接,并将pJM05-ΔVirB4质粒转化进入S17-1大肠杆菌中;通过双亲接合转移将大肠杆菌S17-1中的pJM05-ΔVirB4质粒转移到特利波契巴尔通体中进行同源臂交换,产生单交换菌株;随后利用质粒中sacB基因表达产物分解蔗糖对自身产生毒性作用,在蔗糖平板中筛选出22个双交换菌株,通过菌落PCR及测序验证,共确定出6个VirB4基因框内缺失菌株;并且测序结果表明这6个菌株在VirB4基因框内缺失了2 136 bp。

This study aimed to establish an in-frame deletion strain of Bartonella tribocorum(Btr)with suicide vector pJM05 based on sacB counter-selection.A 1.4 kb fragment surrounding VirB4 gene was inserted into pJM05 plasmid through one step multi-fragment one step clone kit,resulting pJM05-ΔVirB4 plasmid.This plasmid was then transformed into S17-1 E.coli for conjugative transfer of parents.Single crossover strain of Btr was screened under kanamycin selection,and then spread on chocolate plates added with 5%sucrose to screen the double crossovers.These strains were further detected by kanamycin resistance,PCR and sequencing.Among 22 double crossover colonies selected,6 were confirmed by PCR to be knockout mutants.Sequencing results demonstrated that each of these mutants contained an in-frame deletion of 2136 bp.