鼠伤寒沙门菌spvBC基因编辑株的构建

利用λRed重组系统和pBAD原核表达载体构建鼠伤寒沙门菌spvBC质粒毒力基因修饰菌株,为深入探究沙门菌毒力基因spv的功能和致病机制及宿主抗感染免疫提供工具菌。以pKD4为模板,PCR扩增含spvBC同源臂的卡那霉素抗性基因以构建同源打靶片段,再将其电转入含有质粒pKD46的鼠伤寒沙门菌中进行同源重组,随后将质粒pCP20电转导入阳性转化子,消除卡那霉素抗性基因,PCR鉴定敲除株的构建。PCR扩增含酶切位点的spvBC基因片段,扩增产物与原核表达载体pBAD/gⅢ分别双酶切后连接构建pBAD-spvBC重组质粒,PCR筛选阳性菌落并测序鉴定。将构建成功的pBAD-spvBC重组质粒电转导入spvBC敲除株中,Western blot测定不同浓度L-阿拉伯糖诱导SpvB和SpvC蛋白表达情况。PCR结果表明鼠伤寒沙门菌spvBC基因敲除成功;PCR及测序结果表明pBAD-spvBC重组质粒构建成功,Western blot结果表明13 mmol/L L-阿拉伯糖可诱导SpvB和SpvC蛋白正常表达。λRed重组系统可用于沙门菌质粒上大片段基因的敲除,pBAD原核表达载体可用于沙门菌质粒上大片段基因的回补,丰富了细菌质粒的基因修饰和编辑策略。

Construction of spvBC Gene Editing Strains of Salmonella typhimurium

λRed recombination system and pBAD prokaryotic expression vector were used to construct spvBC gene editing strains of Salmonella typhimurium,which may provide tools for further study on the pathogenic mechanisms and functions of spv as well as its role in host anti-infection immunity.Plasmid pKD4 was used as template,and kanamycin resistance gene containing spvBC homologous arm was amplified to construct homologous linear DNA fragment via PCR.Then the linear fragment was electrically transferred to a recombinant strain of S.typhimurium with pKD46.After the recombination,plasmid pCP20 was electrically transferred to positive colonies to delete the kanamycin resistance gene,and gene deletion was identified by PCR.spvBC gene fragment containing enzyme loci was amplified by PCR.Both the amplified product and the prokaryotic expression vector pBAD/gⅢ were double digested and then ligated to construct recombinant plasmid pBAD-spvBC.Positive colonies were screened by PCR and identified by sequencing.Finally,the constructed pBAD-spvBC recombinant plasmid was electrically transferred to spvBC deletion strain.The expression of SpvB and SpvC induced by L-arabinose at different concentrations was determined by Western blot.PCR results indicated that the knockout of spvBC in S.typhimurium was successful.PCR and sequencing results demonstrated the successful construction of the recombinant plasmid pBAD-spvBC,Western blot results showed 13 mmol/L L-arabinose induced normal expression of SpvB and SpvC.λRed recombination system can be used to knock out large gene fragments on Salmonella plasmid,as well as pBAD prokaryotic expression vector can be used to complement large fragments on Salmonella plasmid,which enriches the gene modification and editing strategies of bacterial plasmids.