鼠伤寒沙门菌spvC基因缺陷变异株的制备

Construction of a spvC gene-deleted mutant in Salmonella enterica serovar Typhi

目的:为进一步研究鼠伤寒沙门菌毒力基因spvC的功能,制备鼠伤寒沙门菌spvC基因缺陷变异株。方法:根据鼠伤寒沙门菌spvC基因序列,设计PCR特异性引物,制备spvC基因缺陷性同源性核苷酸片段,导入自杀质粒pCVD442后再导入鼠伤寒沙门菌野生株,进行同源重组,用PCR观察重组现象,将完全重组的菌株作为spvC基因的缺陷变异株,并通过核苷酸序列分析加以确定。结果:PCR及序列分析证实,缺陷变异株的spvC基因缺失711个碱基。结论:成功构建鼠伤寒沙门菌spvC基因缺陷变异株,为进一步研究其在鼠伤寒沙门菌中的功能奠定了基础。

Objective: To investigate the function of spvC gene,the deletion mutant of the spvC gene was constructed in Salmonella enterica serovar Typhi. Methods: As the genomic information,two pair's primers were designed,upper-and down-stream of the spvC gene to amplify two homologous DNA fragments. The homologous recombinant DNA fragment of the defective target gene was cloned into the suicide plasmid pCVD442 which was then transferred into the target cell of S. enterica serovar Typhi. The recombination was visualized by PCR,and the complete recombinant strain was selected as the spvC gene deleted mutant strain and confirmed by the corresponding sequencing analysis. Results: A deletion of 711 bp of the spvC gene was confirmed by PCR and sequencing analysis. Conclusion: The spvC gene-deleted mutant of S. enterica serovar Typhi was generated successfully,and it was a foundation to study the detail functions of the spvC gene in S. enterica serovar Typhi.