伤寒沙门菌fljA样基因缺陷变异株的制备

Construction of a fljA Like Gene Deleted Mutant in Salmonella Enterica Serovar Typhi

目的:为探讨z66鞭毛抗原阳性伤寒沙门菌的fljA样基因的功能,制备沙门菌fljA样基因缺陷变异株。方法:根据伤寒沙门菌fljA样基因序列,设计PCR特异性引物并在5'末端加接酶BglII位点,扩增fljA样基因上、下游片段,用BglII消化后,定向连接成fljA样基因的缺损性同源性核苷酸片段,先克隆至pGEM T质粒,再经BamHI酶切后导入自杀质粒pGMB151,电转化入伤寒沙门菌(GIFU10007),进行同源重组。用PCR观察重组现象,将完全重组的菌株作为fljA样基因的缺陷变异株,并通过相应的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fljA样基因缺失231个碱基。结论:成功构建伤寒沙门菌fljA样基因缺陷变异株,为进一步研究其在伤寒沙门菌鞭毛抗原表达的调控作用奠定了基础。

Objective: For investigating the function of fljA like gene, the deletion mutant of the fljA like 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 fljA like gene to amplify two homologous DNA fragments. A BglII site was added in 5'-termini of the reverse primer of the upper-stream fragment and the forward primer of the down stream fragment. After amplification and purification, two fragments were digested by BglII and ligated as the homologous recombinant DNA fragment, which contained the defective target gene. Then it was cloned into the pGEM-T easy vector. The homologous recombinant DNA fragment, was cut by BamHI from the vector and cloned into the suicide plasmid pGMB151. It was then transferred into the target cell of S. enterica serovar Typhi GIFU 10007, which was a z66 antigen-positive strain by electroporation. The recombination was visualized by PCR, and the complete recombinant strain was selected as the fljA like gene deleted mutant strain and confirmed by the corresponding sequencing analysis. Results:A 231bp deletion of the fljA like gene was confirmed by PCR and sequencing analysis. Conclusion:The fljA like gene deleted mutant of S. enterica serovar Typhi was generated successfully, and it was a foundation to study the function of the fljA like gene in regulation of flagellin gene expression of S. enterica serovar Typhi.