伤寒沙门菌fljB∶∶lacZ变异株的制备

Construction of a fljB∶∶lacZ mutant of Salmonella enterica serovar Typhi

目的:为研究伤寒沙门菌z66鞭毛抗原编码基因fljB的表达调节机制,建立该fljB基因的β-半乳糖苷酶基因(lacZ)插入变异株。方法:采用自杀质粒介导的同源重组方法进行制备。设计加接KpnⅠ和SalⅠ的特异性引物,用PCR扩增获得fljB基因的上、下游同源性DNA片段,并与用KpnⅠ和SalⅠ酶切pSV-β-半乳糖苷酶载体(pSV-β-galactosidase vector)的片段定向连接,将连接片段克隆至自杀质粒pGMB151,再通过电击法将重组质粒转入伤寒沙门菌,在含X-Gal的蔗糖平板上筛选获得同源重组的变异株。结果:经筛选获得阳性克隆,经PCR及序列分析证实,fljB基因中的763 bp被3550 bp的lacZ片段替代。结论:成功构建伤寒沙门菌flj∶∶lacZ突变株,为进一步研究fljB基因表达调节机制奠定了基础。

Objective： To study the mechanisms of expressional mutant strain of Salmonella enterica serovar Typhi was constructed. regulation of fljB： z66, the fljB∷lacZ Methods： The mutant strain was prepared by the method of homologous recombination mediated by suicide plasmid. The special primers with Kpn I and Sal I sites respectively were designed, the upper-and down-stream homologous fragments of the fljB gene were amplified by PCR, a lacZ cassette from the pSV-β-galactosidase vector was linked with the upper-and doun-stream homologous fragments respectively. The resulting product was inserted via Sma I sites into the suicide plasmid pGMB151. S. enterica serovar Typhi were transformed with recombinant plasmid by electroporation transformation, selected in the sucrose plate containing X-Gal. Homologous recombinants were screened by PCR. Results： A 3 550 bp insertion in fljB gene was confirmed instead of 763 bp deletion by PCR and sequencing analysis. Conclusion： The mutant with fljB：：lacZ have been successfully constructed and it was a foundation to study the expression and regulation mechanism of fljB gene in S. Typhi.