伤寒沙门菌fljB∶∶lacZ变异株的制备.DOCVIP

临床医学论文-伤寒沙门菌fljB∶∶lacZ变异株的制备 【摘要】? 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3 550 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。 【关键词】? 伤寒沙门菌； fljB基因； β-半乳糖苷酶基因； 基因融合 　　［Abstract］? Objective: To study the mechanisms of expressional regulation of fljB∶z66, the fljB∶∶lacZ mutant strain of Salmonella enterica serovar Typhi was constructed. Methods： The mutant strain was prepared by the method of homologous recombination mediated by suicide plasmid. The special primers with KpnⅠ and SalⅠ 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 SmaⅠ 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. 　　［Key words］? Salmonella enterica serovar Typhi; fljB; lacZ; gene fusion 　　伤寒沙门菌(Salmonella enterica serovar Typhi)是一种具有鞭毛、革兰阴性人类致病菌。鞭毛是沙门菌运动的基础，在其侵袭过程中具有极重要的作用。沙门菌鞭毛素基因的表达受鞭毛调节因子FlhDC和FliA促进，并受环境影响，可由RcsB、PhoP、OmpR等多种中央调节因子参与调节［1-5］。长期以来人们一直认为伤寒沙门菌是单相菌株，1981年Guinee首次在从印度尼西亚分离获得的伤寒沙门菌中发现一种新的鞭毛抗原，命名为z66抗原［6］。2004年黄新祥等首次成功克隆了伤寒沙门菌z66抗原的编码基因［7］。最近研究表明，z66抗原的编码基因属于伤寒沙门菌的二相鞭毛素fljB基因，且位于一罕见的线性质粒中［8］。z66鞭毛抗