O139群霍乱弧菌TLC、CTX、RTX基因簇缺失株的构建及鉴定

Construction and Identification of TLC,CTX,and RTX Gene Cluster Deletion Mutant of O139 Serogroup Vibrio cholerae

目的构建O139群霍乱弧菌TLC、CTX、RTX基因簇缺失株。方法PCR扩增TLC上游片段(TLC up)、RTX下游片段(RTX down),克隆入pU18中,再在两者间插入氯霉素抗性(Cm)基因,获得重组质粒pUC18-TLC up-Cm-RTX down。酶切回收TLC up-Cm-RTX down片段,克隆入自杀质粒pDS132,构建重组自杀质粒pDS132-TLC up-Cm-RTX down。通过接合将重组自杀质粒转入O139群霍乱弧菌ZJ199693,20%蔗糖选择培养基(Cm抗性)筛选TLC、CTX、RTX基因缺失株。结果PCR、酶切证明重组自杀质粒pDS132-TLC up-Cm-RTXdown构建正确。PCR证实O139群霍乱弧菌ZJ199693TLC、CTX、RTX毒力基因簇成功缺失。结论成功缺失O139群霍乱弧菌ZJ199693TLC、CTX、RTX基因簇,为进一步研究其功能和疫苗构建打下基础。

Objective To construct TLC, CTX and RTX gene cluster deletion mutant of O139 serugroup Vibrio cholerae. Method The upstream fragment of TLC gene cluster（TLCup）, downstream fragment of RTX gene cluster （RTXdown） and the chloromycetin resistance （Cm） gene were PCR amplified and ligated with the plasmid pU18 to create recombinant plasmid pUC18-TLCup-Cm-RTXdown. Then the fragment containing TLCup-Cm-RTXdown fusion was excised and cloned into the suicide plasmid pDS132 to create the recombinant suicide plasmid pDS132-TLCup- Cm-RTXlow. At last the recombinant suicide plasmid was conjugally transfered into the O139 Vibrio cholerae ZJ199693 and the deletion mutant were grown in a modified LB medium containing 20% sucrose and chloromycetin. Result The recombinant suicide plasmid pDS132-TLCup-Cm-RTXlow was verified by PCR and enzyme digestion. The deletion of TLC,CTX and RTX gene clusters of O139 serogroup Vibrio cholerae ZJ199693 was identified by PCR. Conclusion Construction of TLC, CTX and RTX gene cluster deletion mutant of O139 Serogroup Vibrio cholerae will be benefit for the research on the function of TLC, CTX and RTX gene clusters of O139 serogroup Vibrio cholerae and as the O139 serogroup Vibrio cholerae vaccine candidates.