洪伟;陈学书;吴昌学;郜双林;赵艳;谢渊;陈峥宏;官志忠;周建奖;
HONG Wei;CHEN Xueshu;WU Changxue;GAO Shuanglin;ZHAO Yan;XIE Yuan;CHEN Zhenghong;GUAN Zhizhong;ZHOU Jianjiang;Key Laboratory of Molecular Biology,Guizhou Medical University;Department of Microbiology,School of Basic Medical Sciences,Guizhou Medical University;

• 1:贵州医科大学分子生物学重点实验室
• 2:贵州医科大学基础医学院微生物教研室

摘要(Abstract)：

目的:通过同源重组基因失活方法,构建幽门螺旋杆菌(H.pylori)cag A基因失活突变株。方法:使用分子克隆方法构建包含cagA基因上、下游同源臂及氯霉素转乙酰基酶(CAT)的自杀型cag A基因靶向失活质粒pHG1,经PCR验证后将该质粒电转化入H.pylori 11639菌株中,通过氯霉素抗性筛选出cagA基因失活突变株。结果:pHG1质粒构建成功并转化进入H.pylori 11639菌株,氯霉素筛选获得cagA基因组区段发生重组的Δcag A突变株(Δcag A::gro EL-cat);PCR结果表明,cagA基因突变株构建成功。结论:使用自杀型同源重组质粒pHG1成功构建幽门螺旋杆菌(H.pylori)cag A基因失活突变株。
Objective: To construct H. pylori cagA gene inactivation mutant by using homologous recombination method. Methods: Suicide cagA gene targeted inactivation plasmid pHG1 with the upstream and downstream homologous arms of cagA gene and chloramphenicol transacetylase( CAT)marker was constructed by standard molecular clone method. After PCR validation,pHG1 plasmid was transformed into H. pylori 11639 strain by electroporation and ΔcagA inactivation mutants was screened by chloramphenicol resistance. Results: pHG1 plasmid was successfully constructed and transformed into the H. pylori 11639 strain. ΔcagA: gro EL-cat mutants was obtained by chloramphenicol screening.PCR results showed that cagA gene was successfully constructed. Conclusion: H. pylori cagA gene inactivation mutant strain was successfully construlted by suicide homologous recombination plasmid pHG1.

关键词(KeyWords)： 幽门螺杆菌;质粒;DNA,重组;突变
Helicobacter pylori;plasmid;DNA,recombinant;mutant

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金(No.31560318);; 贵州省科技厅重大专项[黔科合计Z字(2012)4010];; 贵州医科大学博士启动基金[院博合J字(2014)07]

作者(Author): 洪伟;陈学书;吴昌学;郜双林;赵艳;谢渊;陈峥宏;官志忠;周建奖;
HONG Wei;CHEN Xueshu;WU Changxue;GAO Shuanglin;ZHAO Yan;XIE Yuan;CHEN Zhenghong;GUAN Zhizhong;ZHOU Jianjiang;Key Laboratory of Molecular Biology,Guizhou Medical University;Department of Microbiology,School of Basic Medical Sciences,Guizhou Medical University;

Email:

DOI: 10.19367/j.cnki.1000-2707.2015.12.008

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