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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 an inactivated mutant of cag A gene of H. pylori by homologous recombination inactivation. Methods: The suicide cag A gene targeting inactivation plasmid pHG1 containing upstream and downstream homologous arms of cagA gene and chloramphenicol acetyltransferase (CAT) was constructed by molecular cloning method. After validation by PCR, the plasmid was electroporated into In the H.pylori 11639 strain, the cagA gene inactivating mutant was screened by chloramphenicol resistance. Results: The recombinant plasmids of pHG1 were successfully transformed into H.pylori strain 11639. The recombinant plasmid cagA (Δcag A :: gro EL-cat) was obtained by chloramphenicol screening. The results of PCR showed that cagA mutant Build successfully. Conclusion: The inactivation mutant of cag A gene of H. pylori was successfully constructed by using suicide homologous recombinant plasmid pHG1.