幽门螺杆菌cagL基因缺失株的建立

Construction of cagL gene mutant in Helicobacter pylori

目的:构建幽门螺杆菌Ⅳ型分泌系统cagL基因缺失株,为研究cagL基因的功能奠定基础。方法:利用同源重组原理,PCR扩增该基因编码区两侧序列,作为同源臂,构建出带卡那霉素抗性标志的载体,采用电穿孔法将其转化入受体菌中,并经PCR验证后获得了cagL基因缺失株;突变株和野生株分别与胃上皮细胞GES-1共培养后,检测其对CagA蛋白转运的影响。结果:构建cagL基因缺失的自杀质粒,并获得一株cagL基因缺失株;CagA转运实验表明,cagL基因缺失后,导致幽门螺杆菌CagA转运功能的丧失。结论:成功获得了幽门螺杆菌cagL基因缺失株,该基因参与CagA蛋白的转运,是该菌IV型分泌系统中重要组成成分之一。

Objective：To construct the cagL gene deletion mutant of Helicobacter pylori and investigate the function of cagL gene.Methods：We designed and amplified the upstream homologous fragment and downstream homologous fragment of cagL gene through PCR.We constructed the suicide plasmid.Then we introduced the suicide plasmid into Helicobacter pylori by eletroporation and checked the deletion mutant by PCR.We detected the ability of CagA translocation through performing the coculture of Helicobacter pylori,the deletion mutant and gastric epithelial cell respectively.Results：We constructed the suicide plasmid successfully and got the cagL gene deletion mutant.Through CagA protein translocation assay,we detected that cagL interrupted the translocation of CagA in the deletion mutant.Conclusion：We constructed cagL deletion mutant of Helicobacter pylori.This study suggests that cagL gene,a component of CagA translocation,may be an important component of type Ⅳ secretion apparatus of Helicobacter pylori.