幽门螺杆菌Cag致病岛hp0523基因的缺失及其对CagA蛋白转运能力的影响

Construction of hp0523 gene mutant in Helicobacter pylori Cag-PAI and the influence on the ability of CagA protein translocation

【目的】构建幽门螺杆菌Cag致病岛编码的hp0523基因缺失株,为研究hp0523基因的功能奠定基础。【方法】本研究利用同源重组原理,设计并扩增了hp0523基因上下游同源臂片段,构建hp0523基因缺失自杀质粒pBlueKM40-Δhp0523,电击转化进入幽门螺杆菌后,通过抗生素筛选后并经PCR验证无误后,获得hp0523基因缺失株H.pylori11637Δhp0523;采用幽门螺杆菌与胃癌上皮细胞BGC-823共培养后,分析该基因缺失前后对细胞毒性蛋白CagA转运能力的影响;并分析比较了缺失前后,CagA蛋白的表达能力变化;【结果】构建幽门螺杆菌hp0523基因缺失的自杀质粒,并成功获得一株hp0523基因缺失株;CagA转运实验表明,hp0523基因缺失后,可以导致细菌转运CagA蛋白的能力丧失;此外,还发现hp0523基因缺失可以影响CagA蛋白表达。【结论】成功获得了幽门螺杆菌hp0523基因缺失株,初步研究发现hp0523基因是幽门螺杆菌Cag致病岛中重要致病因子之一,参与CagA蛋白的转运,可能是其转运装置中组成成分之一。

[ Objective] To construct the hp0523 gene mutant of Helicobacter pylori and investigate the function of hp0523 gene. [ Methods] We designed and amplified the upstream homologous fragment and downstream homologous fragment of hp0523 gene via PCR method. We constructed the suicide plasmid pBlueKM40-Ahp0523 based on allelic exchange. We introduced the suicide plasmid pBlueKM40-Ahp0523 into Helicobacter pylori 11637 by electroporation and screened the mutant based on antibiotic selection. We checked the mutant using the PCR and gene sequenced. We performed the cocuhure of Helicobacter pylori and gastric cell BGC-823 and detected the ability of CagA＇s translocation and expression via Western blot. [ Results] We constructed the suicide plasmid pBlueKM40-△hp0523 successfully and got the hp0523 deletion mutant. PCR and gene sequenced results showed the gene hp0523 was deleted. The results of CagA translocation assay showed that hp0523 interrupted the translocation of CagA. The comparison between wild-type and mutant showed that hp0523 affected the expression of CagA. [ Conclusions] We constructed the hp0523 deletion mutant of Helicobacter pylori NCTC11637. This study suggests that hp0523 gene is an important virulence factor,which may be a component of the apparatus for CagA＇s translocation.