幽门螺杆菌cag1基因缺陷株的构建与鉴定

Construction and identification of cag1 gene mutant of Helicobacter pylori

目的:构建幽门螺杆菌(Helicobacter pylori,H.pylori)细胞毒素相关基因致病岛(cytotoxin associated gene pathogenicity island,cag PAI)中的第1个编码基因hp0520/cag1基因缺陷株,为进一步研究cag1基因在cag PAI中的功能奠定基础。方法:使用同源重组技术,通过PCR扩增出cag1基因两侧同源臂序列,酶切纯化后分别连接于带卡那霉素抗性标志的载体两端,构建成cag1基因自杀质粒。将该质粒通过电穿孔导入受体野生株中,通过抗性筛选与PCR验证得到cag1基因缺陷株,与人胃上皮GES-1细胞共培养后,与野生株比较,观察细胞形态变化。结果:成功敲除H.pylori cag1基因,获得cag1基因缺陷株;与野生株相比,cag1基因缺陷株破坏细胞的能力降低。结论:成功获得幽门螺杆菌cag1基因缺陷株;cag1基因缺失后H.pylori对GES-1细胞的毒力减弱。

Objective： To establish cag1 gene mutant of Helicobacter pylori（H.pylori） NCTC11637 for exploring the influence of cag1 gene on cytotoxin associated gene pathogenicity island（cag PAI）.Methods： The cag1 gene deletion fragment（upstream and downstream of the open reading frames） was amplified by two pairs of primers designed according to the sequence of cag1 and its upper and down streams.Then electroporation was used to transfect the suicide plasmid into H.pylori.Positive clones were screened out on the basis of kanamycin resistance and confirmed by PCR and DNA sequencing.GES-1 cells were infected with either the obtained cag1 gene mutant or the wild-type strain for 8 h.Morphological changes of infected cells were observed under microscope.Results： We generated and identified a cag1 gene mutant of H.pylori.The cag1 gene mutant caused much less scattering and elongation of GES-1 cells than the H.pylori wildtype strain.Conclusion： The cag 1 gene mutant of H.pylori was successfully obtained,which proved that the absence of cag1 strongly affected the virulence of H.pylori.