Cloning And Sequencing Of VacA, CagA Gene Fragment Of Helicobacter Pylori With Coccoid Form

Objective To clone and expression vacA, cagA gene from coccoid Helicobacter pylori and to infer its possible pathogenisis, and to offer an experimetic refference for anti-H.pylori vaccine preparing.Methods Firstly, coccoid form was obtained from strain NCTC11637 by exposure to antibiotics in subinhibitory concentrations and collected. Secondly, vacA, cagA gene of the coccoid H.pylori were amplified by PCR. After purified, the target fragments were cloned into plasmid pMD-18T, and the recombinant plasmid pMD-18T-vacA, pMD-18T-cagA were transformed into E.coli JM109. The sequence of inserted fragments were analyzed. Thirdly, vacA and cagA gene from recombinant plasmid pMD-18T-vacA, pMD-18T-cagA were digested with restriction enzyme and were inserted into expression vector pET32a (+), respectively. The positive recombinants were transferred into E.coli BL21 and identified by restriction enzyme digestion and PCR. Finally, the genetically engineered bacteria of including pET32a (+)-vacA and pET32a (+)-cagA plasmids were induced by IPTG, respectively, the expression was analyzed by SDS-PAGE and gel dinsitometric scanning.Results The results revealed that vacA gene of 3888bp and cagA gene of 3444bp were obtained from the coccoid H.pylori genome DNA, recombinant plasmid pMD-18T-vacA, pMD-18T-cagA constructed was successfully digested by BamH I +Sac I , and the products of digestion were identical with the predicted one,respectively. Sequence analysis also showed that the homology of coccoid and the reported original sequence were 99.8% and 99.7%, respectively. Plasmid pET32a (+)-vacA and pET32a (+)-cagA could express a specific 156kDa and 148 kDa protein in E.coli BL21, respectively, and the VacA protein accoutered for 15.5% of the total protein of recombinant bacterial.Conclusion The present data indicate that coccoid H.pylori contain complete vacA, cagA gene, and could synthesize its protein, respectively, which may be related to the disease relapse and transmission when coccoid H.pylori recover virulence under suitable conditions.