Cloning and expression of vacA gene fragment of Helicobacter pylori with coccoid form.

摘要

BACKGROUND: Helicobacter pylori (H. pylori) can transform, in vivo as well as in vitro, from dividing spiral-shaped forms into nonculturable coccoid forms, whose importance in disease transmission and antibiotic treatment failures is unclear. The aim of the present study was to clone and express the vacA gene of coccoid H. pylori and to infer its possible pathogenesis. METHODS: Firstly, coccoid form was obtained from strain NCTC 11637 by exposure to antibiotics in subinhibitory concentrations and collected. Secondly, vacA gene of the coccoid H. pylori was amplified by PCR. After being purified, the target fragment was cloned into plasmid pMD-18T, and the recombinant plasmid pMD-18T-vacA was transformed into E. coli JM109. The sequence of inserted fragment was analyzed. Thirdly, vacA gene from recombinant plasmid pMD-18T-vacA was digested with restriction enzyme and was inserted into expression vector pET32a (+). The positive recombinants were transferred into E. coli BL21 and identified by restriction enzyme digestion and PCR. Finally, the genetically engineered bacteria including pET32a (+)-vacA plasmids were induced by IPTG, the expression was analyzed by SDS-PAGE and gel densitometric scanning. RESULTS: The results revealed that vacA gene of 3888bp was obtained from the coccoid H. pylori genome DNA, recombinant plasmid pMD-18T-vacA constructed were successfully digested by BamH I +Sac I, and the product of digestion was identical with the predicted 1. Sequence analysis also showed that the homology of coccoid and the reported original sequence was 99.8%. Plasmid pET32a (+)-vacA could express a specific 156kDa protein in E. coli BL21, and the protein accounted for 15.5% of the total protein of recombinant bacterial. CONCLUSIONS: The present data indicate that coccoid H. pylori contains complete vacA gene, and could synthesize its protein, which may be related to the disease relapse and transmission when coccoid H. pylori recovers virulence under suitable conditions.