摘要
AIM: To clone and sequence the cagA gene fragment of Helicobacter pylori ( H pylori) with coccoid form.METHODS: Hpyloristrain NCTC11637 were transformed to coccoid form by exposure to antibiotics in subinhibitory concentrations. The coccoid Hpyloriwas collected, cagA gene of the coccoid H pyloristrain was amplified by PCR.After purified, the target fragment was cloned into plasmid pMD-18T. The recombinant plasmid pMD-18T-cagA was transformed into E.co/iJM109. Positive clones were screened and identified by PCR and digestion with restriction endonucleases. The sequence of inserted fragment was then analysed.RESULTS: cagA gene of 3 444 bp was obtained from the coccoid H pylorigenome DNA. The recombinant plasmid pMD-18T-cagA was constructed, then it was digested by BamH I+SacI, and the product of digestion was identical with the predicted one. Sequence analysis showed that the homology of coccoid and the reported original sequence H pyloriwas 99.7%.CONCLUSION: The recombinant plasmid containing cagA gene from coccoid Hpylorihas been constructed successfully.The coccoid Hpyloricontain completed cagA gene, which may be related to pathogenicity of them.
AIM:To clone and sequence the cagA gene fragment of Helicobacter pylori(H pylon)with coccoid form. METHODS:Hpyloristrain NCTC11637 were transformed to coccoid form by exposure to antibiotics in subinhibitory concentrations.The coccoid Hpyloriwas collected,cagA gene of the coccoid Hpyloristrain was amplified by PCR. After purified,the target fragment was cloned into plasmid pMD-18T.The recombinant plasmid pMD-18T-cagA was transformed into E.coli JM109.Positive clones were screened and identified by PCR and digestion with restriction endonucleases.The sequence of inserted fragment was then analysed. RESULTS:cagA gene of 3 444 bp was obtained from the coccoid Hpylori genome DNA.The recombinant plasmid pMD-18T-cagA was constructed,then it was digested by BamH I+Sac I,and the product of digestion was identical with the predicted one.Sequence analysis showed that the homology of coccoid and the reported original sequence H pylori was 99.7%. CONCLUSION:The recombinant plasmid containing cagA gene from coccoid H pylorihas been constructed successfully. The coccoid H pylori contain completed cagA gene,which may be related to pathogenicity of them.
