Construction of vacA-ctxB prokaryotic expression vectors to Helicobacter pylori / 重庆医科大学学报

ABSTRACT

Objective:To lay a foundation of expressing VCTB fusion protein and preparation the oral vaccine for prophylaxis and therapy of H.pylori infection,the prokaryotic expression vectors contained the fusion gene vacA toxic subunit of H.pylori and cholera toxin subunit B(ctxB)was constructed.Methods:The high homogeneity gene fragment was found in comparing VacA toxic subunit gene of domestic typical H.pylori strains.The primers were designed according to vacA and ctxB sequences in the gene bank.VacA toxic subunit gene was amplified by PCR as the template of DNA genome of H.pylori and cloned into plasmid pQE30,which was plasmid pQE30-vacA.The ctxB gene was amplified by PCR as the template of pET32(?)+-ctxB plasmid.The purified ctxB gene was inserted into pQE30-vacA to construct expressing plasmid pQE-vctB of containing vacA and ctxB genes.pQE-vctB was transformed into prokaryotic E.coli Top10.The recombinant plasmid of bacteria cultivated was extracted and purified,and cutted with the incision enzyme to evaluated.Results:The sequence of vacA gene amplified was about 723 bp and the sequence of ctxB gene was about 372 bp.They were consistented with the anticipated length of the DNA.The expressing plasmid pQE-vctB was conformitied with objective gene inserted into pQE30.vctB fusion gene sequenced as 1 092 bp by the sequencing was conformitied with the genebank,and encodes polypeptides of 364 amino acid residues.Conclusion:The prokaryotic expression vectors contained vacA and ctxB fusion gene was constructed successfully and lay a foundation of expression VCTB fusion protein.