Construction and expression of prokaryotic expression plasmid of vacA gene of coccoid helicobacter pylori

Objective To construct pr okaryot ic expression plasmid of vacA gene of coccoid Helicobacter pylori.Methods VacA gene was digested with restr ictio n endonucleases from recombinant plasmid pMD-18T-vacA and was inserted into expression vector pET32a(+)by subclone technique,then the recombinants were transformed into E.coli BL21 and ident ified by restriction endonucleases digestion and PCR.Then the geneticaly engineered bacteria of including pET32a(+)vacA plasmids were induced by IPTG,the expression product was analyzed by SDS-PAGE and densitometric scanning.Results The positive recombinant plasmids pET32a(+)vacA were identified by restriction endonucleases dig estion and PCR,in accordance with the expected results.Sequence analysis showed that the vacA gene homology of coccoid.H.pylori and reported in GenBand was 99.2%.Plasmid pET23a(+)vacA could express a specific 156KD a protein in E.coli BL21,the protein accouterd for 15.5% of total protein of recombinant bacterial.Conclusion The prokar yotic expression plasmids,which contain vacA gene of coccoid H.pylori are successfully constructed.Plasmid pET32a(+)vacA can express specific protein in E.coli BL21.This work will help study ing the biologic character of VacA protein and DNA vaccine against H.pylori infection.