Construction and characterization of bivalent vaccine candidate expressing HspA and Mr18000 OMP from Helicobacter pylori

AIM: To construct a recombinant vector which can express outer membrane protein (OMP) with Mr18 000 and heat shock protein A (HspA) from Helicobacter pylori(H.pylori)in E.coli BL21, and to exploit the possibility for obtaining the vaccine conferring protection from H. pylori infection.METHODS: The target gene of HspA was amplified from H.pylori chromosome by PCR, and then inserted into the prokaryotic expression vector pET32a (+) by restrictive endonuclease enzyme kpn Ⅰ, BamH Ⅰ simultaneously. The recombinant vector was used to sequence, and then together with pET32a (+)/Omp18, digested by restrictive endonuclease enzyme Hind Ⅲ and BamH Ⅰ simultaneously. pET32a(+)/HspA and Omp18 were recovered from 1% agarose gel by gel kit, and ligated with T4 ligase by BarmH Ⅰ digested viscidity end. The recombinant plasmid of pET32a(+)/HspA/Omp18 was transformed and expressed in E. coli BL21 (DE3) under induction of IPTG. After purification, its antigenicity of the fusion protein was detected by Western blot.RESULTS: Enzyme digestion analysis and sequencing showed that the target genes were inserted into the recombinant vector, composed of 891 base pairs, encoded objective polypeptides of 297 amino acid residues. Compared with GenBank reported by Tomb et al, there were 1.3 %and 1.4 % differences in obtained H. pylori nucleotide sequence and amino acid residues, respectively. SDS-PAGE analysis showed that relative molecule mass (Mr) of the expressed product was Mr 51 000,Mr of protein expressed by pET32a (+) was about Mr 20 000, and soluble expression product accounted for 18.96 % of total bacterial protein.After purification with Ni+2-NTA agarose resins, the purification of recombinant fusion protein was about 95 %. Western blot showed that recombinant fusion protein could be recognized by the patients′ serum infected with H. pylori and anti-Omp18 monoclone, suggesting that this protein had good antigenicity.CONCLUSION: The gene coding for H. pylori Mr18 000OMP and HspA was cloned and expressed successfully. The re