Cloning And Expression Of 1188 Gene Of Helicobacter Pylori And Identification Of Immunogenicity Of The Recombinant Protein

Objective:Choose Hp1188 as a target to obtain the purified recombinant Hp1188 by genetic engineering and then evaluate its immunogenicity and protective efficacy against H.pylori infection both in vitro and in mouse model, finally to determine the feasibility of rHp1188 in H.pylori vaccination.Methods:①The hp1188 gene was amplified from helicobacter pylori NCTC 11637 strain by PCR. The PCR product was inserted into prokaryotic expressing plasmid pQE30 and confirmed by DNA sequencing. The recombinant plasmid pQE30-hp1188 was transformed into E.coli DH5αand was induced for expression with IPTG.②The recombinant protein was purified by Ni2+-NTA agarose and analyzed by SDS-PAGE, Bradford quantification, western blot and double diffusion assays.③ELISA method was used to measure Hp1188-specific antibodies in sera of H.pylori infected patients.④H.pyloni infected mouse model was established and applied in oral vaccination rHp1188 and CTB. The bacterial colonizing density was determined by H. pylori cultivation histological examination with Giemsa staining. HE-stained slides were observed for gastric histopathological changes in immunized mice.Results:①A recombinant plasmid pQE30-hp1188 was constructed. DNA sequence analysis showed one open reading frame of 810bp with a 98% sequence homology with that in Genbank, which coded for polypeptides of 269 amino acids. SDS-PAGE showed that the molecular weight of expressed protein rHp1188 was about 30 kD. The level of soluble expression was above 47% of total cell protein. After purified with Ni2+-NTA, the purity of rHp1188 was above 90% with the concentration up to 1.0mg/ml. Double diffusion test and western blot analyses confirmed that the antibody titer reached 1:16 with fine specificity against hp1188. Western blot showed it could be recognized by rubbit antibodies against H. pylori whole cell.②The indirect ELISA based on rHp1188 was successfully established to assay the anti-Hp1188 in serum, and its sensitivity and specificity was 87.5% and 86.7% respectively.③The infection rate and colonization of helicobacter pylori in the prevntion group was much lower than that of the positive at the same period of infection(P<0.05). Reliefs of the local chronic inflammation in gastric tissues of the mice were also observed in the prevention group.Conclusion:The recombinant expressing vectors pQE30-hp1188-DH5αare successfully constructed. The recombinant Hp1188 protein is highly expressed. The recombinant protein possesses fine antigenicity, and immunization with the recombinant protein is able to inhibit adhesive capacity of H. pylori in vivo.