Construction Of Recombinant Vaccine Candidate Expressing HspA-UreB Fusion Protein Of Helicobacter Pylori And Its Immunocompetence Study

Helicobacter pylori (H.pylori) is the principal cause of chronic active gastritis, peptic ulcer and a significant factor for gastric cancer. Once this infection is established, it persists for decades and the bacteria are rarely eliminated from the stomach despite specific immune responses to infection. The current treatment for H.pylori infection is based on antibiotics together with a proton pump inhibitor or antacid, which leads to eradication of the bacteria in more than 90% of the cases. However, the eradication treatment has several disadvantages, such as adverse effects of antibiotics, emergence of antibiotic-resistance strains and high cost, especially on patients in developing countries where the prevalence of this infection is still high. So, vaccination is the only way to eradicate H.pylori infection all over the world. It need a kind of safe and effective vaccine.It is demonstrated that Urease B subunit (UreB) and Heat shock protein A subunit(HspA) could stimulate body to raise immunoresponse against challenge of H.pylori. Further more, the activity of Urease can rise more than four times when expressing together with HspA.Gene encoding the structural A subunit of heat shock protein (hspA) and B subunit of urease (ureB) of H.pylori amplified by Polymerase Chain Reaction(PCR) were inserted into prokaryotic expression vector aftersequenced with the direction of hspA-ureB. The fusion protein HspA-UreB was expressed by E.coli. The immunogenicity and immunoreactivity of fusion protein HspA-UreB was assayed by immuning animal and Western blot and this kind fusion protein was purified by using of affinity chromatography . Objective:To construct vaccine candidate expressing HspA-UreB recombinant fusion protein ofH.pylori and analysis the immunogenicity and immunoreactivity . To supply a basis for the study of recombinant gene engine vaccine ofH.pylori.Methods and Results:1. Clone and sequence analysis of hspA gene and ureB gene ofH.pylorihspA gene and ureB gene were amplified by PCR from clinical strain MEL-HP27 and international strain NCTC11637 chromosome DNA. hspA gene and ureB gene were inserted into the clone vector pNEB193 separately, and then were transformed into the TB1 E.coli strain. The recombinant plasmid was detected by Ampicillin antibiotic and blue-white screen and digesting plasmid by restriction endonucleases and specific PCR. The sequence of hspA gene and ureB gene was determined and analyzed by biological software Omiga2.0.It was showed that: (l)The recombinant plasmid of hspA was digested into two fragments which are clone vector fragment(2.7kb) and hspA gene fragment (0.35kb) . 0.35kb hspA gene fragment could be amplified by specific PCR. It was demonstrated that recombinant plasmid of hspA gene (pNHA ) was constructed successfully. (2) The recombinant plasmid of ureB was digested into two fragments which are clone vector fragment (2.7kb) and ureB gene fragment (1.7kb) . 1.7kb ureB gene fragment could be amplified by specific PCR. It was demonstrated that recombinant plasmid of ureB gene (pNUB ) was constructed successfully.Gene sequencing results: (1) hspA gene fragment was 357bp long. It encoded the polypeptide of 118 amino acid residues, corresponding to calculated molecular masses of 13 kDa. The homology in nucleotide acid of hspA gene between H. pylori strains of MEL-HP27 and NCTC11637 was 97.48% and the homology in putative amino acids was 97.46%. hspA sequence of MEL-HP27 had high homology from 95.76% to 97.46% as compared to the nucleotide reported in Genbank and putative amino acids homology was from 95.76% to 97.46%. (2) ureB gene fragment was 1710bp long. It encoded the polypeptide of 569 amino acid residues, corresponding to calculated molecular masses of 63.5 kDa. The homology in nucleotide acid of ureB gene between H.pylori strains of MEL-HP27 and NCTC11637 was 97.67% and the homology putative amino acids was 99.47%. ureB sequence of MEL-HP27 had high homology from 96.08% to 98.30% as compared to the nucleotide reported in Genbank and...