Expression Of Helicobacter Pylori KatA And The Fusion Protein Of KatA With E Coli Heat-labile Enterotoxin B Subunit

In order to study the role of H. pylori catalase as candidate antigen of genetic engineering vaccines in preventing against H. pylori infection, we cloned the KatA DNA fragments amplified by PCR from genome of Chinese Helicobacter pylori clinical isolate(No.98010) into prokaryotic plasmid pET-28a to constructed a recombinant vector. The immunogenicity of KatA expressed by the recombinant vector in E.coli BL21 was investigated with inoculated rabbits. Afer that, ltB and katA fusion gene was spliced by overlap extension. LTB and KatA fusion protein was expressed by corresponding recombinant vector. Our work might provide a preparation of H. pylori vaccines on the base of KatA and LTB. The main results were as following:1. The gene encoding the KatA of H.pylori was obtained from Chinese Helicobacter pylori clinical isolate. The full length of H. pylori catalasegene consisted of 1518bp encoding 505 amino acids. Compared with KatA of four H. pylori strains accessed in Genbank, the indentities of nucleotides was up to 95% and their similarities of deduced amino acids was above 97%.2. An excellent catalase protein expression vector pET-28a-katA was successfully constructed.3. The optimal conditions of KatA expression in recombinant engineering-bacterium pET-28a-katA/E.coli BL21 were as follow: IPTG concentration was 500μmol/L, culture medium OD₆00 was 0.8 around and induction time 4⁶ h. Under these conditions, KatA production could reach to 27.4% of total bacterium protein. 4. The technique of recombinant KatA purification was established. Western blot proved that the recombinant KatA was recognised by anti-Hp serum. The sequences of N-terminal 5 amino acids were in accordance with those of native KatA.5. Recombinant KatA could effectively stimulated the production of anti-KatA IgG in rabbits, the antibody could react with KatA in various H. pylori strain. These suggested that the recombinant KatA be conservative antigen and posses a good immunogenicity.6. KatA-LTB fusion protein expression vector pET-28a-KL wasobtained, which could effectively expressed the fusion protein in E.coli BL21. The recombinant fusion protein posses good immunoreactivities and mucosal adjuvant properties, could not only react with KatA and LTB, but also bind to GM1 ganglioside.