Cloning And Expression Of The UreA Gene Of Helicobacter Pylori And The Preliminary Study On Its Immunogenicity

Helicobacter pylori (HP) is a pathogenic bacterium leading to many digestive tract diseases in human beings, has an extensive infectivity and these infectious diseases are difficult to be healed clinically. It is a promising way to use immune protection against Hp infection.Objective To investigate the immunogenicity and cross immune protection of urease sub-unit A antigen of Helicobacter pylori strain 26695, the ureA gene was cloned and subcloned into prokaryotic expression vector, and the ureA protein was expressed and purified, and the purified protein was used to immunize domestic rabbit for antiserum against ureA protein. Both rabbit and patient posstive antisera were used to perform antiserum cross reaction and immune protection between animal and patient antibodies with Enzyme-linked immunosorbent assay (ELISA). This experiment was performed for this aim to probe a possibility of using uerA immunogenicity in prevention and theropy disease from Helicobacter pylori infection via specific animal antibodies or monoclonal antibodies.Methods (1) UreA gene fragments were amplified by polymerase chain reaction (PCR) technique from HP genome DNA as a template. The PCR product was cloned into pMD 18-T vector and transformed into E. coli strain DH5a for desired gene identification. The ureA fragments were inserted directly into expressing vector pBV220-IL1 to construct pBV220-IL1-ureA, and the recombinant plasmid was sequenced and bioinformatic analysis.(2) The expression of pBV220-IL1-ureA was induced by 42℃temperature, the result was analysed with SDS-PAGE, the ureA identification was tested by means of Western-blotting, and ureA protein was separated and purified with SDS-PAGE cutting gel method.(3) The purified protein, mixed with complete and incomplete Freund adjuvant, was used to immunized domestic rabbit for obtaining antiserum against ureA and the specific bindings of ureA immunogenic protein with the antisera from immunized rabbit and patient were checked by ELISA. The cross protection between rabbit and human antisura was tested using competive binding reactions with ureA protein.Results (1) A 714 base pairs fragment was obtained by PCR amplication and it shared 99% homology with the knowable ureA gene sequence of HP strain 26695 in GenBank data.(2) The prokaryotic expression vector pBV220-IL1-ureA was correctly constructed and the fusion prokaryotic ureA protein about 41kDa was induced by the heat shock at 42℃temperature and purified with gel cutting method.(3) The purified ureA protein was used to immunize domestic rabbit and the rabbit antiserum on the concentration about the 1:1.28 X 104 titre. The competitive cross protections between rabbit antiserum and patient antiserum were identified by ELISA and showed perfect competition between two antisura.Conclutions (1) The recombinant plasmid pBV220-IL1-ureA was constructed successfully which was confirmed to be correct by sequencing. The homogenicity of nucleic acid and that of amino acid was high with Genbank data. (2) The ureA protein was expressed, purified and used to immunize animal. The experimental data revealed nicer immunogenicity. (3) The specific binding between ureA protein and rabbit antibody and Competition experiments showed that rabbit antiserum and human sera with cross reaction could make a competitive immune protection. This experiment was made a basis for further research on prevention and theropy diseases from Helicobacter pylori infection via polyclonal antibodies or monoclonal antibodies.