| Helicobacter pylori is a microaerophilic, spiral and gram-negative bacillus first isolated from human gastric antral epithelium in 1982. It is now recognized as a human-specific gastric pathogen that colonizes the stomachs of at least half the world's population, and there are approximately new infected thousands people annually. In some individulals, their infections are associated with the development of peptic ulcer, gastric adenocarcinoma, mucosa-associated lymphoid tissue (MALT) lymphoma and primary gastric non-Hodgkin's lymphoma, moreover with extradigestive diseases. This organism was recently categorized as a class I carcinogenetic factor by the World Health Organization, and direct evidence of carcinogenesis was recently demonstrated in an animal model. Although there are many methods in eradication H. pylori infection, such as bi-, tri- pharmic therapy, the definitive curative effects were acquired, a serial of antibiotics was used to lead to resistant H. pylori produced, meantime medical side effects, patient's endure andcompliance were challenged. Because of those mentioned above, the interests of scientists in H. pylori vaccine have been significantly increasing, many investigators have been investigating H. pylori vaccine to reduce and prevent H. pylori infection, eradicate diseases associated with H. pylori infection. Immunization against this bacterium represents a cost-effective strategy to reduce global H. pylori-gastric cancer and peptic ulcer rates. To date, H. pylori vaccine candidate antigens identified include the urease enzyme, VacA, and so on. Mr18 000,26 000 outer membrane proteins and HspA are outer membrane proteins of H. pylori, and the vaccines prepared with HspA was used to inoculate Balb/c mice, 70~80% of experimented mice were protected from H. pylori infection. In order to acquire a better immunocompetent effect, some investigators suggested that bi-valuable antigens vaccine was possibly superior to single antigen. So in this study, we constricted recombinant vectors containing genes encoding outer membrane protein (OMP) with Mr18 000,26 000 OMP and HspA from H. pylori respectively, expressed in prokaryotic expression E. coli BL21, and DH5a (PREP4), and explored the possibility obtaining the vaccine conferring protection from H. pylori infection and a diagnostic reagent kit quickly detecting H. pylori infection.PART ICLONING AND EXPRESSION OF RECOMBINANT PLASMIDS OF 18kDa, 26kDa OUTER MEMBRANE PROTEIN GENES OF H. PYLORIObjective: To construct recombinant vectors containing genes encoding outer membrane protein (OMP) with 18kDa, 26kDa from H. pylori, express in prokaryotic expression E. coli BL21, and DH5a (PREP4), and explore the possibility obtaining the vaccine conferring protection from H. pylori infection and a diagnostic reagent kit quickly detecting H. pylori infection respectively. Methods: The target genes encoding Mr18 000,26 000 OMP of H. pylori wereamplified respectively from H. pylori chromosome by PCR. After objective genes and prokaryotic expression vector pET32a (+), pQE30 digested with BamH I,Hind Ⅲ simultanously, the purified objective genes were inserted into the compatible sites of prokaryotic expression vectors pET32a (+), pQE30 respectively, by using T4 DNA ligase at a molar ratio of 4:1 at 4℃ overnight. The recombinant vectors were identified by PCR and restriction endonuclease enzyme digestion. The recombinant vectors were correspondly transformed and expressed in E. coli BL21 (DE3), DH5a under induction of IPTG. The expression products were analyzed by SDS-PAGE and identified through Western blot test. Induced recombinant fusion proteins with large scale were purified with Ni2+-NTA agarose after sonicated by ultrasonic wave. The antigenicities of recombinant fusion proteins expressed in E. coli BL21, DH5a were investigated by ELISA or western blot and immunized BALB/c mice.Results: Enzyme digestion analysis and sequencing showed that the target genes had been subcloned into recombinant vector respectively, but...
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