Experimental Studies On Helicobacter Pylori Multicomponent Vaccine Fused With Intra-molecular Adjuvant

Helicobacter pylori (Hp) is a gram negative, spiral, microaerobic bacterium that colonizes human gastric mucosa and infects a large amount of population worldwide, leading to specific gastric diseases. There is a dilemma existing in anti-infection therapy because the single-drug therapies hardly eliminate Hp, while the costs of multi-drugs are expensive. The most crucial thing is that the current therapies give low healing rate, and can not prevent reinfection. Besides, the increasing of drug resistance strain makes anti-Hp treatment more and more complex. Therefore, the effective immunization of vaccine provides a promising way against Hp infection.The study was on the basis of the established fusion protein HspA-HpaA-UreB414 (rHHU) developed by our laboratory, aiming at constructing a fusion gene with hhu fragment and E.coli heat-labile enterotoxin subunit B (ltB) gene and obtaining the multicomponent vaccine fused with intra-molecular adjuvant LTB. Purified the recombinant fusion protein and then immunized the Mongolia gerbils orally with target protein as well as other compared vaccine protocols. The study provides a certain extent of experimental evidence for a new form of Hp vaccine.The study has been involved in the following aspects:1. The gene fragments hhu and ltB were cloned respectively by PCR from the conserved plasmid pET-28a-hhu and pET-28a-ltB. We combined ltB fragment with hhu gene by SOE PCR (splicing by overlap extension), setting ltB gene ahead of hhu gene, which constructed a fused gene form lhhu. The lhhu gene was constructed into prokaryotic expression vector pET-28a(+) by restriction endonucleases NcoⅠand XhoⅠ, the positive recombinant was transformed in E.coli BL21(DE3) and E.coli JM109(DH3) respectively.2. plhhu/BL21 and plhhu/JM109 were induced with IPTG. at 25℃, 37℃, 42℃respectively. It was confirmed by SDS-PAGE and western blotting with different antibodies...