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 Nco I and Xho I , 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 antibodiesthat rLHHU had two bands of the newly expressed protein which were close in molecular weight. It was speculated that the smaller one is the degradation product.3. The hhu gene and ltB gene was fused again in another form, setting ltB gene after hhu gene and a flexible linker GGGSGGGS was introduced into them. The hhul gene was constructed into pET-28a(+) and the positive plasmid phhul was transformed in E.coli BL21(DE3). After induced with IPTG at 37°C, the rHHUL protein was expressed and confirmed by SDS-PAGE and western blotting, showing an approximate molecular weight 55.2KD of target protein, and the expressing ratio was indicated as 20% of total bacterial protein by UVP scan.4. The secondary structure, three-dimensional structure of rHHUL protein and the flexibility, hydrophilicity of linker were predicted by computer programme.5. The expressing form of fusion protein was proved to be inclusion body by examining the ultrasonic ingredient of engineering bacteria. The isoelectric point of target protein was predicted by Anthe_ 5.0. After washing of inclusion body and affinity chromatography purification by AKTA-explore 100 system, we got the target fusion protein with purity of more than 80%.6. The fusion protein rHHUL retains an immunoreactive of every component involved in it as well as a binding activity with GMlganglioside.7. 56 Mongolia gerbils were divided into four groups randomly, then immunized orally with 150ug multicompotent protein fused with LTB (rHHUL), multicompotent protein without LTB (rHHU), multicompotent protein mixed with LTB (rHHU + LTB) and PBS respectively on 0,7,14,28 day. 10 days after last immunization, gerbils were challenged with 10 CFU Hp strain. 30 days after challenge, the samples of serum and gastric tissue of every group were collected. The level of serologic specific IgG assessed by ELISA indicated that immunized groups have a significant high level compared with PBS control group (p<0.001). Hp culture, rapid urease test (RUT) and special PCR were used to evaluate the immune protection on stomachs samples and the result showed the protective efficacies were 91.5%, 74.6% and 84.3% respectively.To sum up, vaccine plays a crucial and promising role in controlling the worldwide infection of Hp. It is important to investigate Hp multicomponent protein fused with intra-molecular adjuvant, which undoubtedly settled a foundation for the further developingof gene engineered vaccine against Hp.