Expression Of Helicobacter Pylori HspA And UreB In Silkworm Pupae And Identification And Evaluation Of Their Immunological Activity

Helicobacter pylori(H. pylori)is a gram negative bacteria that has the exceptional ability to colonize the mucus and mucosa l of our gastric and duodenal. It is present in more than half of the world's population ,In 20% of infected people, chronic infection will lead to clinical symptoms ranging from chronic gastritis and peptic and duodenal ulcer disease to gastric cancer and mucosa-associated lymphoid tissues lymphoma and is considered as a class I carcinogen by the World Health Organization. There is a higher prevalence in the developing countries The currently used eradication strategy focuses on treatment of those individuals that display overt disease symptoms with a combination of two to three antibiotics and a proton pump inhibitor. It has become a standard therapy with eradication rates of 80% and subsequent clinical improvement. this therapy is effective, but remains the increase in antibiotic resistance, re-infection, patient compliance and high cost limit the application of this strategy for the control of H. pylori infection,both prophylactic and therapeutic vaccination of humans against H. pylori infection could be an effective and economic approach to control infection A number of vaccination strategies, which have been demonstrated in animal models, result in a significant reduction of H. pylori colonization on challenge. Most of these strategies for prophylactic and therapeutic vaccination have been based on the use of selected antigens known to correlate with the pathogenesis of H. pylori infection. Many studies have demonstrated that H. pylori in infected mouse stomach can be cleared by oral immunization with H.pylori urease B subunit(UreB). Heat shock protein A subunit (HspA) of H.pylori can be used as a vaccine candidate because of its strong antigenic properties. 1. Expression of EGFP and UreB / HspA of Hp in silkworm pupae by Bac-to-Bac/BmNPV baculovirus expression system(BES)Bombyx mori baculoviru(snuclopolyhedrovirus,BmNPV) expression system(BES) has a lot of advantages such as high expression efficiency and low cost.In order to express HspA and UreB of H.pylori in silkworm pupae. We cloned hspA and enhanced green fluorescence protein (egfp) genes into vector pFastBacDual to form donor vector pFastBacDual-(EGFP)(HspA)and cloned ureB gene into vector pFastBacDual to form donor vector pFastBacDual-UreB ,then they were transformed into E. coli BmDH10Bac to obtain the recombinant Bacmid-(EGFP)(HspA) and Bacmid-UreB respectively by screening and identification. Their DNA were used to transfect BmN cells and generated the recombinant baculovirus BmNPV-(EGFP)(HspA) and BmNPV-UreB. A specific protein band of HspA and UreB were detected in silkworm pupae haemolymph at 96 h post-infection,which were around 13kDa and 62kDa by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis.The results showed that HspA and UreB have been expressed successfully in silkworm pupae. The expression level of UreB and HspA determined by ELISA were 0.49mg/ml and 0.52mg/ml, respectively in silkworm pupae haemolymph.2. Optimum acquisition time of pupae wxpressing HspA was determined by a non-fusion co-expression way of EGFP and target proteinIn order to determine the time course of expressed recombinant proteins in silkworm pupae and the optimum acquisition time. The traditional way is that haemolymph is harvested every 12 h beginning at 72 h post-infection. Subsequently, the haemolymph is analysed by SDS-PAGE or ELISA to detect the time phase of expression of target protein, According to this,to detect the time of collection of silkworm pupae.It is very difficult to track the time phase of protein expression by real-time way.In order to achieve visualization and make a real-time track for the level of expression of target protein. we use a dual expression baculovirus to express the EGFP and target HspA gene under the control of P10 and Pph promoters respectively by a non-fusion way. The results indicated that a direct correlation with the intensity of green fluorescence and the level of recombinant protein expression. It demonstrated that it is impossible to determine the optimum harvest time of silkworm pupae in accordance with the intensity of green fluorescence.3. Evaluation of protective and therapeutic efficacy against H.pylori infection in mice by oral administration of UreB and HspA expressed in silkworm pupaeH.pylori infection remains a significant global public health problem. Oral vaccine against this infection appears to be a preferable strategy. Therefore, on the basis of constructing successfully the mice model of infection of H.pylori.We investigated the effect of immuno-protection agaist H.pylori infection by oral immunity of HspA and UreB that were expressed in silkworm and evaluated their therapeutic efficacy. Mice immunized with rUreB,20% of animals (n = 10) were H pylori positive by the biopsy bacterial culture and PCR test. 10% of animals were H pylori positive by the biopsy urease test. The study demonstrated that a recombinant subunit antigen could induce an immunoprotective response against gastric H.pylori infection.