Clone And Expression Helicobacter Pylori Gene Hp0410 And Immunogenicity Study Of HP0410

1. Research background and proposalIt had long been believed that mental stress and life style are the reasons for chronic gastritis and stomach ulcer until 2005 when two Australia scientists J Robin Warren and Barry J Marshall have discovered Helicobacter pylori (Hp) and proved that this bacteria is the main factor leading to gastritis and peptic ulcer and been awarded Nobel physiological and medical prize due to their outstanding work. Helicobacter pylori that lead to chronic gastritis and stomach ulcer cause the most spread infection in worldwide and it is also closely related to gastric adenocarcinoma and stomach mucosa related B cell lymphoma. How to prevent Hp infection effectively is the focus problem, which is concerned by a large number of researchers.Presently, the most frequently therapy of gastritis and stomach ulcer adopted is the antibiotic method. However this method is likely to be ineffective as Hp usually developing resistance to the drug and if Hp is not totally exterminated the diseases will soon recur in 70%-80% patients. Some researchers also believe another reason of recurrence is that Hp in patient's mouth can be transported into his stomach. Due to the inefficiency of antibiotic therapy, vaccine is considered to be the most effective way in controlling Hp infection. The hot points of vaccine research are Urease subunit A (UreA) or B (UreB), CagA and VacA, Hot shock protein 60 (Hsp60) etc. But so far, these vaccine candidates show several shortcomings: Ure A and Ure B can not induce overall and stable protective immune response; Cag A and Vac A are highly variable and whether Vac A is essence in Hp infection is still incertitude; Hsp 60 is high homogeneous compared with the human's so that it may not induce protective immune response and probably can cause inflammation. Further study is needed in order that more rational vaccine candidate of Hp can be found out. Adhesin is essential in the infection of Hp. As it locates on the surface of Hp and has a strong antigenicity, it would be an ideal protective antigen of Hp and the specific antibody binding to adhesin could probably inhibit the infection of Hp and eradicate it. Presently the research concerning adhesin focus on two types of adhesin: blood group antigen-binding adhesin (BAb) that can bind to the Le~b antigen of human cell's surface and N-acetylneuraminyl-lactose-binding haemagglutinin (NLBH). NLBH is 1.4kb long and contains 30RF (ORF1～3). The ORF2 is the hpaA gene containing 783bp and coding Helicobacter pylori adhesin A (HpaA) that is indispensable in the adherence of Hp for hpaA muted Hp cannot infect mice. HP0231 is a hypothetical protein according the analysis of ORF of Hp genome while HP0410 is a putative homologue protein of HpaA, which may probably well be the subunit of N-acetylneuraminyl-lactose-binding haemagglutinin (NLBH) a kind of protein functioning in adhering of Hp. Until now the specific function of HP0410 and HP0231 have not been identified yet as well as their antigenicity and prospect of being rational vaccine candidates of Hp.In this work our main purpose is to predict the features of HP0410 and HP0231 such as second structure and epitope according to the bioinformatic analysis, then to express and purify the more rational antigen as vaccine candidate according to the results, thirdly to scan the sera of stomach ulcer patients by using the recombinant protein in order to confirm that the protein can induce immune response in human body and to study whether the protein could become a new method of Hp infection diagnosis via immunohistochemistry, fourthly to study the epitopes of the protein by phage display technology and then to study the interaction between the protein and cell, finally to study the immune-protective effect of the protein by constructing multi-epitope recombinant vaccine. 2.Methods1. DNA primers of Helicobacter pylori (Hp) gene hp0231 and hp0410 were designed according to the Hp strain 22695's DNA sequence which was released in GenBank of NCBI. These primers were used to amplify the hp0231 and hp0410 from the extraction of Hp NCTC11639's genomic DNA via PCR. The production were cloned into pMD18-T and then transformed in E. Coli TOP10 to amplify. The sequences of these two genes were analyzed by bioinformatics softwares.2. hp0231 and hp0410 were cloned into pGEX-4T-1 and successfully induced it to express. Large amount HP0410 was induced and purified.3. The recombinant protein was used to scan 29 monoclonal antibodies. The antibody was examined by sensitive-special experiment and used in immunohistochemistry. 85 sera either from patients of gastric ulcer or gastritis and 90 from healthy people were used in ELISA to determine whether the protein HP0410 could induce specific antibody in human infected by Hp.4. Then the antibody and the sera of patients have been used to construct a M13 phage library of HP0410. By sequencing the DNA of phage, we got the sequences of simulated epitopes of HP0410.5. From the cell model, we have measured a series concentration of IL-8 and GRO-a secreted by SGC7901 stimulated by HP0410 in different time and quantity. We used cell ELISA to study whether HP0410 could attach to the cell's surface directly.3.Results1. The results of bioinformatics shows that the two proteins are out-membrane protein and all have significant antigenicity, furthermore their homology to be low in compared with other species so that it looks unlikely they could lead to cross-recation and self immune response.2. The recombination bacterias could express soluble proteins at high level. A monoclonal antibody (E018) was obtained by scanning 29 monoclonal antibodies of Hp.3. The outcome of immunohistochemistry is positive and the results of ELISA showed that 94% sera of patients were positive while the relevant number in healthy people was 74%.4. We obtained 8 epitopes from the phage library scaned by E018 and 6 epitopes scaned by sera. The results of cell model and cell ELISA suggested that HP0410 probably has no inflammation function and the antigen determinant of E018 may be the site of HP0410 adhering to cell surface.4. Conclusion1. Our work suggests that HP0231 and HP0410 are hot-prospect vaccine candidates of Hp.2. The antibody of HP0410 widely existed in patients. HP0410 could induce humoral immunity in vivo.3. The homogenous analysis of the epitopes suggested that they simulated spatial epitopes of the high antigenicity segments according to the bioinformatics of HP0410.4. Cell experiments suggested that HP0410 could not lead to inflammation so that it might become a safe vaccine of Hp.