Prediction Of Urease Subunit B Epitopes Of Helicobacter Pylori And Epitope Screening By Phage Display

Background and Objective Helicobacter pylori (Hp), a microaerophilic, spiral and gram-negative bacillus is recognized as a human-specific gastric pathogen that colonizes the stomaches of at least half of the world's populations. Most infected individuals are asymptomatic. However, in some subjects, the infection causes acute, chronic gastritis or peptic ulceration, and plays an important role in the development of peptic ulcer and gastric adenocarcinoma, mucosa-associated lymphoid tissue (MALT) lymphoma and primary gastric non-Hodgkin's lymphoma. This organism is categorized as a class I carcinogen pronounced by the World Health Organization. Recently, epidemical research found out the relations between infection of Hp and coronary artery disease, arteriosclerosis, idiopathic thrombocytopenic purpura, diabetes, iron deficiency anemia, chronic urticaria and bronchitis.Urease is a main virulence factors of Hp which facilitates the colonization of Hp. It buffers the acid environment by hydrolysing urea intoNH₃ and CO₂. Urease may reach as much as 10% of total bacterial protein. The large subunit of Urease, Urease B subunit (UreB), is where the activity center of urease located. The conservation of UreB among the diiferent species of the Helicobacter genus and its highly antigenic property have drawn the attention of most researchers to develop vaccine based on this molecule. The study arounded using UreB as a vaccine agent indicated that immune response could be induced in some extent by immunization of this subunit, but the results from different groups on colonization clearance were not in consensus. Vaccines composed of a single protein antigen usually elicited lower immune protections, and have limited application.In the last few years, a novel vaccine strategy constructing multiple antigenic peptides (MAP) on one carrier molecule was developed and might be a promising way to conquer the dificiency of the subunit vaccine. Screening efficient epitopes is the first step for developing MAP vaccine. Studying the epitopes could not only reveal the molecular mechanism and feature of immune response induced by protein antigen, but also could be applied for investigation of novel molecular vaccine, serological detection. In this study, we intend to screen the Th and B cell epitopes on the UreB molecule of Hp, and lay a foundation for developing the MAP vaccine.Methods Ni-NTA chromatography was applied to extract the target recombinant expression product rUreB and routine intracutaneously immunization method was performed to prepare rabbit anti-rUreB serum. The epitopes of B and Th cell epitopes in UreB molecule were predicted and analyzed using bioinformatic technique. The major B and Th cell combined epitope peptides of UreB were selected to construct their phage display systems. PEG/NaCl precipitation method and SDS-PAGE were used to extract the recombinant phage and identify the target recombinant PIII(rPIII). By using commercial anti-whole H.pylori IgG or rUreB antiserum as the first antibody, Western blot assays were established to identify as well as to screen the epitope peptides.Results The output of rUreB was approximately 30% of the total bacterial proteins and the purified rUreB showed a single band in SDS-PAGE gel. rUreB antiserum was harvested from immunized rabbit. In comparison with the corresponding sequences in GenBank, the similarities of nucleotide and putative amino acid sequences of the cloned ureB gene were 96.88%⁹7.82% and 99.65%⁹9.82%. Four epitope peptides UreB230, UreB322, UreB479 and UreB527 were selected from the result of online prediction and successfully expressed in their recombinant M13 phages. Western blot assays using the different first antibodies displayed similar positive results and the reactive intensities in the assays of both UreB322 and UreB527 were remarkably stronger than those of UreB230 and UreB479.Conclusion In this study, purified recombinant UreB and rUreB antiserum was havest. For identification of epitopes, phage display systems of the major UreB B and Th cell combined epitope peptides are successfully constructed in this study. The applied bioinformatic techniques can be used to efficiently predict antigenic epitopes. UreB322 and UreB527 are confirmed to be the efficient antigenic epitopes of UreB that can be used as candidate epitopes for developing MAP vaccine of H.pylori.