Cloning, Expression And Immunological Analysis Of Fragments Of Urease B Subunit Of Helicobacter Pylori

Helicobacter pylori (H. pylori) is strongly associated with many gastrointestinal diseases and plays a role in various extragastric diseases, categorized as a grade-I carcinogen by International Agency for Research on Cancer. H. pylori infects more than 50 percent of the world's population, and in developing countries more than 80 percent. There are drawbacks in current treatment of H. pylori infection with a combination therapy with two different antibiotics together with proton pump inhibitor,including high costs ,poor patient compliance ,increased risk of developing antibiotics resistance , and furthermore ,unable to protect against reinfections. H. pylori vaccination might decrease the incidence rate of H. pylori-associated diseases .The main therapeutic vaccine candidates against H. pylori so far are the whole bacterial cell and the whole cell lysate or virulence factors of H. pylori such as Urease, Hsp (heat shock protein), Vac (vacuolating cytotoxin), CagA (cytotoxin associate antigen), Nap(neutrophil-activating protein), HpaA (adhesion A )and so on alone or in different combinations with various adjuvants. These vaccines candidates against H. pylori have been evaluated in experimental animals and several of these studies have suggested that vaccination is possible to induce protective immunity .Proteantigen induce immune response by its epitopes, and specific cell responses in host are primely induced by immunodominant epitopes. Urease is the most affluent protein in H.pylori,and it is a colonization and virulence factor of H.pylori.Urease has conservative gene sequences,and its B subunit (UreB) is avirulent being as the prime protective antigen against H.pylori. Expression of the fragments of UreB would help study the epitopes, especially the immunodominant epitopes of UreB, therefore would promote clarification of the effective components of UreB and development of a more effective H. pylori vaccine.The aims of this study were to clone , express and analyze the immunological characteristics of fragments of UreB by economically reliable genetic engineering techniques,which would provide an experimental material for protective epitope-screening; and to establish a cytologic experimental method of screening immunodominant epitopes of H.pylori loaded by DC2.4 cells in C57BL/6 mouse model,therefore my work would make foundations for fine analysis epitopes of UreB,and make further guide to a more effective protecttive vaccine against H.pylori.Methods1. The protein structure and entire gene sequence of UreB of H.pylori was analyzed by the DNASTAR and DeepView/Swiss-PdbViewer 3.7 (SP5) softwares; based on which, five truncated fragments of UreB were designed,named as U1 (1-185aa),U2(98-294aa),U3(179-380aa),U4(292-476aa) and U5 (375-569aa), covering the entire gene sequences without omitting any epitope of UreB.2. U1, U2 , U3,U4 and U5 were were amplied by PCR with a UreB recombinant that previously made in our laboratory as the template, then the products were constructed in the prokaryotic expression vector pET-11c (+);the recombinant plasmids pET-11c -U1,pET-11c -U2, pET-11c-U3, pET-11c-U4 and pET-11c-U5 were double digested by NdeⅠ/EcoRⅠ,with the sequence-consistency analyzed in comparation with the information in GenBank.3. pET-11c-U1,pET-11c-U2,pET-11c-U3,pET-11c-U4 and pET-11c-U5 were transformed into Escherichia coli BL21(DE3), then the products were induced by IPTG.SDS-PAGE analysis was used to confirm the expression of the recombinant fragments ,and Western-Blot analysis was used to evaluate the antigenicity of the recombinant fragments .4. BABL/C mice were immunized with coarsely purified UreB fragments and PBS control, respectively by oral route of 100μg/mouse.The antigens were emulsified in Complete Freund's Adjuvant (CFA) at 1:1 ratio. These were followed by three orally immunizations of the immunogens dissolved 1:1 in Incomplete Freund's Adjuvant (IFA) at one-week intervals. 7 days after the last immunization, antiserum was separated to analyze the immunogenicity of the five fragments by indirect ELISA.5. In vitro Th1-type cell responses of C57BL/6 mouse cells were determined: C57BL/6 mice of different gender were immunized with H. pylori sonicate and PBS control by subcutaneous route of 100μg/mouse. The antigens were emulsified in CFA at 1:1 ratio. These were followed by three subcutaeously injections of the immunogens dissolved 1:1 in IFA at one-week interval. 7 days after the last immunization, mice lymph-node cells and spleen lymphocyte were separated and stimulated by PMA plus ionomycin, then the percentage of IFN-γ-secreting CD4+T cells was evaluated by flow cytometry analysis.6. In ordrer to eliminate the influence of trypsogen, DC2.4 cells were cultured without trypsogen treatment, and the morphous of them were observed by the inverted microscope and the expression of MHC-Ⅱand CD86 molecules were evaluated by flow cytometry analysis.7. In order to optimize the role of DC2.4 cells as antigen presenting cells,the optimal approach of antigen loading and the maturation of DC2.4 cells were determined: UreB in combination with LPS and TNF-αwere used to stimulate DC2.4 cells,and the cell suspension of DC2.4 cells were acquired at different time during the culture,then the expression of MHC-Ⅱand CD80 molecules on DC2.4 cells were evaluated by flow cytometry analysis,after which mature DC2.4 cells loaded with UreB antigen were acquired.8.In order to acquire a bulk culture of UreB specific T cells, in vitro UreB-loaded mature DC2.4 cells were cocultured with the spleen lymphocytes from H.pylori immunized mice at a ratio of 1:1, 1:2 ,and 1:5 respectively, with 1×105 spleen lymphocytes per a well in total volume of 200μL in 96-well round culture plate. After cocultured in 37℃,50% CO2 incubator for five days, IL-2(100 U/mL)were added in,and the DC2.4 cells were cultured for two days,the cell suspension was acquired.9.In order to determine the optimal response ratio of mixed cell response ,UreB specific T cells were cocultured with UreB-loaded mature DC2.4 cells at a ratio of 1:1, 1:2 ,and 1:5 respectively, then the percentage of IFN-γ-secreting CD4+ T cells was evaluated by flow cytometry analysis.Results1. Five fragments of U1 (1-185aa),U2(98-294aa),U3(179-380aa),U4(292-476aa) and U5 (375-569aa) were cloned successfully; five recombinant plasmids of pET11c-U1,pET11c-U2,pET11c-U3,pET11c-U4 and pET11c-U5 were cloned and constructed,and the sequences of all the five fragments consisted with the information in GenBank.2. SDS-PAGE analysis showed that the recombinant proteins U1, U2, U3, U4 and U5 were successfully expressed in Escherichia coli BL21,with the protein molecular weight as 20.0kDa, 21.0kDa,22.0kDa,21.0kDa and 22.0kDa respectively;Western Blot assay showed that all of the five fragments U1,U2,U3 ,U4 and U5 had good reactivity with UreB specific polyclonal antibodies. Indirect ELISA assay showed that all of them have good immunogenicity .3. Flow cytometry analysis analysis showed that the number of IFN-γ-secreting CD4+ T cells was the largest in the spleen lymphocytes of male C57BL/6 mice.4. Flow cytometry analysis analysis showed that there were low level of MHC-Ⅱexpression and high level of CD86 expression on DC2.4 cells with trypsogen treatment; both of the expression of MHC-Ⅱand CD86 molecules were lower on the DC2.4 cells without trypsogen treatment.5. Flow cytometry analysis analysis showed that after DC2.4 cells were loaded with UreB for 24h, then UreB-loaded DC2.4 cells were stimulated by LPS and TNFαfor additional 12h,there was a high expression level of CD80 and MHC-Ⅱmolecules on DC2.4 cells .6. After the number of IFN-γ-secreting CD4+ T cells were evaluated by flow cytometry analysis, the optimal response ratio of mixed cell response was determined: in vitro,UreB specific T cells were cocultured with UreB-loaded mature DC2.4 cells at the ratio of 1:2 ,then the bulk culture of UreB specific T cells were acquired in the seventh day,then the UreB specific T cells were cocultured with UreB-loaded mature DC2.4 cells at the ratio of 1:2 ,the number of IFN-γ-secreting CD4+ T cells was the largest .Conclusion1. Expression of the fragments of UreB would promote clarification of entire epitopes of UreB.2. DC2.4 cell, which is an immature and immortal cloned DC cell line, could effectively process and present UreB to specific T cells.3. Cytological experimental method of screening UreB immunidomiant epitopes were established, holds promise for the analysis of immunodominant epitopes and the discovery of novel protective epitopes of UreB.