Study Of Recombinant Mycobacterium Smegmatis Live Vector Vaccine Expressing The 26KD Outer Membrane Protein Of Helicobacter Pylori

Background: Helicobacter pylori (H. pylori) is a spiral-shaped, gram-negative bacterium. H. pylori induces chronic inflammation of the stomach mucosa, causing chronic gastritis and peptic ulcer. Moreover, H. pylori infection is related to gastric mucosa-associated lymphoid tissue lymphoma and to an increased risk of gastric cancer,it has therefore been designated as a grade I carcinogen by the World Health Organization in 1994. Due to the high prevalence of H. pylori, the prevention and treatment of H. pylori infection has become a hot spot in the field of medical micro-organisms and gastroenterology. The current treatment against H. pylori infection consists of a combination therapy with two different antibiotics together with a proton pump inhibitor or/and bismuth.However, there are many drawbacks associated with this treatment such as antibiotic resistance, recurrence, reinfection and high cost. So, Vaccination of humans could be an effective and economic approach to prevent and treat H. pylori infection all over the world. However, the protein vaccines of H. pylori have poor immunogenicity, most vaccines need strong mucosal adjuvant to induce protective immune. But mucosal adjuvant have strong toxic side effects, which limit its further development. A recombinant live vector vaccine which provide a new way for the study of H. pylori vaccines can be an effective solution to this problem .Mycobacterium smegmatis(M. smegmatis) is a species of rapid growing mycobacteria from soil. It can propagate one generation every 1-3 hours. It is a non-pathegent bacterium and effective cell immunity adjuvant. With the development of molecular biology and genetic engineering technique, many exogenous proteins can be expressed in M. smegmatis, and had good immune effect .It provides a new way for live vector vaccine of H. pylori.Omp26 is an outer membrane protein of H. pylori, its molecular weight is 26000, The Omp26 not only can be used as an antigen for detecting H. pylori infection, but also have immunogenicity. The Omp with small molecules (in particular, Mr 18 000 and 26 000) not only have an important value for screening gastric cancer and gastric cancer with high-risk population, and can induce immune response in animal or human to eradicate and prevent H. pylori infection.In this study, the Omp26 gene of H. pylori were cloned into the E.coli–mycobacterium shuttle vector , the recombinant vectors were transformed into M. smegmatis by electroporation, the positive strains of the recombinant M. smegmatis(rM.smegmatis) were identified by restriction map,PCR and western blotting. Plasmid pEGFP was transformed into rM.smegmatis by electroporation,the stability and toxic side effect of rM.smegmatis vaccine were observed. The rM.smegmatis vaccine strains were orally administrated to the BALB/c mice or H. pylori-infected BALB/c mice with gastric tube, the prophylactic or therapeutic effect against H. pylori and immune mechanism of the rM.smegmatis were investigated as a fundament for the development of H. pylori vaccine. H. pylori live vector vaccine provide a new method for preventing and treating H. pylori infection.This research can be divided into four parts:Part 1: Construction of recombinant M. smegmatis live vector vaccine expressing outer membrane protein 26kDa antigen of H. pyloriObjective: To construct the E.coli–mycobacterium shuttle expression vector of outer membrane protein 26kDa antigen (Omp26) of Helicobacter pylori and express in M. smegmatis by gene-engineering, and screen recombinant M. smegmatis live vector vaccine expressing outer membrane protein 26kDa antigen of H. pylori, which would lay a foundation for prophylaxis and therapy of H. pylori infection.Methods: Omp26 gene was amplified from pET32a-Omp26 by PCR and cloned into plasmid pMD18-T, after identification by PCR ,enzyme digestion analysis and sequencing, Omp26 was subcloned into E.coli-mycobacterium shuttle vector ( pLA71,pLA73,pJMas, pJHsp70) that were digested with KpnI and NotI resulting in E.coli-mycobacterium secretive expressing and non-secretive expressing shuttle plasmid. The recombinant vectors were transformed into M. smegmatis by electroporation. The rM. smegmatis was identified by restriction map and PCR, and the expressing product was detected by SDS-PAGE and western blotting.Results: 594 bp DNA fragment was amplified from pET32a-Omp26 by PCR, sequencing showed that the target gene was Omp26 at length of 594 bp. As compared with gene HP AY033499 in GenBank, cloned Omp26 gene sequence homology was up to 98.8%. Omp26 was subcloned into E.coli-mycobacterium shuttle vector, E.coli-mycobacterium secretive expressing and non-secretive expressing shuttle plasmid were successfully constructed. The recombinant vectors were transformed into M. smegmatis by electroporation, then the rM. smegmatis were identified through kan gene resistance screening, restriction map, PCR technique. Omp26 were secreted into culture supernatant in pPL71-Omp26 and pPL73-Omp26 with signal peptide sequence, and this secreted protein could be recognized by anti-H. pylori antiserum. Otherwise, the protein was expressed in a non-secreted way in the pJMas-Omp26 and pJHsp70- Omp26 due to lack of the coding region of the signal peptide. Conclusion: Recombinant shuttle plasmid pPL71-Omp26, pPL73-Omp26, pJMas-Omp26 and pJHsp70- Omp26 were successfully constructed . Recombinant M. smegmatis strains expressing Omp26 of H. pylori were successfully constructed.Part 2: Stability , security and colonization of rM. smegmatis In vitro and vivoObjective: To study the stability of rM. smegmatis in vitro and vivo, security and colonization in vivo.Methods: The pEGFP vector was then transformed into the rM. smegmatis by electroporation. Twenty colonies were randomly picked to observe fluorescence and take swift extracting plasmid to determine the stability of the recombinant plasmid. The mice were sacrificed 4 weeks and 8 weeks after orally immunized with rM. smegmatis, stability and safety of rM. Smegmatis were evaluated by histology, viable bacterial counts and plasmid extraction.Results: The stability of the rM.semgmatis in vitro was studied, the result showed that the rM. semgmatis is stable when it growed with and without Kan resistence . It could contiune propagate for 20 generation. The rM. semgmatis could stably colonize in the gastric mucosa, spleen, lung and liver for 4 weeks after immunization by intragastric administration, and no complications were observed.Conclusion: Green fluorescent protein could be used as a molecular marker in the investigation of screening recomninant vaccine, rM. smegmatis is a particularly attractive vector for the delivery of heterologous antigens with high stability and low toxicity.Part 3: The prophylactic effect of rM. semgmatis live vector vaccine against H. pylori infectionObjective: To evaluate the prophylactic effect of rM. semgmatis against H. pylori infection in animal model, and study the mechanism of prophylactic protection.Methods: 2×107 CFUs of rM. smegmatis strain were orally administrated to the BALB/c mice with gastric tube. At the same time, the PBS and M. smegmatis were set as the control groups, respectively. 4 weeks after immunization a batch of mice in each group were killed, and their serum, stomach and spleen were kept for future use. The remaining were attacked twice by H. pylori SS1 and then killed 4 weeks later. The immunoprotection effect of the gastric mucosa against H. pylori infection were evaluated by the rapid urea assay, H. pylori quantitative cultivation and histological examination. Multiplication of mouse spleen lymphocyte was detected by MTT; The expression of cytokines in stomach and spleen were analyzed by RT-PCR 4 weeks after immunity and challenge. The levels of the H. pylori specific serum IgG, IgA, IgG1 and IgG2a were detected by ELISA analysis.Results: The number of H. pylori in rM. Smegmatis(pPL73-Om26) were much lower than that of the PBS and the M. smegmatis groups. Meanwhile, the reduction of H. pylori Colonization and decrease of chronic gastritis were observed in rM. Smegmatis(pPL73-Om26). Oral immunization with rM. Smegmatis(pPL73-Om26) have significant proliferation of spleen lymphocyte after the stimulation of ConA and H. pylori antigens. The specific serum IgG , IgA, IgG1 and IgG2a antibodies were significantly induced with Oral immunization of rM. smegmatis(pPL73-Om26). RT-PCR of cytokines revealed that M. smegmatis and rM. smegmatis(pPL73-Om26) group had relatively high levels of mRNA for IFN-γand IL-2, but the levels of IFN-γand IL-2 in PBS group were negative 4 weeks after immunity. After attacked by H. pylori, the levels of IFN-γand IL-2 mRNA expression in rM. smegmatis group were significantly lower than that of the PBS and M.smegmatis group (P<0.05). While the level of IL-4 mRNA expression in rM. smegmatis group were significantly higher than that of the PBS and M. smegmatis group (P<0.05). Other cytokines such as IL-6, IL-12 and IL-10 were not detected in all groups.Conclusion: The rM. smegmatis by intragastric administration not only can reduce the H. pylori colonization, but also relieve the gastric inflammation reaction and prevent H. pylori infection. The mechanism of prophylactic protection was that rM. smegmatis vaccine induced Th1 and Th2 balanced immune response. Part 4: The therapeutic effect of rM. semgmatis live vector vaccine against H. pylori infectionObjective: To evaluate the therapeutic effect of rM. semgmatis against H. pylori infection in animal model, and study the mechanism of therapeutic protection.Methods: H. pylori infected BLAB/c mice model was constructed, four weeks after infection, 2×107 CFUs of rM. smegmatis(pPL73-Omp26) or M. smegmatis were orally administrated to the H. pylori infected mice, using PBS as negative control. Mice in each group were killed four weeks after immunization, the therapeutic protection against H. pylori infection were evaluated by the rapid urea test, H. pylori quantitative cultivation and histological examination. Multiplication of mouse spleen lymphocyte was detected by MTT; The concentrations of Th1 cytokines (IFN-γ, IL-2, IL-12) and Th2 cytokines (IL-4, IL-6, IL-10) in stomach and spleen were assessed by RT-PCR. The levels of the H. pylori specific serum IgG, IgA, IgG1 and IgG2a were detected by ELISA analysis.Results: Oral therapeutic immunization with rM. smegmatis (pPL73-Omp26) induced a significant reduction in the bacterial load in the stomachs of H. pylori-infected mice, which was comparable to the reduction induced by oral immunization with PBS or M. smegmatis. And the gastric histology of mice infected by H. pylori revealed less severe inflammatory infiltration and gastric damage. Oral immunization with rM. Smegmatis(pPL73-Omp26) have significant proliferation of spleen lymphocyte after the stimulation of ConA ,which was comparable to the proliferation induced by oral immunization with PBS or M. smegmatis .The levels of specific serum IgG , IgA, IgG1 and IgG2a antibodies were significantly increased after oral immunization with rM. smegmatis (pPL73-Omp26) (p< 0.001). RT-PCR of cytokines revealed that mice immunized with rM. smegmatis (pPL73-Omp26) resulted in increased levels of IFN-γ,IL-2 and IL-4 in the stomach and spleen lymphocyte compared to control infected mice (p < 0.05), the cytokines IL-6,IL-12 and IL-10 were not detected in all groups.Conclusion: H. pylori infected BLAB/c mice model was successfully constructed in this study. The rM. smegmatis by intragastric administration could reduce the H. pylori colonization, relieve the gastric inflammation and treat H. pylori infection.These results showed that the rM. smegmatis vaccine could induce Th1 and Th2 cell-mediated immune responses, and boost antigen-specific Th 1/ Th 2 balanced type humoral immune response.