| Salmonella enterica(SE) is a facultative intracellular pathogen that is capable of causing disease in a range of hosts. The vaccine is an effective way to prevent SE entering food chain. Compared with the inactivated vaccine, attenuated vaccine on the prevention of SE in intestinal colonization and systemic infection had better effect. Hence, it is tremendously important to develop an attenuated vaccine to control this disease.In the special host environment of nutrition deficiency, SE can survive and avoid being cleared by the host immune system, it is attributed to many related virulence genes in bacterium and their interaction each other. SE invasion of the host cells is an important sign of SE infection, hil A gene is a dominant player in the control of gene expression which is required during the invasion of host cells. Cpd B can hydrolyze c AMP acting as second message in bacterium. This terminates c AMP-conducted biochemical effects which crucially regulate many cell activities. To further attenuate the virulence of SE, SM6△cpx R/lon/hil A mutant and SM6△cpx R/lon/hil A/cpd B mutant was constructed based on the SM6 △ cpx R/lon double-deletion mutant with Red homologous recombination system. Preliminary study of biological characteristics of mutant strains, their pathogenic test, immune tests and challenge tests laid the foundation for the reseach and development of genetically engineered attenuated live SE vaccine. This study includes:(1) Construction of triple-deletion mutant strain and fourfold-deletion mutant strainWith Red recombination system,the chloramphenicol resistance gene flanked by homologues of the hilA gene was amplified by PCR. The PCR products were electro-transformed into SM6△cpx R/lon strain with p KD46.After hil A gene was replaced by the chloramphenicol gene, the recombinant was constructed(SM6 △ cpx R/lon/hil A::Cat). Then, the resistance gene was eliminated using temperature-senstive plasmid p CP20,finally the hil A gene was completely deleted by this procedure. Through identification by PCR and sequencing, the mutant strain SM6△cpx R/lon/hil A was successful constructed. And using the same method, cpd B gene was deleted based on the SM6△cpx R/lon/hil A mutant.(2) The biological characteristics of deletion mutant strainsThe obtained triple-deletion mutant and fourfold-deletion mutant were cultivated serial passages in vitro and identified by PCR, the results indicated that these two deletion mutants were genetically stable. Successively cultivate the deletion mutants and the wild type strain in LB medium. The growth curves showed that there were no significant differences in the proliferation ability between the triple-deletion mutant and SM6, however the fourfold-deletion mutant grew relatively slowly. That demonstrated that the lack of hil A gene had no influence on the growth speed of bacteria. On the contrary, the lack of cpd B gene affected bacteria growth. The utilization ability of 95 kinds of carbon sources was determined by automatic microbial identification system, these bacteria showed decrease ability with the order of SM6△cpx R/lon/hil A/cpd B, SM6△cpx R/lon/hil A, SM6.(3) Pathogenicity test of the deletion mutantsThe result of median lethal doses(LD50) of SM6△cpx R/lon/hil A, SM6△cpx R/lon/hil A/cpd B and SM6 showed that SM6△cpx R/lon/hil A was at least 215 times higher than that of the parental strain SM6 and 1931 times for SM6△cpx R/lon/hil A/cpd B strain. Intraperitoneal innoculation of seven-day-old SPF chickens with SM6△cpx R/lon/hil A, SM6△cpx R/lon/hil A/cpd B and SM6 respectively, two chickens from each group were sacrificed at two day intervals to collect their liver, spleen and intestine in order to determine their cloning and persistence. The amount of bacteria recovered from the liver, spleen and intestine of mutants-innoculated chickens was significantly lower than that of SM6-infected chickens; in fact, the mutants SM6△cpx R/lon/hil A and SM6△cpx R/lon/hil A/cpd B was detected less in the liver, spleen and intestine ten days postf innoculation, respectively, while a large number of parental strain SM6 persisted in the spleen, cecum and intestine. This showed that in chickens, the SM6△cpx R/lon/hil A and SM6△cpx R/lon/hil A/cpd B mutants had reduced colonization abilities compared with the parental strain.(4) Immunity test of the deletion mutantsAfter optimization of the vaccination paths and doses, the SPF chickens were immunized in following procedures: Group Ⅰ(SM6△cpx R/lon/hil A, the immune dose of 1×108 CFU/mL), Group Ⅱ(SM6△cpx R/lon/hil A/cpd B, the immune dose of 1×109 CFU/m L), Group Ⅲ(inactivated vaccine), and control group(PBS). The animals in each group were immunized 3 times with 2 week intervals, and challenged with SM6 on day 35. The serum antibody titers were determined using enzyme-linked immunosorbent assay(ELISA) every week. Two weeks after each immunization, dynamic changes of CD4+ and CD8+ T cell subsets were detected by flow cytometery. IL-2, IL-6, IFN-γ were detected by ELISA kits. And survival rates, internal organ pathological changes, and the immune protection efficacy of vaccine were assessed.The serum antibody levels of SM6△cpxR/lon/hil A/cpd B and inactivated vaccine groups were significantly higher than that of SM6△cpx R/lon/hil A group, and three experimental group antibody levels were higher than that of the control group. The SM6△cpx R/lon/hil A/cpd B and inactivated vaccine groups can produce strong humoral immune response and no obvious difference between these two groups. The secretion of IL-2, IL-6 and IFN-γ were all rised in all immunization groups. In collaboration with the assay of CD4+ and CD8+ T cell subsets, these results revealed that SM6△cpx R/lon/hil A and SM6△cpx R/lon/hil A/cpd B vaccine could induce better cellular immune response. Seven days after challenge, the bacterial loads in cecum, liver and spleen of SM6△cpx R/lon/hil A/cpd B were lower than that of groups of SM6△cpx R/lon/hil A and inactivated vaccine. In the protection test, chickens from SM6△cpx R/lon/hil A/cpd B and inactivated vaccine groups had no obvious symptoms observed after challenge, which was better than the SM6△cpx R/lon/hil A group with three chickens showing clinical symptoms. In the control group, there were two chickens dead and ten chickens with clinical symptoms after challenge. In general, the results revealed that SM6△cpx R/lon/hil A/cpd B manifested high protective efficacy to chickens challenged with SE through activating humoral immunity and cellular immunity. The work laid theoretical foundation for the development of attenuated vaccines. |
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