Construction Of Spvb. Spvc Gene-deleted Mutants In Salmonella Typhimurium And Its Function On Symptoms And Immune Response

Objective:Since we had constructed spvB/spvC gene-deficent strains, now we constructed the spv B.spvC gene-deficent strains to make the experiment more credible. The wild strains and spvB/spvC/spvB.spvC defect strains infect BALB / c mice respectively. Analyze the effect of spvB, spvC genes by observe the symptoms and immune response between the infected mice. Therefore provide theoretical and experimental evidence to study the pathogenic mechanism of the spvB/spvC, and provide means for cell infection model orvaccine development with the follow research. Methods:1. Acording to the suicide plasmid-mediated homologous recombination constructe spvB.spvC Gene-Deleted Mutants in Salmonella Typhimurium. Check outthe Salmonella spv gene sequence(include spv ABCD) in the GeneBank database, designed two pairs of specific primers for spvB/spvC, and plus restriction sites on the 5 ’end of the primer. Amplify nucleic acid fragment of spvB upstream and spvC downstream by PCR, connecte them and clone them into to the suicide plasmid pCVD442(pCVD442-ΔspvB.spvC), which was transduced into the target cell of the wide type strain of Salmonella typhimurium for homologous recombination.Observe the reorganization by PCR, The mutant spvB.spv C was selected by specific PCR, choose the stable recombinant strain considered complete spvB.spvC mutant gene defects, the sent to DNA sequencing.2. Study the effect of spvB, spvC genes by observe the symptoms and immune response between the infected mice. All BALB / c mice were given free access to food and water, room temperature(20-22 ℃), humidity(50%-55%). They were randomized to PBS、STM.211、STM.211-ΔspvB、STM.211-Δspv C、STM.211-ΔspvB.spvC group, and were infected with 0.2ml PBS、STM.211、STM.211-ΔspvB、STM.211-ΔspvC、STM.211-ΔspvB.spvC respectively by intraperitoneal. Observed the infection symptoms(the change of the mental status、movement、diarrhea、 weight、hair). Upon infection 1 d、3 d、5 d、7 d、9 d,5 mices in each group were randomly selected to remove eyes to get blood. Then, text IL-10, IL-12, IFN-γ of the Serum by ELISA. At the same time, isolate liver and spleen to observe the pathological changes of target organ with the general view or under the microscope.Results:1. A stable spvB.spvC gene deleted mutants in Salmonella typhimurium(STM.211- ΔspvB.spvC) was constructed successfully, which 1965 bp was deleted and confirmed by PCR and DNA sequencing.2. The first day of the infected mice appear to different levels of aggregation curled, exercise less, listlessness, coarse hair, some mice showed irritation, but no diarrhea, And the most severe symptoms within infected 3-5 days. The infected symptoms of group STM.211 、 STM.211- Δ spvB and STM.211- Δ spvC was significantly serious than the PBS group, but there was no significant difference between STM.211-ΔspvB.spvC and PBS group. After Salmonella infection, IFN-γ and IL-12 secretion was significantly increased, while IL-10 increased to a lesser extent, resulting in Th1 responses advantage. But the density of cytokine was different between the groups. The IFN-γ and IL-12 in group of STM.211、STM.211-ΔspvB and STM.211-ΔspvC were significantly lower than STM.211-ΔspvB.spvC(p<0.05); while IL-10 was significantly higher than STM.211-ΔspvB.spvC group(p <0.05); but there was no statistically significant difference between STM.211 group, STM.211-ΔspvB group and STM.211-ΔspvC(p> 0.05). Conclusion:1. Construction of spvB.spvC Gene-Deleted Mutants in Salmonella Typhimurium by suicide plasmid-mediated homologous recombination, and provide means for cell infection model orvaccine development with the follow research.2. spvB/spvC gene that inhibits the TH1 cytokines(IFN-γ and IL-12) secretion but promote the TH2 cytokines(IL-10) expression, trend to TH2 shift. This shows that spvB/spvC gene can conversion the types of immune response, which will help the bacteria resistant and evade the immune defenses, increasing worse infection outcome.