Construction Of Salmonella Enteriiidls ClpP Gene Deletion Strain And The Analysis On Its Virulence And Immune Effects

Avian Salmonellosis is one of the important diseases of poultry industry, it can cause thechicken diarrhea, serious impact on production performance, even causing death, and it can passthe poultry products into the food chain and affect the health of human beings. The vaccine is aneffective way into the food chain to prevent Salmonella enteritidis. Compared with the inactivatedvaccine, attenuated vaccine on the prevention of Salmonella enteritidis in intestinal colonizationand systemic infection had a better effect. At the same time, Salmonella infection spectrum isbroad, can be used as a live vector carrying and expression of exogenous genes, and constructionof a multivalent vaccine. These applications are based on the right of attenuated Salmonellafoundation, so it is necessary for us to research the Salmonella virulence related genes functionand assessment of deletions of genes related to the attenuation level.The test of the use of lambda phage RED recombination system, construction of chickenSalmonella enteritidis ClpP gene deletion strains of SM6/^ΔClpP. By scanning electron microscopyto observe the changes in bacterial morphology before and after the ClpP gene deletion; UsingBiolog MicroStation system on carbon source metabolism were measured, draw the conclusion:ClpP deletion mutant of Tween40, Tween80, β-methyl-D-glucoside, Succinic Acid Mono-MethylEster, D-gluconic acid, α-hydroxybutyric acid, L-raffinose, D, L-lactic acid, D-lactobionic acidlactone, D-Glucosaminic Acid and L-glutamic acid metabolic changes.In order to study SM6/^ΔClpP and SM6virulence changes, measured by SM6/^ΔClpP and SM6on chicken LD50, respectively, of1.33×1010cfu/2.51×109cfu/only; On mouse monocytemacrophage invasion ability of J774.1a, SM6/^ΔClpP decreased about48times; SM6/^ΔClpP strainadapted to high salt and low pH capacity decreases；Using SM6/^ΔClpP and SM6on chicken attack,SM6/^ΔClpP strains in vivo colonization time should be shorter than wild strain.With the construction of the SM6/^ΔClpP and parent strain SM6, to SPF chickens were lowdoses （5×106cfu/only）, at the same time, set the PBS control group.707-day-old SPF chickenswere divided into test group Ⅰ (SM6/^ΔClpP group,30), test group Ⅱ（the parent strain of SM6,30）and control group （group PBS,10）, a total of2immunocompetent, at intervals of two weeks. Afree weekly after the wing vein blood collection, separation of serum, to Salmonellalipopolysaccharide （LPS） as the coating antigen, using indirect ELISA for the detection of serumIgG. Free two weeks and two free two weeks and two free three weeks after stimulation withSalmonella lipopolysaccharide （LPS） and concanavalin A （ConA）, MTT assay spleen lymphocytestimulation index; kit for the detection of the whole blood and spleen of IL-2of IL-6andIFN-gamma secretion.Three weeks after the second immunization, SM6five times the medianlethal dose of virus attack. Virus attack a week later, killing not death of chickens, the rate ofprotection combined with chicken and visceral pathological changes to evaluate the protective effect of Salmonella attenuated immune. The test results show that test groupⅠ and testgroup ⅡLPS antibodies are at a high level; with LPS and ConA as irritants, the SI values of thegroupⅠand groupⅡwas significantly higher than that in control group, IL-2, IL-6and IFNgamma in cytokine levels also tended to rise.