Study On Outer Membrane Protein Of Salmonella

Salmonella, Gram-negative bacteria，can cause zoonotic disease. Salmonella outermembrane protein can induce macrophage apoptosis, bacterial resistant, and as a virulentfactor plays an important role in the pathogenesis. The distributions of btuB, mipA, nmpC,pagC were studied and outer membrane protein BtuB, MipA, NmpC, PagCimmunogenicity was detected. The function of Salmonella typhimurium gene mipA wasevaluated by deletion this gene.Firstly, the distributions of btuB, mipA, nmpC, pagC were studied. Our laboratoryhave detected four immunogenicity outer membrane proteins BtuB, MipA, NmpC, PagCby reverse genomics, immune proteomics in previous study.The genes of coding proteinsare btuB, mipA, nmpC, pagC. Through PCR technology, the distributions of btuB, mipA,nmpC, pagC were detected in141intestinal bacteria strains.The results showed that btuB,mipA gene were detected in38Escherichia coli strains,15Shigella strains and88Salmonella strains; Howere, nmpC and pagC were not detected in Escherichia coli,Shigella, and only part of Salmonella strains carried these two genes；More importantly，nmpC was not detected in Salmonella typhi. This study laid the foundation for typhoidclinical diagnosis and new vaccines.Secondly，the immunogenicity of outer membrane proteins BtuB, NmpC, PagC wasdetected. Outer membrane proteins NmpC, PagC and his-NmpC, his-PagC were purifiedthrough anion exchange method and immobilized metal ion affinity chromatography andhigh purity proteins have been obtained. First of all, NmpC, PagC combined withaluminum adjuvant and chitosan, mice were immunized. The experimental results showthat there was no significant difference between antibody titers of the aluminum adjuvantgroups and chitosan groups; Second, his-BtuB, his-NmpC, his-PagC combined withaluminum adjuvant, mice were immunized with mixed groups and found that IgG titerswere significantly different; Third，NmpC, PagC, his-NmpC, his-PagC were combinedwith aluminum adjuvant. It shows that the histidine tag has immunogenic. Comparedantibody titers of histidine tag groups with no histidine tag groups， difference wassignificant after immunization of mice.Thirdly, the fuction of Salmonella typhimurium mipA was studied. mipAhomologous mutations box was constructed via molecular biology techniques. Combinedwith the λ-Red phage recombination system, Salmonella typhimurium mipA wasreplaced by the kanamycin gene.50013△mipA::kan mutant,50013mipAH strain and50013△mipA::kan mutant,50333mipAH strain were successfully constructed. Through the serological experiments, physiological and biochemical identification, the resultsdisplay that mipA deletion did not affect the physiological and biochemicalcharacteristics of bacteria. Virulence evaluation experimental results show the invasionability of50013△mipA::kan mutant is much more stronger than50013wild-type strainand50013mipAH strain, meanwhile the invasion ability of50013mipAH strain than50013wild-type strain was weakened. It has been proved that the mipA plays animportant role in bacterial invasion.