Studies On Erysipelothrix Rhusiopathiae Virulence Genes And The Immunoprotection Of Recombinant Proteins And Development Of An Indirect ELISA Based On RSpa

To investigate the biochemical characters, antimicrobial susceptibility, major virulence genes and pathogenicity of11porcine Erysipelothrix rhusiopathiae strains identified presently from tissue samples collected from2010to2012in pig farms from Human province. The biochemical characteristics and antibiotic susceptibility of the bacteria were determined with VITEK2automatic bacteria identification system and Kirby-Bauer method, respectively. Six major virulence genes (CPS-A, CPS-B, CPS-C, SpaA, RspA and RspB) and16S rRNA of the bacteria were amplified and sequenced, and the sequences were analyzed with software DNAstar and MEGA5.0. The virulence of the bacteria was determined by the lethal dose50(LD50) test on mice. Among the results of42biochemical reactions by VITEK2system,36were the same for all strains and6were different, while difference between strains isolated from different years was not observed. In the antibiotic susceptibility test, the11strains showed same susceptibility to10of the13different antibiotics, and there were sight differences between the strains with the rest3antibiotics. The six virulence genes investigated were found in the genomes of all strains, with nucleotide sequence identities of99%-100%and98%-100%to those genes of the reference strains Fujisawa and ATCC19414, respectively. For the sequence of16S rRNA gene, homology of100%was observed between the present strains and those from Sweden and Uruguay. The LD50of the present strains were from5CFU to136CFU, with no difference between strains isolated from different years.The gene encoding4lipoprotein (BMP-1,BMP-2,MBP-a and MBP-b) and1the surface protein (SPBa) were amplified from the single colony of erysipelothrix rhusiopathiae by using PCR technique. The amplified product was inserted into pET-28a vector of restriction enzymes EcoR I and Sal I sites to build recombinants. Plasmids were identified by useing agarose gel electrophoresis, restriction enzyme digestion, sequencing analysis, and then transformed to E. coli BL21(DE3) strain for expression. A large number expressed product was purified by nickel ion affinity chromatography column to obtain the purified protein. Analysising the nature of the protein and immunogenicity with SDS-PAGE, western-blot and immune protection test in mice.The SDS-PAGE electrophoresis analysis showed all the5recombinant protein were soluble expression, western-blotting results showed that rBMP-1and rSPBa-N had significantly Reactogenicity. The result of mice immune protection test showed that all5recombinant proteins cannot induce a immune protection in mice against erysipelothrix rhusiopathiae. However, rBMP-2and rSPBa-N could patial delay the speed of the death.Synthetic LTB gene sequence was inserted into pET-28a vector of restriction enzymes Nco I and EcoR I sites to build recombinant plasmid pET-28a-LTB, and then BMP-2and SPBa-N gene was inserted into the pET-28a-LTB of the EcoR I and Sal I sites to build the plasmid pET-28a-LTB-BMP-2and pET-28a-LTB-SPBA-N, respectively. Plasmid pET-28a-LTB-BMP-2and pET-28a-LTB-SPBA-N were transformed into E. coli Rosetta(DE3) and BL21(DE3) to induced expression. Expressed product was purified by nickel ion affinity chromatography column to obtain the purified protein, but rLTB-BMP-2is not linked to the column so cannot be purified. The SDS-PAGE electrophoresis analysis showed all the2recombinant protein had soluble expression, western-blotting results showed that rLTB-BMP-2and rLTB-SPBa-N had significantly reactogenicity. The result of mice immune protection test showed that3group (rLTB-BMP-2recombinant protein supernatants, Freund’s adjuvant with mix protein, aluminum hydroxide adjuvant with mix protein) could patial induce a immune protection in mice against erysipelothrix rhusiopathiae.We established the indirect ELISA methods based on SPA for swine erysipelas infection, and explored and optimized conditions of the test. A number of serum were detected by SPA-ELISA and the SE/MR antibody detection kit. The result showed that the sensitivity was97.2%and the specificity was67.86%because of the high cutoff value of the reference kit. The earliest antibody response was detected at14days post-vaccination (dpv) by SPA-ELISA and SE/MR kit. The SPA-ELISA showed good consistency with the commercial indirect ELISA kit.