Construction Of SrtA Gene Knock-Out Mutant Of Sstreptococcus Suis Type2 And Analysis Of Its Virulence

Streptococcus. suis type 2 is a Gram-positive bacteriam, and is an important zoonatic pathogens of different disease, such as meningitis, septicemia, which became a substantial threat to human health especially those involved in pig industry. A total of 35 capsular types have been identified, but types 1/2,1,2,7,9,and 14 can also caused disease, Among them, the serotype 2 has always been considered the most virulent and the most frequently isolated serotype from diseased animals. Important S. suis virulence associated factors include the polysaccharidic capsule, extracellular proteins including muramidase-released protein, extracellular factor, and a hemolysin. The pathogenesis of the infections not completely understood, we researched the function of Sortase in Streptococcus suis type 2, identification of srtA gene possessed in Streptococcus suis type 2, and knock-out mutant of srtA gene was constructed by allelic replacement. A reduction the virulence of the srtA mutant compared to parent in animal model systems of infection. And prokaryotic express and analyze antigenicity of Sortase A of S. suis 2 05ZYH33, which laid the foundation for the further pathogenesis.1 Homologous analysis of Streptococcus suis type 2 srt gene and prokaryotic expression of srtATo elucidate the distribution of srt gene in the genome of S. suis 05ZYH33, prokaryotic express and analyze antigenicity of Sortase A. In study, Homologous of genes that encoding sortase family members were analyzed, six genes were found to be homologous to srt of S. suis 05ZYH33. A new putative srt gene was found compared to the isolated strain from Japan. All putative srt genes in the genome of 05ZYH33 were analyzed, The predict SrtA and Sortase-like proteins were members of sortase family class A, while SrtBCD and SrtE belonged to class B. The srtA gene of S. suis was cloned and expressed in E.coli DH5α, the recombinant protein showed specific antigenicity by analysis of SDS-PAGE and Western blot. Mice were immunized with recombinant protein ,the antibody titer in blood serum reached 1:6400 by ensure of indirect ELISA after four times immunization with recombinant protein.2 Construction of srtA gene knock-out mutant of Streptococcus suis type 2To research the function of Sortase of Streptococcus suis type 2 virulent strain 05ZYH33. Recombinant gene knock-out vector was constructed consisting of Spc~r cassette with flanking homology regions to the target genes, and the isogenic srtA-deficient mutant was screened by allelic replacement. PCR analysis and Southern hybridization confirmed that the coding sequence of srtA were replaced completely by spc~r cassette in mutant 05ZYH33△srtA. Then the biological characteristic of 05ZYH33△srtA were examined in vitro. A reduction the virulence of the srtA mutant compared to parent in animal model systems of infection, which laid the foundation for the further Pathogenesis.3 Developmend and characterization of monoclonal antibody against sortase protein of Streptococcus suis type 2In this study, hybridoma cell lines secreting monoclonal antibody against sortase protein of Streptococcus suis type 2 were generated. 6-8 weeks olds BALB/c mice were immunized endermicly with purified recombinant sortase protein, splenocytes from the immunized mice were fused with SP2/0 and indirect ELISA was used screen hybridoma cells. 1 hybridoma cell lines 3D2 secreting antibody against Sortase protein of SS2 were established, westernblot analysis confirmed that the McAb only reacted with sortase protein of SS2.