Prokearyotically Cloning, Expression, Characterization And Immunologic Properties Study Of Important Proteins From S. Suis 2 Highly Virulent Strain

Streptococcus suis is an important zoonotic pathogen which has been associated with a wide variety of infections in swine, such as meningitis, septicemia, arthritis and pneumonia. This organism has also been isolated from humans with meningitis or endocarditis and Toxic Shock Syndrome. Several putative virulence factors of S. suis 2 have been described, but are not well characterized which inhibite the prevention and cure. The experiments studied Enolase, Suilysin and Hemolysin of S. suis 2 05ZYH33 strains isolated from patients of Ziyang Sichuan province in 2005.1 Molecular cloning and characterization of Enolase from S. suis 2A highly homologous eno gene was unveiled by genome-wide mining. Prokearyotically cloning, expression and purification Enolase from 05ZYH33. Whole cells Enzyme-linked immunosorbent assay and Fluorocytometric analysis were developed to give confirmation about Enolase localization on the surface of 05ZYH33 strain. Adherence assay implicated Enolase involve in the S. suis 2 adhesion. Plaque Forming Cell experiment indicated that Enolase acts in the suppression of the specific host immune response. Western-blot experiment demonstrated clearly it shares strong specific antigenicity. Indirect ELISA provid an efficient Eno-based method for detection of antibodies against S. suis 2. The results exhibited the properties of Enolase which may play pivotal roles in the severe infection of S. suis 2.2 Molecular cloning and characterization of Suilysin from S. suis 2S. suis 2 05ZYH33 suilysin was amplified and inserted into pET32a. The yield of fused protein came to its highest peak following 4h of induction with 1mmol/L IPTG at 37℃. The fused protein mainly existed in inclusion body. After being treated with carbamide and purified by chelating chromatography, Suilysin showed clear hemolytic activity. The enzyme activity was affected by temperature, DTT,β-Mercaptoethanol, H2O2 and proteinase K. Further, in an animal model system, we demonstrated that the mice immunized with Suilysin become to be protected against lethal doses of bacteria which means the successful application of proteomics may as a technique for identifying vaccine candidates.3 Molecular cloning and characterization of hemolysin from S. suis 2Bioinformatics was adopted to analyze the S. suis 2 05ZYH33 hemolysin sequence and compaired with other related proteins. These studies found that hemolysin have both signal and transmembrane segments, and consisted with DUF21, CBS and CorC-HlyC. A highly homologous of Hemolysin with other hemolytic proteins was unveiled. Further, prokearyotically cloning hemolysin gene from S. suis 2 05ZYH33. The results would lay the roots for the future research of the function of Hemolysin.