Prokaryotic Expression Of A Fragment Of Extracellular Factor From Streptococcus Suis Type 2 And Determination Of Its Immunoprotection Potency

Streptococcus suis type 2 (SS2) is an important zoonotic pathogens. Extracellular factor (EF) is an important virulence factor of SS2.EF gene fragment from 1423th to 2203th bp was amplified by PCR with genomic DNA of HA9801 as template. HA9801 is an isolate of Streptococcus suis type 2. The PCR product was cloned into pMD 18-T vector and the recombinant construct named as pMD 18-T/ef was transformed into host strain E.coli JM109. Positive clones(white clones) were picked among Blues ones. Then the recombinant plasmid pMD 18-T/ef was extracted from positive clone, identified by sequenceing, PCR and endonuclease restriction analysis. Results showed the EF fragment was inserted in correct orientation with correct sequence. Then the ef gene fragment was cut off from pMD 18-T/ef by EcoR I and Xho I, and subcloned into expression vector pGEX-4T-1 producing recombinant plasmid pGEX-4T-1/ef. Subsequently pGEX-4T-1/ef was transformed into E.coli DH5α. Positive transformants were collected by Blue-White clone selection and pGEX-4T-1/ef was extracted for verification. All analysis including sequenceing, PCR and endonuclease restriction digestion showed that the target gene was cloned into pGEX-4T-1/ef in designed direction with right sequence. Then EF was expressed by culturing host cell E.coli DH5αand inducing with IPTG at a final concentration of 0.5mM. The following SDS-PAGE analysis showed that EF was expressed at a high level mainly in the form of inclusion bodies existing in the cytoplasm. Western blot revealed that there existed antigen epitopes on the expressed EF fragment. So subunit vaccine was prepared with the expressed EF fragment. Groups of BALB/c mice were immunized and subseguently challenged with HA9801 at 1.0×109CFU/mL. Relative protection rate of mice reached 44.4% indicating EF was an immunogenic protective antigen of SS2.