Identification Of New Protective Antigens Of Streptococcus Equi SSP. Zooepidemicus

Streptococcus equi ssp. zooepidemicus (SEZ) belongs to the betahemolytic Group C streptococci, which can cause disease both in animals and humans. In china, S. zooepidemicus is the main pig pathogen, which could cause meningitis, septicemia, purulent arthritis, endocarditis and sudden death. It is also a substantial threat to human health, especially those involved in the pig industry. Although a varity of vaccines against Group C streptococci have been commercialized up till now, the effect is not always satisfactory. We need to develop a safe and efficient new vaccine.An immunoproteomic approach has been used to screen the cell wall-associated proteins of S. zooepidemicus ATCC35246.10 immunogenic proteins were successfully identified, most of which have not been formally reported in S. zooepidemicus. We need to further identify that whether these immunogenic proteins could stimulate the body to generate protective immune responses. In this study, specific primers were designed and synthesized based on the sequence of the identificated proteins published on the genbank. The target gene were amplied form genomic DNA of S.zooepidemicus strain ATCC35246 by polymerase chain rection(PCR), and cloned in the proper orientation into the expression vector pET-32a(+) or pET-28a(+), and then the recombinant plasmids were transformed into E. coli BL21 (DE3). Identified by PCR and restriction enzyme digestion of recombinant plasmid, positive clones were picked. Recombinant fusion proteins were expressed in BL21 after induced by IPTG., and SDS-PAGE analysis showed that recombinant proteins were consistent with the expected value. Western-blot showed that the fusion proteins could be recovered from the swine antiserum against ATCC35246. The result indicates that fusion proteins have part of a natural protein antigenicity.The recombinant fusion proteins were purified by affinity chromatograph, and emulsionized by isovolumic ISA206. The ICR mice were immunised respectively through hypodermic inoculation, and mice inoculated with S. zooepidemicus ATCC35246 and PBS emulsified in the same adjuvant respectively were served as control. Detect IgG antibody titers in sera by ELISA, and opsonic activity by in vitro opsonophagocitic assay. The result indicated that effective immunological resposes were induced in mice, the IgG titers in sera increased signigicantly, and showed good complement-dependent opsonophagocitisis. After the booster immunization, each group was challenged intraperitoneally with a 5LD50 of log-phase S. zooepidemicus ATCC35246. The results showed that the protective rates of experimental group were significantly higher than the PBS control group, and the protective rate of recombinant EF-G protein was 50%, while mice vaccined with recombinant TF,TK,GAPDH,PPH respectively were 37.5%. So the recombinant proteins showed immune protective potency in mice and that's the base of subunit vaccines in the future.