Cloning And Expression Of The Fragment Of Epf Gene Of Streptococcus Suis Type 2 And The Immune Protection Test In Mice

Streptococcus suis type 2 is a important cause of many different diseases such as pneumonia, meningitis and arthritis. It not only impaires pig industry, but also affects human health.. The pathogenicity of Streptococcus suis type 2 is associated with its' virulent factors, including extracellular factor protein(EF). Strains of EF" phenotype were frequently isolated from healthy pigs. Strains of EF+ phenotype were frequently isolated from diseased pigs and Strains of EF phenotype were isolated at a high frequency from human patients. It suggests that EF was an important virulent factor. However, the precise role of EF in the pathogenesis had not been established. According to the analysis of DNAStar software, the fragment of epf gene was cloned and expressed. The protection of animal test described the efficacy of the recombinant proteins in preventing infection and disease. The expression study support further research about the structure and function of EF and the candidate for a subunit vaccine.1 Cloning and expression of the fraction of extracellular protein factor gene of Streptococcus suis type 2According to the epf gene in Genbank, the fragment of epf gene containing epitopes was selected by the analysis of DNAStar software. Then one pair of primers was designed and the endonuclease of EcoR I and Xho I was added to the end of primers. After cloning and expression, the recombinant protein of molecular weight of 53,000 was demonstrated by SDS-PAGE. It showed that the fragment has antigenical epitopes by Western blot.2 Recombinant protein purification and sera preparationThe recombinant protein of EF of Streptococcus suis containing a His-tag sequence was purified by metal chelation chromatography under native or denaturing conditions. After purification, a dominant protein band with 53,000MW was detected in SDS-PAGE gel. Rabbits were immunized by the purified protein emulsified with Freund's adjuvant. The antibodies titer were 1:1000, 1:2000 respectively detected by indirect enzyme-linked immunosorbent assay.3 Immune protection in mice by recombinant proteins of MRP and EF ofStreptococcus suis type 2Two recombinant proteins of MRP and EF(rMRP and rEF) containing epitopes were expressed. Balb/c mice were immunized with the above recombinant proteins emulsified with Freund's adjuvant both or alone. The inactivated bacterin was the control. After the second immunization, the mice were challenged with SS2-H strain and gained the antisera of rMRP/rEF and the bacterin. The results showed that the anti-rMRP and anti-rEF titres of rMRP/rEF were both 1:2000 by indirect enzyme-linked immunosorbent assay, the anti-rMRP titre of the inactivated bacterin was 1:400 while anti-rEF titre wasn't detected. The protection rale of rMRP/rEF and the inactivated bacterin reach 80%(8/10) and 90%(9/10) respectively, while mice vaccined wilh eilher rMRP or rEF alone were less well protected and it were 1/5, 0. The results also demonstraled that there was not less than one virulenl factors of the protective antigen of Streptococcus suis type 2.