| Streptococcus suis serotype 2 and Streptococcus equi subsp.zooepidemicus are the important pathogenies of diseases in pigs. Streptococcus equi subsp. zooepidemicus cause arthritis and septicimia in pigs,and Streptococcus suis serotype 2 is a major cause of meningitis, septicemia, arthritis, and bronchopneumonia in young pigs and can cause meningitis in humans.In many countries ,much attention were paid to Streptococcus suis serotype 2 and many studies were foucus on virulence factors, extracellular protein factor(EPF), muraminidase-released protein(MRP) were two main virulence factors .On the base of the appearance of MRP or EPF, Streptococcus suis serotype 2 could be divided into several types:MRP+EF+,MRP"EF\MRP+EF\MRP*EF\MRFEF+, MRP+EF ,MRP~-EF* el al.In the epidemiological studies in Jiangsu regin of China, MRP+EF+ was correlated to high virulent strains.And it was very important to diagnose, prevention and cure,eradication programs the disease caused by Streptococcus suis serotype 2 that detecting the virulent factor and more studying the function of MRP and EPF in the pathogenesis.At present,the reseach also focused on the studying on two virulent factors,but,we know little about the reaction between MRP and EPF.At the same time,there is not effective vaccine for this disease.The aim is to amplify the gene of mrp and epf respectively. After verified positive immunity, the fragments of mrp and epf linked by PCR was cloned into pET-32a again. By the above method with IPTG, we obtained the recombinant protein,the rabbits were immunitied with this protein to identified if the protein has good protection rates.This research was the base of further studying the structure of virulent factor and subunit vaccine.Now,there are many methods of detecting the antibodies against two type of Streptococcus suis. In this experiment,the whole cell the ultrasonic antigen the capsular polysaccharide ( CPS ) of Streptococcus equi subsp zooepidemius and Streptococcus suis type 2 were extracted and used as antigen for developing an indirect enzyme-linked immunosorbent assay(ELISA) to detect the antibodies against SS2 and SEZ.Six-week-piglets immunized with two value vaccine of Streptococcus suis ,setting negative controls(non-immunity), detected the antiobodies against SS2 and SEZ and challenged by CY and SS2-1 every other month respectively in order to find the relation between the antibodies level of CPS and the protection rates.1. Cloning and expression of one section of muramidase-released protein and extracellular protein factor gene of Streptococcus suis serotype 2 and its protectionAccoding to the epf and mrp gene in GenBank .the fragment of epfand mrp gene containing epitopes were selected by analysis of DNAStar software.then two pairs of primes were designed ,the endonuclease of Nco 1 and EcoR 1 was added to the end of mrp primers , EcoR I and Xho I was added to the end of epf primers. The recombinant plasmids pET32a-mrp and pET32a-epf were constructed and transformed into E.coli BL21 , the fusion protein of MRP about 43 kD and EPF about 47kD were expressed by IPTG. The purified fusion protein was analyzed by westen blot with the produced serum. After verified positive immunity, the fragments of mrp and epf linked by PCR was cloned into pET-32a again. By the above method with IPTG, we obtained the recombinant protein consisted of rMRP and rEPF with molecular weight 74kD.Next, the antigenicity analysis by Western-blot showed the recombinant protein had the conserved epitopes of MRP and EPF respectively.2.Recombinant protein purification and research on the mechanism of coordinated action between two virulent factorThe recombinant protein of MRP-EPF(rMRP-EPF) of SS2 containing a His-tag sequence was purified by metal chelation chromatography under native condition ,after purification ,a dominant protein band with 74kD was detected by SDS-PAGE. In order to further determine antigenicity of recombinant proteins (rMRP ,rEPF and rMRP-EPF), we immunized rabbits immunized by the purified protein emulsified with Freund's adjuvant...
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