Preparation Of Recombinant Surface Proteins Of Streptococcus Agalactiae And Development Of Preliminary Detection Method For The Bacterium

Cattle mastitis is one of the world’s most common and most damaging diseases, it not only influences the amount of milk production and causes economical losses, but also influences milk’s quality. As one of common pathogenic microorganisms of the cattle mastitis, Streptococcus agalactiae is the main problem in dairy industry with great concentration. As a result, it is essential to develop a rapid and effective test method for improving the quality and safety of dairy product and cow business, as well as benefit to human’s health. Three S. agalactiae surface proteins (Rib, Sip, Alpha) were recombinant expressed in this study and the polyclonal antibodies were prepared. These studies are useful for the researches on test strip and multivalent vaccine.In this study, the ATCC standard bacteria of six seratypes were recultivated and the genome DNA were extracted. With the specific primers designed according to the gene sequences of three surface proteins of Rib, Sip and Alpha, fragments of453bp,1302bp and699bp were amplified using the genomic DNA as templates and cloned into pMD18-T vector for sequencing. The sequencing results indicated that the right gene fragments were obtained. Gene fragments of rib and sip were subcloned into pET26b expression vector and gene fragment of alpha was subcloned into pGEX4T-1vector. After being induced by IPTG, three recombinant proteins with molecular weight of18kDa,61kDa and52kDa were successfully expressed. However, Rib and Alpha formed inclusion bodies. After being expressed in larger scale and denaturing and renaturing procedures were performed if inclusion bodies were formed, the three proteins were purified with affinity resins and identified by MS. The similarities of the3recombinant proteins with Rib, Sip and Alpha reached99.99%,100%and100%, respectively. The polyclonal antibodies were prepared through immunizing rabbits and the titers were480000:1,640000:1and320000:1, respectively. With the antiserums, indirect ELISA methods were used to test the ATCC bacteria and the results demonstrated that all the polyclonal antibodies exhibited affinities and specificities to the bacteria.