Immune-protective Effect Of CBP And CPS From Streptococcus Agalactiae In Tilapia

Tilapia is the main freshwater aquaculture fish in the south of China and has the advantages of strong adaptability,fast growth and high fecundity.But in recent years,Streptococcus agalactiae disease has become the biggest threat to tilapia farming and have seriously hampered the healthy development of tilapia aquaculture.Immunization prevention has become an effective method for prevention and treatment of tilapia S.agalactiae disease by improving the immunity of fish,therefore,the research and application of efficient vaccines have caused more and more attention.The surface components of S.agalactia play an important role in the adhesion of pathogens and hosts,often own good immunogenicity,so these surface components can be used as candidate antigens for vaccines.In this study,we expressed the surface protein Choline-binding protein(CBP)and extracted Capsular Polysaccharides(CPS)of S.agalactiae into vaccine to explore whether they can protect tilapia against S.agalactiae infection.The results of this study contain as follows:1.Cloning,sequencing and prokaryotic expression of Streptococcus agalactiae CBP geneWe cloned CBP gene and the size was 1515 bp,coding 504 aa.According to bioinformatics analysis,amino acids encoded by CBP gene were highly conserved among S.agalactiae,conservative even up to 98%-100%.CBP contains two domains,GBS Bsp-like repeat domain and Src Homology 3(SH3)domains,respectively.CBP is the surface protein without a signal peptide or a transmembrane region.Recombinant expression plasmid containing the CBP genes was constructed by prokaryotic expression vector p ET-28a(+),and transformed into Escherichia coli BL21(DE3)for prokaryotic expression.The recombinant protein was successfully induced to express by IPTG and the soluble fraction was purified to obtain the recombinant protein by affinity chromatography.2.Extraction,detection and identification of Streptococcus agalactiae capsular polysaccharidesWe used CTAB organic solvent precipitation method to extract capsular polysaccharides of S.agalactiae,used phenol extraction to remove the protein from CPS,used low concentration ethanol precipitation to remove nucleic acids from CPS,used the anthrone method to determine the concentration and analyzed the molecular weight and composition of monosaccharides.The results showed that,Capsular polysaccharide was composed of glucose and galactose and its molecular weight was distributed between 40000 D-600000 D.Protein content in the extracted capsular polysaccharide samples was not detected.In this study,We have successfully extracted the higher purity and concentration of capsular polysaccharides.3.Protective effect of CBP and CPS vaccines in tilapia against S.agalactiaeCBP protein vaccine and CPS vaccines were prepared.Nile tilapia was twice immunized by intraperitoneal injection and challenged by S.agalactiae strain.The results showed that the relative percent of survival(RPS)of CBP group,CPS-TT group,CPS-CBP group and CPS group were 56.58%,69.03%,57.21%,42.79%,respectively.ELISA analysis showed that serum antibody titers appeared to increase in different degrees,and the antibody titer of CBP group significantly higher than CPS group,indicating that the vaccine can stimulate the fish to produce specific antibodies.The results of this study informed that the CBP vaccine and CPS vaccines have immune-protective effects and can improve the resistance of tilapia to Streptococcus agalactiae.