Screening Of The Immunogenic Proteins Of Streptococcus Agalactiae In Tilapia

Streptococcus agalactiae,Gram-positive bacteria,can infect humans and variety of vertebrates,is also one of the major bacterial pathogens of tilapia.In recent years,there have been prominent problems of streptococcus agalactiae,which seriously threatens the safety of water products of tilapia and the healthy development of the aquaculture,meanwhile,current vaccine immunization provides new ideas and means for the prevention and cure of this disease,but the indistinction of the antigenic component of the vaccine restricts the development of new vaccines.Therefore,screening the immunogenic protein of streptococcus agalactiae is vital to the vaccine development and the test diagnosis of the pathogen.This study uses immunoplatmic techniques for screening,identifying immunogenic proteins of HN016 strains of streptococcus agalactiae,producing tilapia with the above recombinant protein,and evaluating the immunization effect,aiming to provide material and theoretical basis for the research and further progress in the invention of subunit vaccine of streptococcus agalactiae.This study successfully extracted the cell wall protein of streptococcus agalactiae and prepared the infectious sera of tilapia,9 recombinant plasmid were successfully constructed,and 6 recombinant proteins were successively induced and expressed(r Gap,r NADP-gly3,r PMCA-type,r ATP-Clp,r BMP-family,r Fts H).The above six recombinant proteins were experienced on tilapia and the results showed that:The relative immune protection rates were reached at 86.7%,93.3%,60.0%,66.7%,60.0%and 73.3%at the attack dose of the medium concentration(1×10~7 cfu/tail),while the relative immune protection rates were reached at 90.9%,72.7%,90.9%,63.6%,81.8%and 72.7%at the attack dose of low concentrations(1×10~6 cfu/tail).At the same time,the immune protection effect of recombinant protein was evaluated by ELISA serum antibody detection,tissue germ,fluorescent quantitative PCR,tissue pathological slice observation and other experiments.Serum titer detection found that the above six recombinant proteins can induce tilapia to produce high titers of antibody levels.Tissue carrier volume detection found that after attacking,the amount of germs in spleen,liver,head kidney,body kidney,brain,and tail vein whole blood of tilapia has declined significantly in the immunome group of 6 recombinant protein.Selecting the r Gap and r NADP-gly3 immunization groups with higher immune protective rate,which can produce high antibody levels,and with better removal ability of pathogens to conduct pathological observation and discovered that the pathological change in r Gap and r NADP-gly3immunization groups were significantly reduced in the spleen,liver,head kidney,body kidney,brain tissue and intestinal tissue.Finally,the variation on gene expression of immunocompetence different groups were detected by fluorescent quantitative PCR and found that the expression of natural immunocyte cytokines of tilapia in r Gap and r NADP-gly3 immunome was significantly inhibited,and the expression of hematurity factors was increased;while the amount of molecular expressions such as CD4,CD8,and Ig T of obtaining system exhibits an increase in varying degrees.In summary,this study successfully screened 6 bacteria protective antigens of streptococcus agalactiae,which provided tilapia with superior protective antigen.The research conclusions above will provide an important scientific basis for research and development of subsequent ribs subunit vaccines,while these new identified immunogenic proteins are also of great significance for the study of new diagnostic reagents.