Molecular Cloning, Expression And Immune Protective Effect Of FbsA And α-enolase Gene From Streptococcus Agalactiae

Tilapia is the main freshwater aquaculture species in the south of China andStreptococcus agalactiae disease is the current constraint of tilapia aquacultureindustry development. The prevention and control of epidemic Streptococcusagalactiae has become the most thorny issue in the tilapia aquaculture industry, andalmost all antibiotics are difficult to achieve the desired control effect. In order tosolve this problem, the research on subunit vaccines with efficient cross protection formultiple strains and serotype has caused more and more attention. As the bacterialsurface related proteins，they usually have better immunogenicity and often served ascandidate vaccine gene. In this study, based on the domestic and foreign research onStreptococcus agalactiae surface related proteins, we cloned and expressed thesurface related protein FbsA、α-enolase、GapC and made FbsA and α-enolase intovaccine to explore whether them can protect tilapia against Streptococcus agalactiaeinfection. The results were as following:1.According to the research on Streptococcus agalactiae surface related proteins, wecloned the ORF of FbsA, α-enolase and GapC gene and the size were1041bp,1308bp,1011bp and coded346aa,435aa,336aa respectively. Based on the bioinformatics analysis, we found that the C terminal of FbsA has a cell wall anchorLPXTG motif.Besides, the extracellular region has13repeat containing16aminoacids and the N terminal has a signal peptide and transmembrane structure with35amino acids. α-enolase is an enzyme involved in carbohydrate metabolism ofcytoplasm and it can catalyse the dehydration of2-phospho-D-glycerate tophosphoenolpyruvate. GapC protein has the3-glyceraldehyde phosphatedehydrogenase (GAPDH) activity and it is a key enzyme of prokaryotic andeukaryotic organisms of the glycolytic pathway. Both of the two proteins lack signalpeptide and transmembrane domains.2.We used the E. coli BL21(DE3) expression system to express the recombinantproteins pET28-FbsA、pET28-α-enolase and pET28-GapC which has removed of thesignal peptide and transmembrane structure and added six his-tagged and themolecular weights were38.2KD、49.4KDand38.3KD respectively. SDS-PAGEanalysis revealed that the recombinant proteins FbsA、α-enolase were soluble. Therecombinant protein GapC was unsoluble. Purified the expressed protein FbsA andα-enolase by Ni-NTA Agarose for his-tag andultrafiltrated.3.In the immunization protection experiment, the RPS of pET28-FbsA andpET28-α-enolase were40.63%,62.50%respectively. After the primary immunization,we tested the tilapia’s respiratory burst, lysozyme activity and antibody titers. Wefound that, after immunization, the respiratory burst and serum lysozyme activit ofimmunization groups were significantly improved compared with the PBS controlgroup and ELISA assay tested corresponding antibody. The results of this studyinformed that the recombinant protein rFbsA and rEnolase has strong immunogenicityand protection effect and could be used as tilapia Streptococcus subunit vaccinecandidate genes.