Prokaryotic Expression And Immunogenicity Analysis Of LrrG Protein Of Streptococcus Agalactiae Isolated From Tilapia

In recent years, tilapia aquaculture has been badly damaged by streptococcusis.Control and prevention of streptococcusis is a major issue for sustainable aquacultureof tilapia. Streptococcus agalactiae is one of the major causative agents, which hascaused severe economic losses in aquaculture of many species of freshwater, marineand estuarine fish worldwide. Vaccine is one of the main developing strategies toprevent fish streptococcusis. Some surface proteins of S. agalactiae have beenreported and proved to be ideal antigen candidates of protein vaccines, including LrrGprotein. LrrG protein is one of the conserved surface proteins of S. agalactiae, whichexists in all kinds of serotype of S. agalactiae strains. The LrrG protein of S.agalactiae isolated from human has been verified to protect mice from S. agalactiaeinfection. However, it is not known whether the LrrG protein from fish S. agalactiaeisolates possesses the similar effect. In order to obtain LrrG protein and explore itsimmune protection in tilapia, in this study we designed the specific primers andcloned the LrrG gene sequence from tilapia virulent S. agalactiae strain according tothe reported LrrG gene in GenBank of S. agalactiae from human. We obtained theLrrG protein by prokaryotic expression, and immuned healthy tilapia with LrrGprotein. The main results and contents are as follows.1. Characteristics of the cloned LrrG gene and the encoded LrrG protein ofStreptococcus agalactiae isolated from tilapiaThe sequencing results showed that the ORF of the cloned LrrG was2361bp inlength and encoded786amino acid and contained3conserved LRR (Leucine-richrepeat) domains. The colis accounted for68.83%in its secondary structure. NCBIBlast indicated that the homology of LrrG gene sequences of S. agalactiae fromtilapia and human was98.48%. DNAstar showed that the relative molecular weightof the LrrG protein was88.5141kDa, the theoretical of isoelectric point was9.26,and the molecular formula was C3951H6343N1095O1184S12.LrrG protein could formantigenic epitope based on the analysis of three indexes, hydrophilicity, surfaceprobability and antigenic index. This suggests that the LrrG protein may havepotential immunogenicity.2. Construction of recombinant expression plasmid and prokaryotic expression ofthe LrrG protein The LrrG fragment has been subcloned into pET-32a(+) vector and expressed inE.coli BL21(DE3) by IPTG induction at22℃. SDS-PAGE showed the molecularweight of expressed protein was108.9kDa, which was equal to the expected proteinsize. The recombinant protein existed as soluble protein and inclusion bodies. Thesoluble protein and the inclusion bodies were highly purified by Ni-chelating affinitychromatography and the concentration could reach3.40mg/mL and3.19mg/mL byultrafiltration. Both the soluble protein and the inclusion bodies after purified weredetermined by Western blot, which showed single and clear band.3. Immunogenicity analysis of LrrG protein of Streptococcus agalactiae isolatedfrom tilapiaPreliminary experiment showed that the relative percent survival (RPS) of tilapiaimmunized with LrrG protein against tilapia streptococcusis was69.28%. ELISAanalysis indicated the serum antibody titer of post-immuned tilapia at the4th weekswas1:800. These findings provide useful information for further investigation toevaluate the potential value of LrrG protein as genetic engineering vaccine for tilapia.Polylactic-co-glyconlicacid (PLGA) microparticle vaccines containing LrrGprotein were prepared by double emulsion-solvent evaporation method. The averagediameter of PLGA-LrrG protein microparticle was4.5μm, the encapsulationefficiency was38.54%, and the drug loading was1.98%. The encapsulationefficiency, drug loading and cumulative rate of drug-release, as well as themicroparticle diameter were all suitable for oral administration. The healthy tilapiawere immunized with PLGA-LrrG protein microparticle by intraperitoneal injectionor oral administration. The RPS of0.5μg/g and1μg/g PLGA-LrrG proteinmicroparticle group by intraperitoneal injection were77.89%and77.81%respectively, which were higher than2μg/g group. However the RPS of1μg/gPLGA-LrrG protein microparticle group by oral administration was77.54%, higherthan other groups obviously. The results suggest that the RPS of intraperitonealinjection is higher than that of oral administration generally.The antibody level (OD450nm) and lysozyme activities from immune groups hadsignificantly higher level than those of PBS control groups, and intraperitonealinjection groups were higher than oral administration groups. The antibody level andlysozyme activities of the empty PLGA microparticle group were similar to PBScontrol group, which showed LrrG protein could trigger specific and nonspecificimmune response of tilapia. This study shows that PLGA-LrrG protein microparticlevaccine can be applied to prevent tilapia from S. agalactiae infection. Better immuneprotection can be induced by oral administrated1μg/g PLGA-LrrG proteinmicroparticle, which is relatively simple and feasible. These findings lay a foundation for further study on using of LrrG protein as genetic engineering vaccine for tilapia.