Prokaryotic Expression And:Immunogenicity Analysis Of C5a Peptidase Of Streptococcus Agalactiae Isolated From Tilapia

Streptococcus agalactiae is a gram-positive bacterium which was distributedwidely in the world. It could cause the aquatic animals, mammals and even humanbeings suffering from sepsis, pneumonia and meningitis. S.agalactiae has become themajor pathogen of many different kinds of fishes, especially warm-water cultured fish.Currently, it is mainly prevalent in farmed tilapia in southern China, which has causedheavy economic losses. In this study, the scpB gene of S.agalactiae ZP-N strain wascloned, and the distribution of ScpB protein epitopes was analysised, using thebioinformatics and molecular biology technique. And the recombinant protein whichhas biological activity was obtained by means of prokaryotic expression. At the sametime, the immunogenicity of the protein was studied. This study aimed at thedevelopment of a scientific and effective multi-combined vaccine of S.agalactiae andprovides new measures for prevention and control of streptococcicosis of tilapia. Themain contents and results of this study are as follows.1Cloning and construction of recombinant expression plasmid of scpB gene of S.agalactiae isolated from tilapiaS.agalactiae surface proteins can stimulate the host to produce a protectiveimmune. The protein C5a peptidase is widespread in a variety of serotypes ofStreptococcus strains and has a good immunogenicity. It is a possible protein vaccinecarrier that could induce a protective immune response by itself. However, ScpB, theimmune epitopes and the role of virulence groups of S. agalactiae scpB gene from fishare still unclear. In this study, the scpB gene is amplified from genome DNA ofS.agalactiae isolated from tilapia by PCR with specific primers. Restriction analysis andDNA sequencing confirmed that the scpB gene is a length of3405bp, encoding1134amino acid, which is highly conserved and revealed a surprising degree of homologyamong strains isolated from other mammals. Molecular analysis of scpB gene was madeby bioinformatics tools such as DNAstar, Clustal X2.0, MEGA5.05, ExPASyProtParam, NCBI Protein blast, NCBI Conserved Domain and so on. Results showedthat the encoding amino acid contained3Catalytic triad,7Putative active site,2 Specific hits, and4superfamilies (2Peptidases-S8-S53，1DUF1034, and1FlgD-lg).Moreover, it was discovered that C5a peptidase has a mount of epitopes. So it suggeststhat the protein may have strong immunogenicity. And then the PCR product wasinserted into the multiple cloning sites of pET32a(+) and the constructed recombinantplasmid pET32a(+)/scpB was transformed to E.coli BL21(DE3) for expression. Thepositive colony which was identified by PCR and digestion (EcoRⅠand XhoⅠ)showed that the recombinant plasmid pET32a (+)/scpB was constructed successfully.2Prokaryotic expression and immunogenicity analysis of C5a peptidase ofS.agalactiaeIn this study, scpB gene was amplified from the genomic DNA of the S.agalactiaestrain isolated from disease tilapia farmed in Guangdong province, China. The scpBgene contains a2799bp Open Reading Frame (ORF), encoding932amino acids. Themolecular mass of the deduced amino acid sequences was103Ku. Blast analysisshowed that it shares high similar identities with scpB sequences of human GBSregistered in GenBank. Prokaryotic expression vector pET32a (+) was used to constructa recombinant expression vector pET32a (+)-scpB, and then the recombinant weretransformed into E.coli BL21(DE3) strains. The recombinant expression vector pET32a(+)-scpB was selected and then induced to express by0.5mmol/L IPTG for8h at37℃.The expressed recombinant protein was purified by nickel chelate affinitychromatography and ultrafiltration tube enrichment. SDS-PAGE analysis and Westernblot showed that a specific band of protein about124Ku in molecular weight wasobtained. The recombinant strain can produce high expression of ScpB protein, and theexpressed fusion protein mainly existed in a form of inclusion body. To analyze theimmunogenicity of the recombinant protein, the purified fusion protein and Freund sadjuvant were mixed according to a certain proportion to produce vaccines. Threeimmune dosages,1μg/g(F1),3μg/g(F3) and5μg/g(F5) were used in this process. Fourweeks after immunity, tilapia were challenged by artificial infection of GBS ZP-N strain,which has been previously isolated and confirmed to be tilapia pathogen by ourlaboratory. The recorded relative percent survivals (RPS) of the vaccinated groupsranged from69.99%to89%, of which group F5has the highest RPS. The lysozymeactivities, superoxide dismutase (SOD) activities and antibody level (OD450nm) are testedeach week until the end of the experiment. The results showed that after the immune,there are significantly higher lysozyme activities, superoxide dismutase (SOD) activities and antibody activity (OD450) in the vaccinated fish than those in the control group (P <0.01). The results of this study showed that recombinant protein ScpB has a strongimmunogenicity and protective effect, which laid a foundation of further study onstructural domain and S.agalactiae polypeptide vaccine development of ScpB.3The immune protection in tilapia against S.agalactiae using C5a peptidasenanoparticles encapsulated with PLGAThe recombinant surface protein ScpB of S.agalactiae was encapsulated inmicrospheres prepared with the biodegradable materials poly D, L-lactide-co-glycolicacid (PLGA) by ultrasonification emulsification. The encapsulated vaccine wasdesigned to intramuscular injected tilapia, and then the relative percent survival (RPS)of the vaccinated groups was calculated. What s more, the lysozyme activities,superoxide dismutase (SOD) activities and antibody level (OD450nm) are tested eachweek until the end of the experiment. The results showed that the loading rate of ScpBin microspheres is2.55%determined by Bradford method and the encapsulationefficiency reaches48.76%. The RPS of the vaccinated groups ranged from66.80%to87.66%, of which group P1has the highest RPS. After the immune, there aresignificantly higher lysozyme activities, superoxide dismutase (SOD) activities andantibody activity (OD450) in the vaccinated fish than those in the control group (P <0.01). These results showed that PLGA was qualified as the vector and adjuvant ofvaccine of protein ScpB, which has better immune effects and a persistence of immuneprotection.