Study On The Flagellin Protein As Immunologic Adjuvant For PRRSV DNA Vaccine

As one of the serious animal diseases, porcine reproductive and respiratory syndrome (PRRS) is characterized by severe reproductive failure in sows, and respiratory distress and high mortality in piglets. Since it was first reported in the United States in1987, PRRS caused huge economic losses to the swine industry in the world. The control of PRRS is achieved by vaccination, but inactivated vaccine and attenuated vaccine are experiencing enormous issues including unobvious effects, potential to revert to virulence and so on. Therefore the development of safe and effective PRRS vaccine becomes a major issue for researchers.As the major protective antigen protein of PRRSV, GP5plays an important role in the body for clearing PRRSV. Protein M is the major envelope protein of PRRSV. Protein M can form disulfide-linked heterodimers with the GP5protein. The ability of GP5induced neutralizing antibodies was promoted with the heterodimers. Protein M can also induce neutralizing antibodies and serum antibodies against PRRSV. GP3is a highly glycosylated envelope protein of PRRSV, and GP3can induce neutralizing antibodies. The flagellin of Salmonella typhimurium is a potent T cell-dependent antigen. Flagellin is a ligand for TLR5suggested its potential as an adjuvant.In order to obtain safe and efficient PRRSV candidate vaccine, we constructed a DNA vaccine co-expressing GP5, M and GP3, and used flagellin as TLR-dependent adjuvant. We evaluated the humoral and cellular immune responses of DNA vaccine in the mouse model. The details were as follows:1. The preparation of monoclonal antibody against GP3protein of PRRSVThe recombinant prokaryotic expression protein GP3was purified by extracting inclusion bodies, and immunized with the BALB/C female mouse. Spleen cells of mice which serum antibody titer reached the standard level are obtained and fused with SP2/0cells. Sixteen strains of monoclonal hybridoma cell lines were screened by ELISA test. Finally ten strains of monoclonal hybridoma cell lines can react with eukaryotic proteins by Western Blotting and IFA.2. Construction of recombinant eukaryotic plasmids co-expressing GP5, M and GP3The synthetic ORF5(synORF5), synORF6, ORF3, CMV promoter and SV40pA were obtained by overlapping PCR. The synORF5-SV40pA, CMV-synORF6-SV40pA and CMV-ORF3-SV40pA were cloned into pVAXl vector by restriction enzyme. The recombinant eukaryotic co-expressing GP5, M and GP3plasmid pVAX-ORF3/5/6was transfected into cells, and GP5and GP3protein were expressed by Western blotting.3. The expression and purification of flagellinFlagellin gene was amplified from Salmonella typhimurium, and cloned into prokaryotic expression plasmid pET-32a. The recombinant prokaryotic expression plasmid pET-32a-Flic was constructed. Flagellin was expressed in Escherichia coli expression system and verified by SDS-PAGE. We obtain purified flagellin after inclusion bodies were refolded4. DNA vaccine and Flagellin adjuvant induced immunity response of mouseCompared with the group lacking of adjuvantug, different doses of flagellin (1OOμ,50μ and10μ) mixed with the DNA vaccine (100ug) were immunized Balb/C mouse. The serum ELIS A antibody of Balb/C mouse was detected after immunized3weeks and6weeks by GP5-ELISA. The serum neutralizing antibody of Balb/C mouse was detected after immunized6weeks by the neutralization assay. The results showed that the DNA vaccine can induce the mouse to produce antibodies against GP5and neutralizing serum antibody against PRRSV.100μ dose of flagellin as a DNA vaccine adjuvant can increase serum antibody and neutralizing antibody. Flagellin can enhance the ability of humoral immune responses induced by DNA vaccine.The cellular immune response was evaluated by ELISA and real-time RT-PCR after immunized6weeks. The results showed that the DNA vaccine can significantly increase the level of IFN-γ(P<0.05). Compared with the group lacking of adjuvant (465.95pg/mL), different doses of flagellin mixed with the DNA vaccine(1034.94pg/mL、893.01pg/mL、793.01pg/mL) can significantly increase the level of IFN-γ secretion (P<0.05). Flagellin can markedly enhance the ability of cellular immune responses induced by DNA vaccine.