ABSTRACT
To screen the best genotypeⅠJapanese encephalitis virus subunit vaccine candidate antigens, the prMEIII gene, the polytope gene and the prMEIII-polytope fusion gene of the GenotypeⅠJapanese encephalitis virus GS strain were cloned into prokaryotic expression vector pET-30a. The recombinant proteins were obtained after the induction and purification. The prepared recombinant proteins were immunized to mice, and the immunogenicity of the subunit vaccine candidate antigens was evaluated through monitoring the humoral immune response by ELISA, detecting the neutralizing antibody titer by plaque reduction neutralization test, and testing the cell-mediated immune response by lymphocyte proliferation assay and cytokine profiling. The recombinant proteins with the molecular weights of 35 (prMEIII), 28 (polytope antigen) and 57 kDa (prMEIII-polytope) induced strong humoral and cellular immune responses in mice. Compared with prMEIII-polytope and polytope proteins, the prMEIII protein induced a significant expression of IL-2 and IFN-γ (P0.05). The study suggests that the prMEIII protein can be used for the development of the Japanese encephalitis virus subunit vaccine.
Subject(s)
Animals , Mice , Antibodies, Viral , Blood , Antigens, Viral , Allergy and Immunology , Encephalitis Virus, Japanese , Allergy and Immunology , Encephalitis, Japanese , Allergy and Immunology , Immunogenicity, Vaccine , Mice, Inbred BALB C , Vaccines, Subunit , Allergy and Immunology , Viral Vaccines , Allergy and ImmunologyABSTRACT
Aim To isolate and purify a novel plasminogen activator(PA)from Gloydius brevicaudus venom(GBV)and study characterization and biological activities of GBV-PA.Methods Affinity chromatography in Benzamidine Sepharose 6B(AC)and Lichrospher C-18 4.6/250 reversed phase chromatography(RPC)were used for isolation and purification;SDS-PAGE was used to detect molecular weight(MW);Disc polyacrylamide gel eletrophoresis was used to measure the point of isoelectric(pI);Chromogenic substrate method was used to observe the biological activities.Results A novel GBV-PA which its purification reached the homogeneity level was isolated and purified from GBV by AC and RPC;The MW of the novel GBV-PA was 3.26?104 and the pI was 5.2;The novel GBV-PA activated human plasminogen specifically and the special activity was 2.87 t-PA IU?mg-1;Moreover,our results indicated that this novel GBV-PA was a serine proteinase which had no affinity to fibrin.Conclusion A novel GBV-PA that can be isolated and purificated from GBV by AC and RPC was proved to be a serine protease and has no affinity to fibrin.