ABSTRACT
Recombinant baculovirus containing sigmaC gene of Avian reovirus was constructed using Bac-To-Bac Baculovirus expression system, and recombinant sigmaC protein was expressed by infecting the sf9 cell with recombinant baculovirus. Firstly, sigmaC gene of Avian reovirus was cloned and inserted into donor plasmid pFastBacHTA to obtain recombinant donor plasmid pFsigmaC. Plasmid pFsigmaC was transformed into E. coli DH10Bac for integration into bacmid vector and the recombinant bacmid plasmid BacmidsigmaC was obtained. Recombinant baculovirus rBacsigmaC was obtained by transfection of the sf9 cells with BacmidsigmaC. Western blot and indirect immunofluorescence assay (IFA) were carried and the results showed that the recombinant sigmaC protein with 37 kDa molecular weight was expressed successfully.
Subject(s)
Animals , Baculoviridae , Genetics , Capsid Proteins , Genetics , Metabolism , Cell Line , Cloning, Molecular , Gene Expression , Genetic Vectors , Genetics , Metabolism , Orthoreovirus, Avian , Genetics , Metabolism , Recombinant Proteins , Genetics , Metabolism , Spodoptera , TransfectionABSTRACT
Infectious bursal disease virus(IBD) causes infectious bursal disease (IBD), which infects bursal of chicken and can evoke immune suppression. This study identified the antigenic epitopes of four McAbs to IBDV VP3(HRB-3F, HRB-7B, HRB-7C and HRB-10E)with pepscan. A set of 17 partially overlapping or consecutive peptides (P1-P17) spanning VP3 were expressed for epitope screening by pepscan. Finally, two antigenic epitopes, 109-119aa and 177-190aa of IBDV VP3, were identified by Western blot and ELISA. The peptides on epitopes could react with IBDV, and they had better immunnogenicity. The sequences of epitopes were compared with that of several other IBDV strains in the same region, and was found they were totally homologous. This study showed the two epitopes were novel conserved linear B cell epitopes on the VP3 of IBDV. This study provides basis for the development of immunity-based prophylactic, therapeutic and diagnostic measures for control of IBD and further for structural and functional analysis of IBDV.
Subject(s)
Animals , Mice , Antibodies, Monoclonal , Allergy and Immunology , Antibodies, Viral , Blood , Allergy and Immunology , Blotting, Western , Capsid Proteins , Genetics , Allergy and Immunology , Metabolism , Enzyme-Linked Immunosorbent Assay , Epitopes , Genetics , Allergy and Immunology , Metabolism , Immune Sera , Allergy and Immunology , Immunization , Immunohistochemistry , Infectious bursal disease virus , Genetics , Allergy and Immunology , Metabolism , Mice, Inbred BALB CABSTRACT
Infectious bursal disease virus (IBDV), the causative agent of a highly contagious disease in chickens, carries a small nonstructural protein (NS). In this study, vvIBDV Gx-VP5 genes were cloned into plasmid pET30a( + ) and expressed in E. coli with IPTG inducing. BALB/c mice were immunized with the purified recombinant fusion protein. SP2/0 myeloma cells and spleen cells of BALB/c mice were fused by PEG(MW1500), three hybridoma cell lines were examined by indirect ELISA and clone for three times by limited dilution, and were named as 4B4, 6D12, 3E8. The subtype of the monoclonal antibodies were IgG1 with a subtype identified ELISA kit, and light chains were kappa. The ascites titers of monoclonal antibodies were 5 x 10(4), 3.5 x 10(4), 3 x 10(4) by indirect ELISA, respectively. Indirect ELISA and Western blot results showed that the monoclonal antibodies only acted with VP5 protein, IF analysis indicated that three monoclonal antibodies acted with IBDV Gt. There were specific fluorescence in detected Vero E6 cells which transient expressed VP5 protein by IFA. Therefore, monoclonal antibodies specific to IBDV VP5 proteins are specific method for detected VP5 proteins, and base on establish stabilize expressed VP5 protein Vero cell lines to research IBDV VP5 protein function.