Expression Of CDV Major Structural Protein In Sf9Cells And Establishment Of Indirect ELISA Assay

Canine distemper (CD) is a highly contagious, acute viral disease that is caused by Canine distemper virus (CDV). CDV infection range continues to expand with the evolution, not only can infect the Canidae, Mustelidae, the Procyonidae family, the Giant panda Branch, Japanese macaques can also be infected. It was reported that CDV nucleic acid detected in Pagets disease patient organizations.Although attenuated vaccine can prevent CD, which often presents an outbreak caused huge economic losses to farmers. A reason of immune failure is lack of screening antibody detection. CDV microneutralization test(SN), long time; the rapid diagnosis of the colloidal gold test strip, expensive;ELISA method can achieve rapid diagnosis bulk. CDV is sensitive to the physical and chemical factors, and it is difficult to obtain the complete antigen. The most promising method is using a baculovirus expression system,expressing major structural activity protein. The nucleocapsid protein of dolphin measles virus (DMV) also showed the antigenie activity.It is using baculovirus expression system to express the major structural protein H,F,N,then establish the detection of CDV indirect ELISA (iELISA) immunoassay for CDV prevention.The complete CDV-H, F, N gene was obtained by RT-PCR to construct the recombinant bacmid the Bacmid-H, the Bacmid-F, the Bacmid-N and transfect into Sf9insect cells.. It was proved successful expression of the H, F, N protein by SDS-PAGE. Western-blot, indirect immunofluorescence results showed reactogenicity. Western-blot did not timely detected bands of F protein reacting with dogs positive serum, however, the F gene measured in the cytosol after expression, protein cleavage bands measured by SDS-PAGE. iELISA methods were established with the CDV N, H-protein, contrast to SN. N antigen, serum were diluted1:40(1.6μg/mL) and1:80. Wells were coated at37℃for2h, the secondary antibody diluted1:2000, the substrate effected10min; N protein did not react with other disease-positive serum, indicating a good specificity.36canine serum samples were detected, sensitivity96.0%, specificity81.8%and compliance rate91.7%. H antigen, serum were diluted1:40(1.5μg/mL) and1:80, and did not react with other disease-positive serum, indicating a good specificity. iELISA sensitivity92.0%, specificity81.8%and compliance rate94.4%.