Establishment Of Borna Disease Virus Nucleoprotein Antibody ELISA Diagnosis Method

OBJECTIVE:This research aim at establish a Borna disease Virus (BDV) nucleoprotein serum antibody diagnosis method. Provide quickly,convenient,economical and accurate BDV cosmically screening method, while also provide support for BDV nucleoprotein mechanism research.METHODS:1,Design primer according to BDV P40 gene sequence, amplify BDV P40 gene to establish a recombinant plasmid. After identification of the plasmid by PCR, we transformed it into E.coli to express recombination BDV nucleoprotein. After purify the protein by His-trap, we analysis its quantity and purity by SDS-PAGE and Bradford method. The immunogenicity was confirmed by a Western-blot assay and component was confirmed by MALDI-TOF mass spectrum.2,We label the recombination BDV nucleoprotein with HRP, that provide enzyme labeled antigen for further sandwish antigen ELISA method.3,We immunize the New Zealand albino rabbit with recombination BDV nucleoprotein, to get anti-rabbit BDV nucleoprotein polyclonal antibodies.4,We use recombination BDV nucleoprotein as coating antigen,while the labeled nucleoprotein as secondly antigen,establishing a sadwish ELISA diagnosis assay method.RESULT:1,The recombinant plasmid was constructed correctly. The result was confirmed by PCR and nucleotide sequence analysis.2,We had successfully expressed the recombination BDV nucleoprotein result in a high purity and good immunogenicity. The SDS-PAGE test indicated the recombination protein after affinity chromatography have a purity more than 90%. The Western-blot showed the purified protein had a good immunogenicity.while mass spectrum showed a high consistency between recombinant protein and natural BDV nucleoprotein.3,Our experiment validate there is a special combining capacity between recombinant protein and antibody,which indicate the recombinant protein could be used as a coating antigen. This ability was confirmed by ELISA test. Recombinant protein have an special combining capacity with monoclonal antibody.This ability is density consistency,while other antibody don't.4,We have established a BDV nucleoprotein antibody sanwish antigen ELISA detection method, and measured some main parameter.CONCLUSION:1,We had express a solubility recombination BDV nucleoprotein in E.coli, this protein have the potential to replace natural BDV nucleoprotein as a diagnosis antigen.2,We had successfully developing a nucleoprotein antibody sanwish antigen ELISA detection method, and measured some main parameter.