Preparation And Identification Of Polyclonal And Monoclonal Antibodies To Borna Disease Virus Nucleoprotein

Objective Borna Disease Virus(BDV) is a neurotropic, nonsegmentednegative-strand RNA virus. Considering an even larger variety of species hasbeen infected experimentally, the host range is likely to include allwarm-blooded animals, which have the obvious neuropsychiatric symptomswith mental and behavioral abnormalities. Previous study suggested thatBorna Disease Virus may related to many diseases, such as humanpsychiatric disease. However,we haven’t found the proof of the definiterelationship between BDV and neuropsychiatric symptom so far. BDVgenome encodes6proteins in which Nucleoprotein and phosphoproteinhave been confirmed that expressed in the nucleus and cytoplasm of infectedcells with high levels. So the two portein can be used as the main target forantigen detection.BDV recombinant nucleoprotein was used to prepare antibodies whichidentified in immunology later. Then these antibodies could provide a basisto establish a few immunoassay methods and deeply explore its pathogenesis.Methods1. The recombinant vector pET14b-p40was transformed intoEscherichia coli BL21and induced by IPTG to prokaryotic express. Therecombinant protein was purified with His affinity chromatography.2. The purified protein was used as antigen to immunize New Zealandrabbits to prepare the polyclonal antibody，which was further identified byindirect ELISA and Western blotting.3. The purified target protein was used as antigen to immunize BALB/c mice.Spleen cells were separate from the immunized mice and fused withthe SP2/0myeloma cells.The positive clones were selected by indirectELISA and chose the hybridomas with high titer to limited dilution cloningand subcloning.Then hybridoma cell line was established to stable secreteanti-BDV nucleoprotein monoclonal antibody. Finally,the monoclonalantibody was identified by ELISA, Western-Blot and IF.Results1. BDV recombinant nucleoprotein was successfully expressed andpurified. Meanwhile,the molecular weight of protein is about40KD byelectrophoresis analysis.2. The Elisa and Western blot identification of polyclonal antibodyindicated that the titer was above1:256000and the antibody could reactspecifically with the BDV nucleoprotein proteins expressed both in prokaryotic and eukaryotic cells respectively.3. One hybridoma cell line was achieved successfully which couldstably secrete specific McAb against BDV nucleoprotein which named2F6E8.The hybridoma cell line was identified to have the advantage ofgood stability.The immunoglobulin heavy chain was IgG2a and light chainwas κ. The titers of Supernatants and hydroperitoneum were1:2430and1:81000respectively which measured by ELISA. WB andimmunofluorescence experiments were illustrated that the monoclonalantibody could specifically bind to recombinant BDV nucleoprotein andnatural BDV nucleoprotein.Conclusion BDV nucleoprotein polyclonal antibody and monoclonalantibody were produced.These antibodies may offer a useful tool for futurebasic study and clinical research of BDV.