ABSTRACT
In order to prepare the monoclonal antibodies (MAbs) by the recombinant phosphoprotein (p24) of Borne dis ease virus (BDV) as immunogen,the spleen cells of immunized balb/c mice with the recombinant BDV p24 were fused with the myeloma cells SP2/0.The hybridoma cell lines secreting MAbs against p24 were obtained after selected by indirect ELISA and subcloned for 3 times.The MAbs were prepared by intraperitoneal injection in mice,purified by affinity chromatography,identified by western blot and immunofluorescence assay (IFA) for specificity.The purified MAbs against BDV p24 from ascites belonged to IgG1 showed a purity of 98％ and 93％,and a titer of 1 ∶ 81 000,and specific reaction with the BDV p24 restructured and expressed in Oligodendroglia cells (OL).The MAbs against BDV p24,with high specificity and sensitivity,were prepared successfully,which laid a basis for the study of diagnostic reagents and pathogenic mechanism of BDV.
ABSTRACT
In order to prepare the monoclonal antibodies (MAbs) by the recombinant phosphoprotein (p24) of Borne dis ease virus (BDV) as immunogen,the spleen cells of immunized balb/c mice with the recombinant BDV p24 were fused with the myeloma cells SP2/0.The hybridoma cell lines secreting MAbs against p24 were obtained after selected by indirect ELISA and subcloned for 3 times.The MAbs were prepared by intraperitoneal injection in mice,purified by affinity chromatography,identified by western blot and immunofluorescence assay (IFA) for specificity.The purified MAbs against BDV p24 from ascites belonged to IgG1 showed a purity of 98％ and 93％,and a titer of 1 ∶ 81 000,and specific reaction with the BDV p24 restructured and expressed in Oligodendroglia cells (OL).The MAbs against BDV p24,with high specificity and sensitivity,were prepared successfully,which laid a basis for the study of diagnostic reagents and pathogenic mechanism of BDV.
ABSTRACT
Objective To investigate the expression and location of Borna disease virus(BDV)nucleoprotein in transfected oligodendrocytes,and then to explore its expression mechanisms in oligodendroglial cell(OL)and their effects on cell proliferation.Methods We used PCR to detect BDVp40 gene fragments in transfected OL,laser confocal microscopy and Western blot method to detect the expression of nuclear protein and its intracellular location in the cell,MTT to detect the influence of cell proliferation by nuclear protein on the OL.Results We had detected the BDVp40 gene fragments in transfected oligodendrocytes.The nucleoprotein can be positioned in the cytoplasm and cell membrane by laser scanning confocal microscope;Western blot results showed that there is nucleoprotein in the constitutive protein,but was not detected in the cytoplasm.MTT tests showed that nucleoprotein expressed in OL can inhibit cell proliferation.Conclusion It is indicated that the transfected OL stablely express of BDV nucleoprotein,and itslocation is in the cell structure of proteins,particularly in the cytoplasm and more richer in cell membrane,that indicating they may play a key role in cell signal transduction or into the cell-mediated viral infection.BDV nucleoprotein inhibit proliferation of OL,which maybe an important mechanism of Borna disease virus persistant infection and produce symptoms.