Expression And Application Of Peste Des Petits Ruminants Virus N Protein And Vesicular Stomatitis Virus N Protein

Peste des petits ruminants featured as fever, stomatitis, diarrhea, and pneumonia is an acute infectious disease caused by Peste des petits ruminants virus (PPRV). The PPRV genome is negative-sense single strand RNA, and encodes 6 structural proteins (N, M, P, L, F and H protein). The coronavirus nucleocapsid (N) is a structural protein that forms the most stable antigenicity of PPRV. After PPRV infection, the antibody at high titer against N protein was produced and maintained for a long time, without virus-neutralizing activity. Vesicular stomatitis (Vesicular Stomatitis VS) is an acute and highly contagious disease caused by vesicular stomatitis virus (VSV), with character of epithelial blisters in the susceptible animal organs like oral mucosa, tongue, lip, nipple and Coronet etc. VSV genome is is a single molecule of negative-sense RNA that encodes five major proteins:G protein (G), large protein (L), phosphoprotein, matrix protein (M) and nucleoprotein. N protein, highly conservative and with cross protection among VSV all type, can protect the virus from RNase degradation and play a key role in the process of viral replication and transcription. So, PPRV N and VSV N proteinexpressed in the E. coli and purified, then were used indirect ELISA for PPRV antibody detection, and for preparation of VSV monoclonal antibodies, respectively.According to the published N gene sequence of PPRV genome in GenBank, a pair of specific primers was designed and synthesized. RT-PCR was used to amplify the 1500 bp length N gene fragment and the fragment were subcloned into pCold I expression vector. The recombinant N protein, expressed by IPTG induction in E. coli, was purified by affinity chromatography and identified with good immunogenicity by Western blotting. Using the recombinant N protein as antigen and optimizing reaction conditions of ELISA, an indirect ELISA detection method was established for PPRV antibodies. The evaluated results were as followed:the foot and mouth disease virus (FMDV), sheep pox virus, pseudorabies virus positive serum were detected as negative; the batch coefficient of variation were less than 10 %; the coincidence rate was up to 98.3% compared with imported small ruminant animal epidemic disease serum antibody detection kit.Based on published VSV genome N gene sequence in GenBank,2 N gene of VSV different serotypes were synthesized respectively. After sequence analysis, design and synthesis one pair of specific primers, N gene fragment with about 1200 bp length was amplified by PCR, and subcloned into pCold I expression vector. The recombinant N protein was induced with IPTG and purified by affinity chromatography, and the recombinant protein has good reactivity with positive serum of VSV detected by Western blotting.6 weeks old female BALB/c mice were immuned with the recombinant N protein as immunogen, and spleen cells from the mice were cloned and fused with myeloma cells SP2/0, Three stable hybridoma cell lines secreted VSV specific antibody, screened by indirect ELISA, identified further by indirect immunofluorescence were obtained.In summary, the recombinant PPRV N protein expressed in prokaryotic cells with good reactivity, was purified and used for establishment of PPRV indirect ELISA antibody detection method. This detection method was evaluated to be with good sensitivity, specificity, repeatability and accuracy, which was a simple, rapid and high-throughput serum detection method for PPRV antibody detection, clinical diagnosis, epidemiology and vaccine immune effect monitoring. The recombinant VSV N protein, expressed in prokaryotic cells with good immunogenicity, was purified and used as antigen to immune mice. Hybridoma cell lines secreted VSV specific antibody was obtained, after the immuned spleen cells fused with myeloma cells SP2/0. It is potential for VSV immunological detection that monoclonal antibody is prudcued and furified from the hybridoma cell line.