Abstract：Canine distemper virus (CDV) is a pathogen of acute infectious disease, which caused fever, diarrhea, pneumonia and central nervous system disorders in Canis and Felis. The phosphorylation protein (P) is important to replication and pathogenicity of CDV. In this study, according to CDV-P gene (GenBank No. AF164967), primers were designed by primer primer 6.0 software. The P gene was cloned by reverse transcriptional polymerase chain reaction(RT-PCR), and subcloned into plasmid of pET-28(a). The pET-28(a)-CDV-P was constructed and identified by enzyme digestion and sequencing. The pET-28(a)-CDV-P was transfected into Escherichia coli BL21 (DE3). The his-CDV-P was expressed in E.coli BL21 (DE3) by isopropyl-β-D-thiogalactopyranoside (IPTG) inducing. High purified protein was obtained by his-band Ni+ affinity chromatography. The mouse anti-CDV-P antibodies were prepared from balb/c mouse immunized by his-CDV-P protein . In CDV infected cells, the expression of CDV-P protein was detected by Western blot, and subcellular localization was detected by indirect immunofluorescence assay using mouse anti-CDV-P antibodies. The results showed that his-CDV-P protein was expressed by inclusion body and its molecule weight was 65 kD by SDS-PAGE. The expression of his-CDV-P protein was more than 35% of total bacterial protein, and up to 60% of total protein after purification by grayscale scanning analysis. Mouse anti-CDV-P antibody specifically recognized the prokaryotic expression products and viral infections products by Western blot. In the CDV infected cells, the P protein expressed in the cytoplasm and nuclei by indirect immunofluorescence assay. The results showed that the P potein had 2 forms (phosphorylation and non-phosphorylation) and CDV-P protein expressed in the beginning phase and late phase of post-infection, and the phosphorylation and expression level of CDV-P were gradually increasing in infection stage by Western-blot. In this study, recombinant protein CDV-P and mouse anti-CDV-P antibodies were prepared, expression and subcellular localization were detected. Therefore, those results provide basic date for studying phosphoprotein P functions and CDV pathogenic mechanism.
