Prokaryotic expression and polyclonal antibody preparation of nonstructural protein N<sup>pro</sup> of bovine viral diarrhea virus

<bold>Objective</bold> Bovine viral diarrhea virus（BVDV）can seriously damage the reproductive performance，respiratory system，digestive function and lactation ability of cattle.The non-structural protein N^pro encoded by the virus is highly conserved and has proteolytic activity，which plays a key role in mediating immune escape.This study was to express BVDV non-structural protein N^pro in prokaryotic cells and prepare polyclonal antibodies，which provided a material basis for further study of the function of N^pro protein. <bold>Method</bold> The N^pro gene sequence of BVDV was searched on NCBI website.The physicochemical properties of N^pro protein were analyzed with online bioinformatics software，including molecular weight，isoelectric point（pI），instability index，aliphatic index and average hydrophilicity.The homologous recombination primers were designed and the target gene N^pro was amplified and cloned into the pET-28a（+）vector to construct the recombinant plasmid pET-28a（+）-N^pro.The correctly sequenced pET-28a（+）-N^pro plasmid was transformed into <italic>E. coli</italic> BL21（DE3）expression competent cells，and the N^pro protein after induced expression and nickel column purification was used to immunize mice for polyclonal antibody preparation.The reactogenicity of polyclonal antibody was identified by Western blot. <bold>Result</bold> （1）The results of bioinformatics analysis showed that N^pro protein had 504 amino acids，which was an extracellular hydrophilic protein.N^pro protein had no signal peptide and transmembrane domain，and its secondary structure was mainly composed of β-sheet and random coil.（2）The prokaryotic expression plasmid pET-28a（+）-Npro was successfully constructed.After double enzyme digestion and gene sequencing，the recombinant protein Npro with a molecular weight of about 18 ku was expressed in <italic>E. coli</italic>.（3）The purified recombinant protein Npro was used to immunize mice to prepare polyclonal antibodies.The results of enzyme-linked immunosorbent assay（ELISA）and Western blot showed that the titer of the polyclonal antibody was 1∶409 600，which had good specificity，reactivity and immunogenicity. <bold>Conclusion</bold> In this study，BVDV Npro recombinant protein with good immunogenicity is successfully expressed and purified in <italic>E. coli</italic>.The prepared high-titer specific polyclonal antibody provides an effective tool for further study of the biological function of the protein.