Prokaryotic expression and polyclonal antibody preparation of nonstructural protein Npro of bovine viral diarrhea virus

Objective Bovine viral diarrhea virus（BVDV）can seriously damage the reproductive performance，respiratory system，digestive function and lactation ability of cattle.The non-structural protein Npro 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 Npro in prokaryotic cells and prepare polyclonal antibodies，which provided a material basis for further study of the function of Npro protein. Method The Npro gene sequence of BVDV was searched on NCBI website.The physicochemical properties of Npro 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 Npro was amplified and cloned into the pET-28a（+）vector to construct the recombinant plasmid pET-28a（+）-Npro.The correctly sequenced pET-28a（+）-Npro plasmid was transformed into E. coli BL21（DE3）expression competent cells，and the Npro 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. Result （1）The results of bioinformatics analysis showed that Npro protein had 504 amino acids，which was an extracellular hydrophilic protein.Npro 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 E. coli.（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. Conclusion In this study，BVDV Npro recombinant protein with good immunogenicity is successfully expressed and purified in E. coli.The prepared high-titer specific polyclonal antibody provides an effective tool for further study of the biological function of the protein.