Prokaryotic Expression of C-terminus Subfragment of Rep Protein and Preparation of Polyclonal Antibody of New-genotype Muscovy Duck Parvoviruss

  Objective   A polyclonal antibody specific to the carboxy-terminus subfragment of Rep protein of the new-genotype Muscovy duck parvovirus (N-MDPV) was prepared.

  Methods   Based on the protein sequence, the C-terminus subfragment region 487–627 aa of Rep of N-MDPV was synthesized to add His-tag. Using seamless cloning method, the segment was introduced into pET-28a(+) vector, transformed into Rosetta (DE3) Escherichia coli, and induced to express the recombinant protein. The recombinant protein was then purified by nickel column affinity chromatography for immunization on New Zealand white rabbits to produce the polyclonal antibody.

  Results   The prokaryotic expression plasmid pET-28a-Rep-487-627 was constructed, purified, and expressed. The recombinant protein was approximately 24 kDa and mainly expressed in a soluble form. The indirect immunofluorescence and western blot showed that the prepared Rep polyclonal antibody could react specifically with the overexpressed N-MDPV in cells.

  Conclusion   The prepared Rep polyclonal antibody exhibited a reaction specificity that recognized the conformational epitopes and linear epitopes of Rep protein.