Preparation of Mouse-derived Polyclonal Antibody for Nonstructural Waterfowl Parvovirus Proteins

Objective The mouse-derived polyclonal antibody that recognizes the nonstructural (NS) proteins of 4 waterfowl parvoviruses (WPVs) was prepared.

Method Primers targeting the conserved sequence regions of the NS proteins of 4 WPVs were designed by multiple sequence alignment. The specific coding sequences containing the conserved regions were amplified and cloned into pET-32a(+) for prokaryotic expression and purification. The purified protein was used to immunize BALB/c mice three times prior to serum collection for expression confirmation by western-blot and indirect immunofluorescence assay (IFA).

Result The sequence alignment showed the conserved region of WPV-NS proteins to locate at between 351-457 amino acids. The ELIAS, SDS-PAGE, and western blot analyses confirmed the expression of a His-NS recombinant protein with a molecular weight of approximately 45kDa. The prepared mouse-derived polyclonal antibody could specifically recognize the NS proteins expressed in the cells infected with the 4 genotypes of WPV and positively within 6–12h post-infection.

Conclusion The broad-spectrum polyclonal antibody against the NS proteins prepared in this study could specifically recognize all 4 genotypes of WPV. It provided a basis for the development of early infection diagnosis in waterfowls, as well as further studies concerning the biological functions of the viral protein.