Optimal expression and antigenic analysis of nucleoprotein of swine influenza virus subtype H3N2.

The complete NP gene was amplified from the RNA of swine influenza virus(SIV) subtype H3N2 by RT-PCR using a pair of primers with specific enzyme digestion site and was cloned into pMD19-Simple-T vector.It was confirmed by restriction enzyme and sequencing,then the NP gene was subcloned into pET32a vector after being digested with specific enzyme.Rosetta(DE3) transformed with recombinant plasmid pET32a-NP was induced by IPTG.The optimal expressing condition was established by adjusting the concentration of induced bacteria and IPTG,and the temperature of induction.The results of SDS-PAGE indicated that the soluble NP protein was expressed effectively,which was 73ku in molecular weight.The immune reactivity and cross-reactivity of NP protein was confirmed by Western-blot after being reacted with the antisera against SIV subtypes H1N1,H3N2 or H9N2,respectively.Polyclonal antibody was produced by inoculating mice with the purified NP protein and reacted strongly with SIV subtypes H1N1,H3N2 and H9N2 in indirect fluorescence assay(IFA).The results of the indirect IFA demonstrated that the recombinant protein had a good immunogenicity.