Recombinant expression of human influenza A virus nucleocapsid protein and its antigenicity analyses

Abstract

To prepare anti-recombinant protein antibody from immunized mice with recombinant nucleocapsid protein (NP) of human influenza A3 (IFV-A3) virus expressed in prokaryotic cell, and to explore the feasibility of utilizing anti-recombinant protein antibody to detect influenza A virus. NP genes of human influenza A virus were analyzed with computer softwares of ClustalX, Antheprot, et al. to determine the antigenicity in conserved regions. Three different partial NP genes were harvested and cloned into pET-28(c) plasmid, the recombinant plasmids were induced to express partial NP segments in BL21 cells. The recombinant proteins were purified with Ni-agarose by affinity chromatography and immunized BALB/c mice. The polyclonal antisera harvested from mice were analyzed with Western Blot and immunohistochemistry assays to detect the reactions with IFV-A. Three recombinant plasmids were expressed with high yield in BL21 cells, about 15-20 mg/L. Western Blot results indicated that the three prepared antisera (1:2000) positively reacted with NP from IFV-A3-infected cells. And immunohistochemistry assays suggested that anti-NP1, anti-NP2, anti-NP3 antisera positively reacted with IFV-A3 or IFV-A1-infected MDCK cells, with titers of 1:640 to 1:1280. The recombinant NP of IFV-A3 would induce polyclonal antibodies with high titers in mice. The polyclonal antibodies would cross-react with IFV-A3 and IFV-A1. It is feasible to predict the antigenicity with systematical bioinformatics analyses and then induce anti-IFV antibodies with high dilutions, and it is possible to be utilized in the early detection and subtyping analyses of IFV-infections.