Purification of influenza C virus polymerase based on huANP32A protein and efficient preparation of monoclonal antibody targeting PB2 protein

Abstract

Influenza C virus is an important respiratory pathogen not only infecting people, but also pigs, dogs, and other animals. Polymerase is central to the replication of influenza C virus and is an important target for studying the mechanism of viral replication. However, there is no commercial monoclonal antibody (MAb) targeting influenza C virus polymerase, which hampers the development of relevant research to some extent. In order to prepare MAb targeting the polymerase basic protein 2 (PB2) of influenza C virus, influenza C virus RNA-dependent RNA polymerase (RdRp, consists of PB1, PB2 and P3) was co-immunoprecipitated with Flag-tagged human acidic nuclear phosphoprotein 32A (huANP32A-Flag) from 293T cells based on the interaction between huANP32A and influenza virus RdRp. The purified RdRp was used as antigen to immunize BALB/c mice. Six positive hybridoma cell lines (7B11-5, 8A4-5, 13D9-6, 8D4-1, 8D4-3, 9F9-4) that stably secrete and recognize PB2 MAb were screened by indirect ELISA and Western blotting. The subtypes of MAb 7B11-5, 8A4-5, 8D4-1 and 8D4-3 antibody were identified as IgG1, the subtypes of MAb 13D9-6 and 9F9-4 were IgG2a and IgG3, respectively. All the light chains of the MAbs were κ chain. A hybridoma cell line 8D4-1 with high titer was further selected to prepare ascites. The titer of mouse ascites antibody was determined to be 1:64 000. Western blotting results showed that the MAb 8D4-1 had a specific immune response with ICV PB2; laser confocal assay showed that the prepared MAb 8D4-1 accurately detected the subcellular localization of PB2 subunits. Moreover, ICV RdRp was highly enriched by ANP32A. The high specific of the prepared PB2 MAb 8D4-1 may facilitate the polymerase detection, structural analysis and mechanism study of influenza C virus.