Expression Of NP Protein And Preparation Of Monoclonal Antibodies Against NP Protein Of H3N2 Subtype Swine Influenza Virus

Swine influenza virus (SIV) composed of 10 proteins is spherical virus particles. Its nucleoprotein (NP) encoded by the fifth fragment is conserved structural protein, and low mutation rate in different subtypes of influenza viruses. Given the high variability of SIV antigen and potential threat to humanity, the preparation of monoclonal antibodies against NP protein is important to develop the diagnostic reagents for the different subtypes SIVs and to understand the structure and function of NP protein.The coding fragment of H3N2 SIV NP gene was amplified using a pair of primers with specific enzyme digestion site by PCR and cloned into plasmid pMD19-Simple-T vector. It was analyzed by restriction enzyme and comfirmed by DNA sequencing, then the NP gene was subcloned into prokaryotic expression vector pET32a. Rosetta ( DE3 ) transformed with recombinant plasmid pET32a-NP was induced by IPTG and the soluble NP protein was expressed effectively,with molecular weight was 73ku.The immune activity of NP protein was confirmed by Western-blot using the specific sera against H1N1,H3N2 and H9N2 subtype SIV. The multi-clonal sera were produced by inoculating the purified NP protein into mice, and then the sera was used to detect H1N1, H3N2 and H9N2 subtype SIV in indirect fluorescence assay (IFA). Above these results of indirect IFA demonstrated that the recombinant protein has good immuogenecity.Meanwhile, to construct the recombinant plasmid pCAGGS-NP, NP gene was sub-cloned into a eukaryotic expression vector pCAGGS. The recombinant plasmid was transfected into 293T cells, and then the expressed NP protein was confirmed by indirect fluorescence assay (IFA) and Western blot.BALB/c mice were inoculated with 100μg of the recombinant plasmid pCAGGS-NP. After the third immunization, Splenocytes from the immunized mice were fused with SP2/0 myeloma cells. Positive hybridoma clones were screened by ELISA. After three cycles of subcloning,a monoclonal hybridoma cell line (3D7) against the NP of H3 subtype SIV was generated. The 3D7 MAb has good specificity. The ascites ELISA titer of the MAb was 1: 105 and belonged to IgM subclass,κchain. The relative affinity of the purified MAb against purified H3 SIV was 1.02×106M-1. Furthermore, we also tested the reactivity of the MAb by detecting the H1N1,H3N2 and H9N2 subtype SIVs in IFA. The results demonstrated that the MAb had good cross-reactivity with these SIVs. The preparation of the MAb against NP protein will provide reagents for diagnosis of SIV and analyzing the epitope of NP protein in the future.