Expression and its antigenicity of NP gene of H3N2 swine influenza virus isolated in He'nan province

ObjectiveTo clone and express nucleoprotein(NP) gene of swine influenza virus(SIV)and to identify NP protein with good antigenicity.MethodsThe NP gene of A/swine/Henan/2/2008(H3N2) was amplified with PCR.After digested by EcoR I and Xho I,the PCR products were cloned into prokaryotic expression vector pET32a and transformed into Rosetta strain.The recombinant vector,named pET32a-NP,was identified with PCR,EcoR I/Xho I digestion and DNA sequencing.After sopropyl-beta-D-thiogalactopyranoside(IPTG) inducing,the recombinant NP was expressed and analyzed with sodium dodecyl sulfate polyacrylamide gel electrophoresis(SDS-PAGE) and Western blot.Serum antibody against NP was prepared by immunizing the rabbits with the purified NP.The recombinant pcDNA3.1-NP was constructed and transfected into 293T cells simultaneously,and the immunogenicity of NP was detected with immunofluorescence.ResultsThe recombinant plasmid pET32a-NP and pcDNA3.1-NP were successfully constructed.SDS-PAGE and Western blot showed that the expressed protein was about 80 kDa and reacted specifically with sera from pig infected with SIV.Immunofluorescence indicated that NP protein immunized sera could be used in the detection of NP protein expression 293T cells.ConclusionThe NP protein of SIV H3N2 was successfully expressed in Rosetta strain and the expressed NP had good antigenicity and could be used as diagnostic antigen and in the development of subunit vaccine of SIV.