Cloning, Sequence Analyses, Prokaryotic Expression And Detection Of Antiviral Activity Of Changbai Porcine Interferon Gamma Gene

According to the sequence of PoIFN-γgene published by Vandenbroeck. K on Genebank, A pair of primers was designed to amplify Changbai porcine interferon gamma gene by RT-PCR with Primer 5.0 software. The product of PT-PCR named PoIFN-γis approximate 530bp in length was amplified from the total RNA of lymphoid cell induced by Con A. The PoIFN-γgene was cloned into the vector pGEM-T and the recombinant was named pGEM-T-PoIFN-γ. Identifications of restriction enzyme, PCR and sequencing indicated that the PoIFN-γgene has been cloned successfully. The gene includes a complete open reading frame which is 501bp in length and encoding a functional protein consists of 166 Aminos. Changbai porcine IFN-γgene was analyzed in comparison with the other PoIFN-γgene published on Genebank and Zang porcine, Chenghua porcine. Gene sequence comparison indicated that Changbai porcine IFN-γ100% identities with S63926, Zang porcine's, Chenghua porcine's respectively. The deduced amino acid sequences were 100% correspondingly. The results affirmed that IFN-γgene of porcine was conservative. PoIFN-γwas inserted in EcoRⅠand Xho Ⅰmultiple cloning sites of the prokaryotic expression vector pET28b using the expression primers designed according to the sequence of PoIFN-γgene. pET28b-PoIFN-γwas identified and analyzed by corresponding restriction endonuclease on the basis of the genetic sites of pET28b and PCR, which was identified as the IFN-γgene of Changbai porcine. Purified pET28b-PoIFN-γwere transformed to E.coli BL21(DE3) and harvested Recombinant engineering strain. Identification with positive clone selection, SDS-PAGE analyses and Western blot analyses showed PoIFN-γgene has been recombinated...