Cloning, Prokaryotic Expression Of Ovine Interferon Gamma Gene And Study On Antiviral Activity Of Its Recombinant Protein

According to the sequence of Ovine IFN-γgene published by C.J.Mclnnes in 1990, A pair of primers was designed to amplify Ovine interferon gamma gene by RT-PCR with Primer 5.0 software. The product of PT-PCR named OvIFN-γis approximate 554bp in length that amplified from the total RNA of lymphoid cell induced by Con A. The OvIFN-γgene was cloned into the pMD18-T vector and the recombinant plasmid was named pMD18-T-OvIFN-γ. Identifications of restriction enzyme, PCR and sequencing indicated that the OvIFN-γgene has been cloned successfully. The gene includes a complete open reading frame which is 501bp in length and encoding a functional protein that consists of 166 Amino acids. Compared with the published IFN-γgene sequence, the homology of nucleotide sequence was 75.2﹪with Human,96.6﹪with Bovine,99.0﹪with Goat,95.8﹪with red deer,83.0﹪with horse,88.8﹪with camel,86.6﹪with pig,82.4﹪with dog,53.9﹪with cat and 32.9﹪with chicken;the homology of amino acid sequence was 61.7﹪with Human,94.6﹪with Bovine,98.2﹪with Goat,92.2﹪with red deer,77.8﹪with horse,86.8﹪with camel,77.8﹪with pig,76.0﹪with dog,39.5﹪with cat and 6.7﹪with chicken.OvIFN-γwas inserted in multiple cloning sites of the prokaryotic expression vector pPROEXHTa, The recombinant plasmid pPROEXHTa-OvIFN-γwas identified by restriction enzyme and PCR. Purified recombinant plasmid pPROEXHTa-OvIFN-γwere transformed to E.coli BL21(DE3),and then harvested in 37℃. The Recombinant engineering strain of BL21(DE3)/ pPROEXHTa -OvIFN-γwas induced by the IPTG.. SDS-PAGE and Western blot analyses showed the OvIFN-γgene has been highly expressed in E.coli BL21. The molecular weight of Expressed fusion protein is approximately 22.6kDa, the fusion protein exists in the form of inclusion body and the content is approximately 48 percent in total protein of BL21.The results of purification of fusion protein indicated the active protein has been obtained by dialysis. Test of antiviral activity for the recombinant OvIFN-γpurified was done with the method of inhibiting CPE on MDBK by BVDV. Results of detection showed purified protein have activity against the BVD virus. The antiviral activity of recombinant OvIFN-γfor BVDV is 1.0×107U/mL.