Cloning And Expression Of The Chicken γ-Interferon Gene And The Preparation Of Polyclonal Antibody Against Recombinant Chicken IFN-γ

Interferon- is an antiviral cytokine produced by activated T cell and NK cell. It upregulated MHC II antigen expression, influenced the cellular and humoral immune response and has pleiotropic regulatory effects on immune system.One gene was amplified by reverse transcription-polymerase chain reaction (RT-PCR) in splenocytes which was stimulated with ConA for 24 hours. The gene was cloned into pUC19 vector and identified with PCR and Eco RI + Hind III digestion. Then, one of the positive recombinant clone was sequenced and analysed. The result showed that its sequence was 35% and 32% identical to equine IFN- and human IFN- respectively, but it only shared 15% homology with chicken type I IFN. The sequence was just the same as the published chicken IFN- gene. So the chicken IFN- mRNA may be expressed in ConA-activated chicken splenocytes and the IFN- Y gene had been cloned from chicken splenocytes successfully.The cDNA of chicken IFN- Y gene was subcloned into Pst I and Kpn I sites of pPROEX橦T vector to construct recombinant plasmid pPROEX橦T-IFN- . The recombinant plasmid was translated into E. coli DH5 and the bacteria was induced with IPTG. It was demonstrated by SDS-PAGE and Western blot that a 22ku protein which was equal to chicken IFN- y protein in molecular weight was expressed in E.coli DH5 . This protein was purified and used to prepare the antibody against chicken IFN- y protein. The result showed that the antibody could react with purified recombinant chicken IFN- protein.