Cloning And Prokaryotic Expression Of Chicken's IFN-γ Gene

IFN-γ is a cytokine predominantly secreted by activated T cells and NK cells, plays important part in anti-infections caused by various microorganisms (viruses, intracellular bacteria and parasites), anti-tumor proliferation and modulate immune function. It is one of main immune regulatory factors in animal body, especially is resistance to virus infections.It is predict great potential as a new type of romising natural adjuvants. So we cloned and expressed CHIFN-γ gene in prokaryoticly cells, which established a foundation for further research and development. Research content: 1. Cloning of CHIFN-γ gene. Total RNA was abstracted from chicken's spleen lymphocytes after being activated by concanavalin A (Con A), CHIFN-γ gene was amplified by Reverse Transcription—Polymerase Chain Reaction (RT—PCR) and ligated into pGEM-T-easy vector.Recombinant plasmid pGEM-T-easy/CHIFN-γ was indentified by endonuclease restriction digestion, PCR and DNA sequencing. The sequence is same to the sequence of CHIFN-γ gene submitted in GenBank. We also found that the result that spleen lymphocytes is activated 6h, 8h by ConA is good. 2. Prokaryotic expression of CHIFN-γ gene .The CHIFN-γ gene was amplified with the primers re-designed beside the ORF of CHIFN-γ gene.The amplified production and pGEX-4T-1 was digested with EcoR I and Xho I .The sequence was inserted into pGEX-4T-1 and the recombinant expression vetor was constructed,then transformed into Escherichia coli BL21.After being induced by IPTG to expess CHIFN-γ.The recombinant fusion protein ananlyzed by SDS—PAGE.The result showed that chicken's CHIFN-γ gene was successfully expressed in prokaryotic cells as a fusion protein with a molecular weight about 43, 000 Dalton.