The Preliminary Study Of Clone,Expression And Activity Of Human IFN-ε

IFN-ε is a novel member of the type interferon. We know little about it' s structure and function. In this research, The total RNA was extracted from four tissues of human's embryo,which were lung,liver,heart and hand, then we compared and analyzed the distribution of IFN-ε mRNA in these tissues; At the same time,the obtained fragment of IFN-ε was inserted into plasmid pBV220,and was transformed into E. coli. DH5 a to express.Finally we obtained the sample,which purity was above 90%. Then it's anti-virus activity was tested.1 , Distribution of IFN-ε mRNA in tissues of embryoThe total RNA was extracted from four tissues of embryo,which were lung, liver, heart and hand by using TRIZOL LS Reagent, and the keepinghouse gene -actin was taken as the inter-control. We analyzed the distribution of IFN-ε mRNA in different tissues by RT-PCR. The results showed: IFN-ε mRNA has high expression in lung and liver of embryo,but it has almost no expression in the tissues of heart and hand.2, Expression and purification of IFN-ε The fragment of IFN-ε was inserted into the plasmid pBV220 after itis digested by EcoR I Pst I, and was transformed to E. coli DH5 .After inducement in 42 C we obtained the desired band ,whosemolecular weight was similar to prospective protein by 12%SDS-PAGE.The newly synthesized IFN-ε is deposited in bacterial cell as inclusion bodies. The process of IFN-ε consist of fermentation and a series of purification steps. Briefly, a production stain of E.coli harboring the IFN-ε gene is grown in fermenter. The synthesis of IFN-ε was under the control of inducible promoter PRPL. The bacterial cell was collected and disrupted to release the intracellular IFN- ε . IFN- ε was then solubilized by using a buffer containing sodium dodecyl sulphate (SDS) and reduced using dithiothreitol (DTT). At last by means of organic extraction, size exclusion chromatography, we obtained the sample, which purity was above 90 %.3 , Anti-virus activity of IFN- εThe anti-virus activity of IFN- ε was tested by using the system of VSV-WISH.The results showed: the anti-virus activity of IFN- e was 2.3 X 104U/ml.