Cloning, Expression Of Mink Interferon-α, γ Genes And Analysis Of Antiviral Activities

Interferon (IFN) is a series of protein that have extensive biological activities. IFN regulates the immunity system, have antiviral activity, and play an important role in the animal's defence system. The research of animal's IFN is just focus on several kinds of animals. Until now, several recombinant interferon products have been used in pig, dog, and chicken. In the fur husbandry, China is one of the developed countries in the world. At present, the genes and espression of mink (Mustela lutreola)IFN have not been reported. During recent years, mink have become more and more important in farmers'lives, become main channel of earn more money. But various virus diseases disturb mink husbandry. One hand, there isn't an efficient medicine to cure it and some viruses become more virulent.On the other hand, the immunized protocol always failed because of the wrong way in ueage and storage. As a result, control of viral diseases of mink needs medicine with safety, efficiency and less side effect, and mink IFN has high clinic value whose biological products is necessary. Mink IFN has wide anti-virus bioactivity and high research value. This research project was consisted with several sections:1. With the reverse transcription chain reaction (RT-PCR), 13 genes of Mink's interferon-alpha (MuIFN-α) and an IFN-γgene of Mink's interferon-gama (MuIFN-γ) were amplified and cloned, from the total RNA in the lymphocytes stimulated with concanavalin A from the peripheral blood of mink. Then the amplified fragment was inserted into the prokaryotic expressing vector pMD18-T and then sequenced. Sequencing results showed that 13 genes of MuIFN-αand IFN-γgene were obtained. Sequence analysis showed that the 13 clones of MuIFN-αwere composed of 564 nucleotides, encoding a mature polypeptide with 187 amino acids and the clones of MuIFN-γwere composed of 501 nucleotides, encoding a mature polypeptide with 144 amino acids.2. The recombinant expression vectores were transformed into the E.coli and induced to express by different temperature. SDS-PAGE assay showed that target proteins, with the molecular weight were 20-21KDa (MuIFN-α) and 19 KDa(MuIFN-γ), could be expressed in a high level and in the E.coli cell, in the form of inclusion bodies.3. The recombinant MuIFN-α2, MuIFN-α12 and MuIFN-γproteins were added to WISH cells, which were subsequently infected by the 100TCID50VSV. The result showed some cytokine activity. The recomninant MuIFN-α2 and MuIFN-α12 activity were up to 0.18×104 IU/mL and 0.3×103 IU/mL, and the recomninant MuIFN-γactivity were up to 0.1×103 IU/mL.Through this study, 13 genes of Mink's interferon-alpha and the gene of Mink's interferon-gama were obtained. All 13 genes of of Mink's interferon-alpha and the gene of Mink's interferon-gama were expressed in E.coli. Besides that, we have completed the activity analysis of this expression product, which leave a good foundation to great scale industrial production.