Cloning，Expression Of Mink Interferon-α,β Gene And Analysis Of Genetic Evolution

Interferon (IFN) is a kind of protein which secreted by the cells, it has a broad-spectrumantiviral resistance, cell division, immunomodulatory activity in the same cell and it affectscell metabolism, growth and differentiation in different ways at least. At present, recombinantinterferon of commercial pigs, chickens, dogs and other products are applied in the market.But the mink interferon abroad has not been reported yet. There are only domestic ChineseAcademy of Agricultural Sciences Institute of specialty of wild animal zoonosis research hasamplified mink interferon alpha13gene (MiIFN alpha) subtypes using the RT-PCR methodfrom the blood lymphocytes of mink in peripheral, the other has not been reported yet. Inrecent years China’s fur animal industry develops rapidly, it is one of internationallydeveloped countries in a few fur animal breeding industry. Especially in Jilin, Liaoning,Heilongjiang, Hebei, Shandong, Inner Mongolia and other regions, mink farming has becomethe main avenue to become rich by local farmer. Nowadays, the mink population has reachedthirty million, fur animal industry is important industry in our country’s rural industrystructural adjustment. In recent years, mink, fox, nytereutes, canine animal as a specialeconomic animal breeding is rising. While the most important viral diseases are the causes ofcanine animal death. Effect of interferon on virus has a broad-spectrum, special antiviraleffects. So, I will research for canine animal interferon from the following several aspects. Inorder to research canine interferon products, I will research from the following several aspectsand laid the foundation to the development of recombinant mink interferon, and analysis acanine animal interferon is wheather applicable to all Canine animal or not. In addition, it canprovide guidance for clinical treatment and management.1.According to the GenBank published on the ferret IFN alpha gene (EU863613.1) and IFNbeta gene (EF581890.1) cDNA sequence, a pair of primers were designed respectively on thereading frame of conserved regions of the outboard ends, I extract RNA from the spleen tissue directly,using reverse transcription to cDNA as a template for amplification of IFN-alpha andIFN-beta gene. DNAStar software analysis of sequencing results show the full-length gene ofMiIFN-alpha and MiIFN-beta gene have received, MiIFN-alpha length is564bp andMiIFN-beta gene is561bp. Comparing mink IFN-alpha gene sequence with GenBank indexedby the ferret (EU863613.1), the homology of nucleotide is94.3%and the homology of aminoacid sequence is89.3%; the homology of ferret (EU863614.1) nucleotide is95%, thehomology of amino acid sequence is90.9%; the homology of ferret (EU863615.1) nucleotideis95.5%, the homology of amino acid sequence is91.4%. Comparing mink IFN–beta genesequence with GenBank indexed by the ferret (EF581890.1), the homology of nucleotide is100.0%and the homology of amino acid sequence is100.0%too; the homology of ferret(EF368208.1) nucleotide is98.2%, the homology of amino acid sequence is98.4%. Inaddition, comparing the amplified genetic sequence with the canine, vulpes, giant panda andnytereutes gene sequence, we found that MiIFN-alpha nucleotide sequence homology isbetween78.1%-99.3%, amino acid sequence homology is between66.3%-98.4%; Canine andvulpes IFN-alpha’s relative is closely(homology is nearly98.0%), Canine and nytereutesIFN-alpha genetic relationship is relatively recent (homologous is98%); But the relationshipbetween mustela and vulpes, nytereutes, canine is relatively far (gene homology is about79.1%, the homology of amino acid sequence is from66.3%to67.9%). MiIFN-betanucleotide sequence homology is from83%to100%, the homology of amino acid sequence isfrom69.4%to100.0%. Mustela and ferret, canine and vulpes IFN-beta is the closest(homology is100%). Caninne and nytereutes IFN-beta relationship is also more recently(homologous is98.4%), but the homology between mustela and vulpes, nytereutes, canine isrelatively far (gene homology is about86%, the homology of amino acid sequence is from73.7%to75.3%).2. Take MiIFN alpha full-length sequences of gene and MiIFN beta full-length sequence ofgene as a template, designing mature peptide gene expression primer according to theexpression carrier enzyme cutting site. Target gene was subcloned into PET32a-MiIFN alpha,PET32a-MiIFN beta prokaryotic expression vector and was expressed in e.coli. Expressproduct SDS-page analysis take conclusion that two kinds of restructuring interferon exist inthe form of inclusion body, MiIFN alpha recombinant protein size is about42KDa and MiIFN-β recombinant protein size is about43KDa.