Preparation And Activity Analysis Of The New Recombinant Porcine Alpha-Interferon

Interferon（IFN）, a kind of cytokines induced by a series of revulsants, exhibitsvarious biological Activities including antiviral，antiproliferation and immunoloregulation.From the first report on its discovery in1957by Isaacs and Lindenmann, a lot ofresearches have been carried out, and most of them are focused on the study of itsproduction in large scale for the application in treatment. IFN, the quickest first defensesystem in the animal body, play an extremely important role in the cure of viral diseases.However, IFN expression level in the body is very low, and can not meet the requirementsof combating diseases. So obtaining IFN is urgent in vitro using genetic engineeringtechniques. Currently interferon has been expressed in the E. coli expression system, yeastexpression system, animal cell expression system, plant expression system and so on. E.coli expression systems has been studied in depth, and has many advantages, such assimple foster condition, easy operation, short culture period, low cost, high yield,and easyto scale. But sometimes the expressed protein can exist in the form of inclusion bodies,which is a big problem. In the study, we constructed a pET32-PoIFNα/BL21（DE3）recombinant strain，successfully express soluble PoIFNα. After purification and WBidentification, the biological activity of recombinant protein is assayed. The main resultsobtained are as follows:Clone primers is designed according to the reported PoIFNαin the GeneBank, and thePoIFNα mature peptide gene is amplified by PCR using the pig hepatic cell genomic DNAas template and inserted into the pET32a vector. Then transformed into E.coli DH5α forcloning itself. After identified by colony PCR, double digestion and gene sequencing,recombinant plasmid is transformed into E. coli BL21and pET32-PoIFNα/BL21（DE3）recombinant strain is successfully selected.After inducing recombinant strains，the product is analyzed by SDS-PAGE proteinelectrophoresis and shows a specific band of3.8×104KDa in line with expectations.Further, The experiment of the sample processed by the reducing loading buffer and non- reducing loading buffer proved that the product PoIFNαexists in the form of activemonomer.The optimum expression condition is tested:22℃,0.5mM IPTG,10h. Under theoptimum conditions, engineering bacteria is induced in the large-scale, and the expressionproduct is purified by nickel column affinity chromatography to obtain a higher purity ofthe recombinant protein. After WB identification, the purified product is dialyzed andfreeze-dried, then4℃saved.100CCID₅₀PRRSV attack the pretreated Marc145cells by the interferon. As a result,the recombinant protein PoIFNα has the antiviral activity, which lays the foundation forfurther development of PoIFNα development laid the foundation.