Cloning, Prokaryotic Expression And Bioactivity Assay Of Canine Interferon-α

With the occurrence of zoonoses, especially the highly lethal viral diseases, the newstrain of the pathogen, and the emergency of mutants, the prevention of livestock diseasesbecomes more and more important.Since Isaacs and Lindanmann discovered the interferon (Interferon, IFN) in1957, themolecular structure, gene expression and biological activity of interferon has been concerningwith people all these years. Interferon is a protein family which is antiviral, affecting cellgrowth, differentiation and regulation of immune function and so on. Interferon as the firstviral defense system of living body, is being one of the important cell factors of the variousbiological bodies. A large number of studies show that the canine interferon possesses abroad-spectrum antiviral activity, anti-proliferative and immunomodulatory functions. It has agood therapeutic effect for many viral diseases of dogs. The Canine interferon as a vaccineadjuvant, it can develop the new vaccine with good immune effect in practices. It will have apositive effect on preventing and curing of canine disease. Preventing and treating the viraldisease of livestock by recombinant interferon is a very effective means.Through analyses of the natural sequence of canine interferon-α gene and variety ofonline soft wares, canine interferon-α gene was designed and synthesized primers byremoving the signal peptide, introduced two restriction sites, amplified the canine α-interferongene from canine liver genomic DNA by PCR, then the gene was cloned into expressionvector pBV220and transformed the E. coli expression system for The recombinantexpression vector construction. By these processes, it gets efficient expression of canineinterferon-α in E. coli DH-5α and the expression product as the form of inclusion body. AfterSDS-PAGE analysis, strip was produced at about18.0kDa, its size match with the theoreticalvalue. Expression products is accounting for32%of the total bacterial proteins.The Denatured inclusion bodies renaturation, firstly, by Sephacryl S-200preliminarypurification; secondly, by optimized redox system; lastly, the denatured sample was diluted torefolding buffer (at4°C) whose final concentration is between100and200μg/mL. Thismethod greatly improves the yield and efficiency of refolding. Refolded protein is furtherpurified by DEAE-FF, the purity achieve at95%. The result of its activity detection byMDCK-VSV and CPE reduction assay is5.56×106U/mg.