Renaturation And Activity Determination Of Recombinant Canine Interferon-γ Inclusion Body By Dilution

Canine interferon-γ, is a effective treatment of viral infection. Recombinant CaIFN-γwasexpressed as inclusion bodies in Escherichia coli. Inclusion body could recover active protein afterrefolding. Protein refolding by dilution is a simple and quick way of renatueration and is the basisof research on other methods of renaturation.Engineering strain containing pJLA605-CaIFN-γwas highly expressed after inducement andthen Utrasonic discruption adding lysozyme. CaIFN-γinclusion body could be dissolved by8mol/L urea effectively after washing and refolding by dilution. Effects of folding enhancer, waysof adding sample, temperature, pH was researched in the renatutation process of recombinantcanine interferon-gamma by dilution. CaIFN-γ, activity could be assayed in Madin-Darby caninekidney (MDCK) cells using the New Jersey strain of vesicular stomatitis virus (VSV) by cytopathiceffect(CPE) inhibition. Application of coomassic brilliant blue method determinates proteinconcentration after refolding. Engineering strain after inducement obtain inclusion body 285mg/Ldissolved by 8mol/L urea effectively. 0.5M L-arginine,0.5M Guanidine-Hcl,2.0M Urea couldsuppress the aggregation of protein during the renaturation process effectively, and 0.5M L-arginineis the best of them. The activity and specific bioactivity was up to 1.35×10⁴U·ml^-1 and3.47×10⁶U·mg^-1 respectively at 4℃, pH 8.0, 0.5 mol·L^-1 L-arginine, 0.15 mg·mL^-1 proein andpulse renturation operation mode.