Pegylation And Activity Analysis Of Recombinant Porcine Interferon

Research Background: Interferon is a sort of broad-spectrum anti-viral andmodulating cell immunity multifunctional protein. Interferon is widely used in clinicalapplications, especially therapy of viral diseases. However, vivo half-life natural ofinterferon is short, it was difficult to suppress viral persistence. It is significative tomake research on long-acting interferon. The study of human interferon has madeconsiderable progress, and polyethylene glycol interferon has been commercialized,such as PegIntron and Pegasys. The research progress of pegylated porcineinterferon was at a slow pace. Currently, there is not yet relevant listed medicines andeffective patents.Research Purpose: To build a simple way about the preparation of pegylatedrecombinant porcine interferon which have better retention activity.Research Methods:The study was based on soluble expression of the recombinantporcine interferon fusion protein. The fusion protein were split by EK enzymes andpurified by nickel column. Then it was made into interferon stock solution. Accordingto the amino acid sequences characteristics of the recombinant porcine interferon,respectively used the relative molecular mass of20kDa linear chain mPEG-ALD(single-methoxy polyethylene glycol propyl aldehyde) and mPEG-SPA(single-methoxy polyethylene glycol succinimide propionate)-modified interferon.After optimizing pegylated conditions of recombinant porcine interferon, a mass ofproduction pegylation were done. After rpoIFNα were completly pegylated,separation and purification mono-PEG conjugates by used cation exchangechromatography. The modify product was detected by SDS-PAGE. Finally researchedpreliminarily their biological activity assay using the cytopathic inhibition method.Research Results:(1)The purity of preparation recombinant porcine interferon wasabove90%. It met requirements of preliminary experimental study.(2)By optimizingthe conditions of PEG-modified interferon, the optimum modification reaction conditions of the PEGylation were selected,as below:①optimum conditions ofPEG-ALD modified IFNα:50mmol/L PB, pH=6.0,4℃,(PEG/protein) molarratio of3:1. the reaction time was24h.②optimum conditions of mPEG-SPAmodified IFNα:50mmol/L PB, pH=9.0,4℃,(PEG/protein) molar ratio of5:1,the reaction time was2h. The rate of the single PEGylation interferon can reach60%.(3)Compare with natural interferon, the biological retention activity rate ofmono-PEGylated interferon reached2.9%.