Expression And Improvement Of Canine And Feline Pegylated Interferon Fusion Protein In Pichia Pastoris

Interferon(IFN) was produced by a human or animal cells, which appeared highly efficient broad-spectrum antiviral activity and immune regulation activity. It was used extremely in antiviral therapy. However,the clinical application is restricted by its short half-life.Serum albumin was the most abundant protein in the plasma of mamallain.The characteristics, such as stability and long half-life, were very suitable for combining with interferon to extend the half-life of interferon. It was important to develop long-activity IFN for decreasing the cost of production and application in.In our experiment, we expressed the fusion protein by chemeric canine serum,feline serum albumin(CSA,FSA) and alpha interferon(IFN-α) with modified former CSA-IFN-α by secreting expression in Pichia pastoris respectively.Firstly, the glycosylation sites of canine and feline IFN-α were removed by amino acid mutated to avoid immunogenicity of IFN-α protein with glycocylation. Secondly, The codon optimum design canine and feline IFN-α were performed to the to improve the level of the expression of exogenous proteins in Pichia pastoris. Finally,the signal peptide of serum albumin was replaced with αA mating factor to avoid the incomplete signal peptide cutting off. The CSA-IFN-α and FSA-IFN-α were identified by N-terminal sequencing respectively.The culture and purification technology were explored. Fusion protein was expressed by Pichia pastoris X33-CSIFN and X33-FSIFN in 5L fermenter. Fusion protein was purified by ion-exchange column chromatography and gel chromatography. The content of X33-CSIFN fusion protein was 839μg/ml, and X33-FSIFN fusion protein was 954μg/ml. The purification rate can reach more than 90% and 80%, respectively.The activity of canine and feline long-activity IFN was detected in vitro. The activity in vitro of the fusion protein was detected by using MDCK-VSV and CRFK-VSV. The result of X33-CSIFN activity is 6.34×105IU/ml. Feline fusion protein had proved its good antiviral activity by measuring.In our studies, The CSA-IFN-α and FSA-IFN-α with high purification rate was obtained by codon optimum design, chimeric expression and purification. A good foundation has been laid for biological activities evaluation and preclinical research of CSA-IFN-α and FSA-IFN-α.