The Research Of Long-activity Interferon Fusion Protein Expressed By Pichia Pastoris

Interferon(IFN) has many functions in antivirus, anti-tumor and immunoregulation. But the short half-life of IFN restricts its clinical application. In human clinic, the fusion protein of Human serum albumin(HSA) and IFN was used to prolong the half-life of IFN, but there is no report of this kind of fusion protein of IFN in animals. Now most of animal’s IFN are expressed in prokaryotic organism, and their activity and cost are not reasonable. The yeast expression system is eukaryotic and the protein expressed is modified and has high activity. The secretive expression of Pichia pastoris can secrete protein in supernatant of culture with less other proteins. This expression pattern is beneficial to purification and reducing cost of objective protein. In this experiment I expressed the fusion protein of canine serum albumin(CSA) and alpha interferon(IFN-a), and explored the possibilities of long-activity IFN with this method.The study consists of four parts. First, construction of expression plasmid. I amplified the CSA’s cDNA from canine tissue using RT-PCR and modified the cDNA. The EcoR I cutting site is inserted into the5’ end of cDNA after removing the terminator codon and the linker and BamH Ⅰ cutting site are inserted into3’ end. The recombinated cDNA is mutated to remove two Bgl Ⅱ cutting sites that unfit for the construction of plasmid. For increasing the expression of yeast, canine IFN-a is optimized and synthesized. In optimized process the initiation codon of IFN-α was removed, the Bgl Ⅱ cutting site is inserted into5’end. and the EcoR I cutting site is inserted into3’ end. The recombinated DNA is connected as CSA-IFN using pUC18vector. Transforming the fusion DNA into pPIC3.5K which is the vector of Pichia pastoris constructs pPIC3.5K-SI that is the fusion protein’s secreted expressive plasmid of canine long-activity IFN within Pichia pastoris. CSA’s inherent signal peptide was used as secreted expression signal peptide of Pichia pastoris to avoid non-completed remove of yeast’signal peptide.In second part of the experiment, the expressive plasmid is transformed into yeast using the methods of protoplasts-transformation and electro-transformation. Comparative analyzing these two methods that provides the references to the next study of laboratory. After transforming, the positive clones of recombinationed yeast were screened by His culture, Mut culture, G418culture and gene PCR.In third part of experiment, the recombinationed yeast was amplified in culture whose carbon source is glycerin. The objective protein was expressed inductively by culture whose carbon source is methanol, and was isolated and purified. In expression process, the best inductive time was determined as96h. The objective protein was purified by the methods of ultrafiltration, affinity chromatography and desalination. Using SDS-PAGE, the fusion protein of canine long-activity IFN "s concentration was determined as252μg/ml.In fourth part of the experiment, the activity of canine long-activity IFN was detected in vitro and in vivo. The activity in vitro of the fusion protein is detected by using Wish-VSV, and the result is2.56×105IU/ml. The transcriptional level of2’5’-OAS which is the fusion associated cytokines which detected using RTFQ PCR. The Pharmacodynamic effects of the fusion protein in vivo reach the peak at8h, and the duration is close to10days.The experiment focus on animal long-activity IFN using the fusion protein of animal serum albumin and IFN. The objective protein has good activity in vitro and in vivo, and has long half-life in vivo. This experiment lays a foundation for the research in developing animal long-activity IFN that has low cost and good function.