The Secretive Expression Of Procine IFN-α In Yeast Pichia Pastoris

Interferon (IFN), a family of cytokines, have activities on interfering with the replication of various viruses, decreasing cell proliferation and modifying immunological processes. The research about IFN indicates, it is a kind of ideal therapy that IFN act on healthy or unhealthy animals to prevent or cure many kinds of diseases infected by virus. However, animal organisms could not produce IFN commonly, many scholars had attempted to use virus infect animals in order to induce leukocyte to produce IFN. As a result, a very small quantity of IFN was produced and also the cost of production was very high, it was difficult to extract and purify IFN protein. With the development of DNA recombination technology, there are also some reports about the expression of recombined IFN protein by prokaryotic expression system, but the expressedproteins often consist in the form of inclusion body= The operations about extracting and purifying proteins are very complicated, biological activity of proteins are not high. Therefore, this research is the first time to attempt to express porcine IFN-a(PoIFN-a) by eukaryotic expression system.Recently, the expression system of methylotrophic yeast has been developed quickly. As a result of its many advantages, such as high expression, high stability, high secretion and so on, many kinds of Protein, including animal, plant, microorganism proteins, have been expressed by this system in the world, but we have not seen any report about yeast expression of PoIFN-auntil now, this research is the first example in the world that PoIFN-a is expressed by methylotrophic yeast system.This research use recombinant plasmid PGEX-IFN as template, mature PoIFN-a gene was obtained by PCR method. The expression vector pPICZaA with the PoIFN-a gene was constructed by restrict enzyme digestion and ligation, then transformed into E.coli JM109 in order to extract recombinant plasmid from JM109. After DNA sequencing was correct, it was transformed into P.pastoris KM71H strain by electroporation. Highly resistant recombinant was isolated by increasing concentrations of Zeocin? positive clone was selected by PCR .The positive clone was induced with methanol . The results of SDS-PAGE and Western Blot showed that the product was recombinant PoIFN-a fusion protein. Biological activity of this fusion protein was detected by VSV/WISH system, the result was 4.104.1 104IU/m=...