Expression Strategies Research Of Antiviral Activities And Amino Acid With No Redundancy Bovine Interferon Alpha A

Interferon (interferon, IFN) is a glucoprotein which secreted by the cells with a broad-spectrum anti-viral, anti-cell division, immunomodulatory activity and in different ways affect cell metabolism, growth. Interferon alpha is famous as the potentant antiviral factors.In our country. In a number of interferon type, broad-spectrum antiviral activity of interferon a is the most significant. In vitro expression of recombinant bovine alpha interferon (Bovine interferon-alpha BoIFN-a) protein can inhibit the vesicular stomatitis virus (VSV), foot and mouth disease virus (FMDV), bovine viral diarrhea virus (BVDV) and parainfluenza virus (PIV), the proliferation and replication of the virus.So far, E. coli and Pichia pastoris is the most universal used host for the production of recombinant proteins. However, most of the recombination protein of E. coli are tapically obtained as insoluble inclusion bodies that need solubilization and refolding. And the problem of the recombination protein of Pichia pastoris is that not stable and in low. So the soluble expression of rBoIFN-a with activity is more valuble for the production and application.In this study, we integrated the beneficial conditions of the active expression of recombinant protein, Created three with antiviral activities of BoIFN-a expression strategy, namely E.coli periplasm secretion expression strategy, E. coli Ssp DnaB intein (SDI) peptide fusion tag expression strategies and Pichia pastoris secretion expression strategy. The first two expression strategies in E. coli expression system,optimalized the codon usage with of BoIFN-a A restriction Enzyme digestion site was amplified by polymerase chainreaction(PCR) from cloning vector pWL-BoIFN-a,subcloned,seamless integration with the pe1B and the SDI into a series of vector,then constructed a series of expression vectors pET-22b-pe1B-BoIFN-a pMal-c2X-SDI-BoIFN-α pColdⅢ-SDI-BoIFN-a et. The result showed that the fusion protein was expressed in the form of activity. In Pichia pastoris, excision signal peptide of natural BoIFN-a A restriction Enzyme digestion site was amplified by polymerase chainreaction(PCR)from Bovine genome that extracted from fresh liver,subcloned into a series of vector. The fragment of BoIFN-a A was digested ligated into the expression vector pPICZaA-BoIFN-a. The result showed that the fusion protein was expressed in the form of activity in Pichia pastoris.Three with antiviral activities of expression strategy for a total of four active BoIFN-a A protein. All recombinant BoIFN-a A protein in this study shaved the antiviral activity in MDBK-VSV and MDBK-BVDV system. After contrastive analysis the protein form, quantity of expression protein, antiviral activity, production cost of E.coli and Pichia pastoris.The result showed that the recombination protein in Pichia pastoris was more suitable for the industrialized development and mass production. Overall, successful expression is of significance for the development and industrialization of the production of new antiviral interferon preparations for the next step to make a meaningful exploration.