Expression In Pichia Pastoris, Research On Physicochemical Characteristics And Biological Activities Of Bovine IFN-α1, IFN-α7, IFN-α14

Interferon (interferon, IFN) is a glucoprotein which secreted by the specific cells with a broad-spectrum antiviral, antitumor, immunomodulatory activity and in different ways affect cell metabolism, they represent the first line of defense against viral infection. IFNs are generally classified into type Ⅰ,Ⅱ and Ⅲ, and the antiviral activity of interferon a is the most significant in a number of interferon types. Researches have found that the recombinant bovine alpha interferon (Bovine interferon-alpha, BoIFN-α) protein expressed in vitro can inhibit the vesicular stomatitis virus (VSV), foot and mouth disease virus (FMDV), bovine viral diarrhea virus (BVDV) and parainfluenza virus three (PIV-3). Pichia pastoris expression system is one of the most rapid development and the most widely used for exogenous protein in recent years. A variety of animal interferon with comparable activities has been expressed in P. pastoris. BoIFN-α multigene family contains10-12subtypes, there are eight subtypes of the multigene family have been reported until now. But, there’s no systemic study about BoIFN-α multigene family and the study of physicochemical characteristics and biological activity of the various subtypes. So, it’s important to carry on the systematic study of bovine IFN-αsubtype.In this study, the recombinant plasmids constructed and stored in our laboratory which contains the complete open reading frame of BoIFN-α1, BoIFN-α7and BoIFN-α14(BoIFN-α1/7/14) were taken as templates, we amplified the mature peptide gene of BoIFN-α1/7/14by PCR, analysis it use the online software, and using Pichia pastoris expression system to achieve secretion expression of recombinant BoIFN-α1/7/14with no redundant amino acid. The physicochemical characteristics and biological activities of the recombinant BoIFN-α1/7/14were studied in detail subsequently.The results showed that the recombinant BoIFN-α1/7/14were expressed efficiently in the form of secretion in Pichia pastoris. The densities of recombinant proteins were0.2mg/mL,0.4mg/mL and0.163mg/mL; the recombinant proteins have some important physicochemical characteristics such as highly sensitive for trypsin, insensitivity to temperature, sensitivity to alkali while resistant to acid. These characteristics are similar to the typical physicochemical characteristics of IFNα; the antiviral activity of recombinant proteins were6.55×106U/mg, 3.50×106U/m and2.55×106U/mg detected in VSV-MDBK system, the recombinant proteins also can inhibit the CPE of IBRV on MDBK and have antiproliferative activity with a certain degree of dose dependent manner. In conclusion, this study provides a theoretical basis for the development of the new bovine antiviral agents which with high antiviral activity and low toxicity.