Prokarotic Expression And Purification Of Porcine Interferon-α Interferon-γ

Interferon (Interferon, IFN) is a protein which secreted by the cells, at least in the same kinds of cells with a broad-spectrum anti-viral, anti-cell division, immunomodulatory activity and in different ways affect cell metabolism, growth. China is a big raiser countrys, all kinds of pig diseases, particularly viral disease seriously impacts the development of pig industry, reduces economic efficiency. Therefor to develop cost-effective anti-viral and immune-enhancing drugs is a very important coping method. Porcine interferon has broad-spectrum anti-viral, anti-tumor and immune regulation, has become a research hotspot in recent years. Pig interferonα,βandγare three kinds of interferon that we known by know, interferon-αbelongs to typeⅠwhich with broad anti-viral spectrum, quick-acting and pluripotent characteristics. IFN-γbelongs to typeⅡ, except for anti-virus effects, also has a strong immune regulatory function. In this study, we use PET-30 vector system to construct prokaryotic expression vector of porcine IFN-αand IFN-γgene, then anti-viral activity of the recombinant proteins were detected. This study is divided into the following two parts: ?(1) Prokaryotic expression and purification of pig interferon-αand interferon-r. Primers were designed according to the gene sequences of pig interferonαand interferon-γin GenBank. Target genes were amplified by PCR; expression plasmids were constructed, after induction, recombinant proteins were purified by using Ni2 + affinity resin chromatography column, thereby provides candidate protein for further study of its antiviral activity.(2) Anti-viral activity test of the recombinant protein. Anti-star-viral replication activity in PK15 cells of the recombinant proteins were detected by using CPEI (cytopathic effect inhibition). The results showed that purified porcine IFN-αand IFN-γhas certain anti-star-viral replication activity. Mixed this two proteins in different proportions to detect the anti-star-viral replication activity, the results showed that porcine IFN-αand IFN-γhave better activity when missed with the ratio of 125:1.