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为实现干扰素 (IFNα m)的高表达与纯化 ,研究其抗病毒、抗增殖活性。应用PCR技术将已构建的IFNα m的高表达基因亚克隆到原核表达载体pBV2 2 0上 ,构建表达质粒 pBV2 2 0 /IFNα m ,转化大肠杆菌DH5α进行表达。表达产物经初步检定以包涵体形式存在 ,包涵体进行变性、复性 ,然后经CM Sepharose FF、DEAE Sepharose FF离子交换层析和Sephacryal HR10 0分子筛纯化 ,获得较纯的干扰素IFNα m。以IFNα 1b为对照 ,在VSV Wish系统上采用细胞病变抑制法 ,测定纯品IFNα m的抗病毒活性。在Hela细胞上用MTT法进行抗增殖实验。结果表明包涵体含量占菌体总蛋白 4 0 % ,纯化的目的蛋白IFNα m纯度在 95 % ,比活高达 1 2 6× 10 7IU/mg ,纯化收率34 4 % ,IFNα m抗病毒活性和IFNα 1b相当 (P >0 0 5 ) ,抗增殖活性高于IFNα 1b(P <0 0 1)。
To achieve high expression and purification of interferon (IFNαm), its antiviral and antiproliferative activity was investigated. The highly expressed gene of IFNαm was subcloned into the prokaryotic expression vector pBV220 by PCR. The expression plasmid pBV220 / IFNαm was constructed and transformed into E. coli DH5α for expression. The expressed product was preliminarily tested in the form of inclusion bodies. The inclusion bodies were denatured and renatured. The purified inclusion bodies were purified by CM Sepharose FF, DEAE Sepharose FF ion exchange chromatography and Sephacryal HR10 0 molecular sieve to obtain pure interferon IFNα m. IFNα 1b was used as a control, and the anti-viral activity of pure IFNα m was determined by using the cytopathic effect inhibition method on the VSV Wish system. Anti-proliferation experiments were performed using MTT on Hela cells. The results showed that the content of inclusion body accounted for 40% of the total bacterial proteins, the purity of the purified IFNα m was 95%, the specific activity was as high as 126 × 10 7 IU / mg, the purification yield was 34 4%, the IFNα m antiviral activity and IFNα 1b was comparable (P> 0.05), and anti-proliferative activity was higher than that of IFNα 1b (P <0.01).