内含肽介导的重组人干扰素α2a的原核表达及其生物学活性

Prokaryotic expression and biologic characteristics of recombinant human interferon α2a protein in intein-mediated system

目的原核表达内含肽介导的重组人干扰素α2a(rhIFNα2a)融合蛋白,并检测其生物学活性。方法以质粒pBV220-IFN-α2a为模板PCR扩增rhIFNα2a基因,将其插入原核表达载体pTWIN1的内含肽Ssp DnaB基因下游的多克隆位点,构建重组表达质粒pTWIN1-rhIFNα2a,转化大肠杆菌BL21(DE3),IPTG诱导表达,表达产物进行SDS-PAGE及Western blot分析,并对裂菌上清的生物学活性进行测定。结果双酶切及测序结果显示,融合蛋白基因已克隆入表达载体中;rhIFNα2a蛋白的相对分子质量约44 300,表达量占全菌总蛋白的30%,在裂菌上清中的表达量占目的蛋白的25%,可与小鼠抗hIFNα单克隆抗体特异性结合;裂菌上清中rhIFNα2a蛋白的活性为5.57×107IU/ml,表明该重组蛋白具有良好的IFN抗病毒活性。结论已成功在大肠杆菌BL21(DE3)中表达了具有生物学活性的可溶性重组rhIFNα2a与内含肽Ssp DnaB融合蛋白,为大量生产IFNα2a提供了新的思路。

Objective To express recombinant human interferon ct2a protein （rhIFNct2a） in CBD （chitin-binding-domain）intein / pTWIN 1 expression system and determine its biological activity. Methods The coding sequence of rhIFN-α2a was amplified by PCR using plasmid pBV220-IFNα2a as a template and inserted into expression vector pTWIN1 fused in frame with an upstream Ssp DnaB intein gene. The constructed recombinant plasmid pTWIN 1-rhIFNα2a was transformed into E. coli BL21 （DE3）, and protein expression was induced with IPTG. The expressed product was identified by SDS- PAGE and Western blot, and the biological activity of lysis supernatant was determined. Results Both restriction analysis and sequencing proved that the fusion gene was cloned into expression vector pTWIN1. The expressed rhIFNα2a, with a relative molecular mass of about 44 300, contained 30% of total somatic protein, and reached an expression level of 25% in lysis supernatant. The expressed product showed specific binding to mouse McAb against hlFN. The rhIFNα2a activity was 5. 57 × 10^7 IU/ml in lysis supernatant, indicating significantly antiviral activity of the recombinant protein. Conclusion Soluble fusion protein of rhIFNα2a and Ssp DnaB, with biological activity, was successfully expressed in E. coli BL21 （DE3）, which provided a new idea for large-scale production of IFNα2a.