人IFN-ε的表达、纯化及生物学特性研究

目的 表达并纯化人IFN-ε,并对其理化特性和生物学特性进行初步研究. 方法 利用PCR技术以人基因组DNA为模板,获得目的片段的全基因序列后,克隆到原核表达载体pET-32a(+)上,构建重组表达载体pET-32a(+)/IFN-ε,转化到大肠杆菌BL21(DE3)中进行表达,并对表达进行了优化.产物的表达形式为包涵体,经过纯化复性后,获得高纯度的活性蛋白IFN-ε.对IFN-ε的理化性质和抗病毒活性、抑制肿瘤细胞增长活性、刺激细胞产生抗病毒蛋白的活性进行了鉴定. 结果 IFN-ε以包涵体形式表达,纯化后纯度大于95%,抗病毒活性为1.2×105 IU/mg,能够抑制肿瘤细胞的生长,并能诱导细胞产生抗病毒蛋白MxA. 结论 成功表达了人IFN-ε蛋白,并证明此蛋白质具有抗病毒和抗细胞增殖的活性.

Study on expression,purification and biological characteristics of recombinant human interferon-epsilon in E.coli

Objective To construct a novel recombinant human IFN-ε,and to analyze its physi-cal,chemical properties and biological characteristics.Methods Human genomic DNA was used as the template to synthesize IFN-ε gene by PCR.The sequence was cloned into plasmid vector pET-32a(+),and the recombinant plasmid pET-32a(+)/IFN-ε was transformed into E.coli BL2l(DE3).The form of ex-pression product was inclusion bodies.After purification and renaturation.high purity active protein IFN-εwas achieved.The final product was tested for its physical.chemical properties and biological characteristics including anti-viral and anti-proliferative acfivities.Results IFN-ε was expressed in inclusion body in E.coli.After the protein renaturation and purification,the purity was more than 95%.The rhIFN-ε protein had a specific anti-viral activity of about 1.2×103 IU/mg.Its anti-poliferative activity is obvious and can in-duce cells to produce anti-viral protein MxA.Conclusion Human IFN-ε protein was expressed successful-ly.and this protein has anti-virus and anti-prolireration activity.