重组早孕因子(EPF)的表达、纯化及鉴定

Expression,Purification and Identification of Recombinant Early Pregnancy Factor(EPF)

目的:获取大量具有良好生物活性的早孕因子(EPF)重组蛋白。方法:从HeLa细胞中提取总RNA,采用RT-PCR的方法扩增EPF cDNA,将该cDNA插入载体pGEX-5X-1,获得重组表达质粒pGEX-5X-1/EPF,将重组表达质粒pGEX-5X-1/EPF转化大肠杆菌BL21后通过IPTG进行诱导表达,然后用谷胱甘肽-琼脂糖球珠亲和层析方法,分离纯化得到纯重组GST-EPF融合蛋白(rEPF)。再用Xa酶切GST,得到纯化的EPF重组蛋白。用SDS-PAGE鉴定其纯度,用Western blotting鉴定其特异性,用玫瑰花环抑制试验(RIT)检测其生物学活性。结果:HeLa细胞总RNA中有效扩增出EPF cDNA。EPF cDNA以正确的阅读框架插入表达载体pGEX-5X-1,经IPTG诱导后高效表达EPF重组蛋白。Western blotting表明其与抗EPF抗体有特异性结合,RIT检测结果显示目的蛋白具有良好的EPF生物学特性。结论:成功构建了EPF的原核表达载体,并纯化获得了具有生物学特性的重组蛋白,为进一步开展EPF蛋白的相关研究奠定了基础。

Objective： To obtain large amounts of recombinant early pregnancy factor（EPF） protein.Methods： The total RNA was extracted from HeLa cells.EPF gene fragment was amplified with RT-PCR,cloned into the expression vector pGEX-5X-1.The recombinant plasmid pGEX-5X-1/EPF was constructed in this way and expressed under the induction of IPTG.The expressed EPF protein was purified by GST affinity chromatography,and its biological activity was detected by RIT.Results： The recombinant plasmid pGEX-5X-1/EPF was successfully constructed,as confirmed by EcoR1/XhoI digested and PCR.Recombinant EPF protein was expressed in E.coli host stain BL21 star and it had good biological activity,which was confirmed by RIT.Conclusion： The cloning and expression of EPF gene laid a foundation of further study on EPF protein.