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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 a large number of good biological activity of the early pregnancy factor (EPF) recombinant protein. Methods: The total RNA was extracted from HeLa cells and amplified by RT-PCR. The cDNA was inserted into pGEX-5X-1 vector to obtain the recombinant plasmid pGEX-5X-1 / EPF. The recombinant plasmid pGEX- 5X-1 / EPF was transformed into E.coli BL21 and then induced by IPTG. Then pure recombinant GST-EPF fusion protein (rEPF) was isolated and purified by glutathione-Sepharose beads affinity chromatography. The GST was further digested with Xa to obtain the purified EPF recombinant protein. Its purity was identified by SDS-PAGE, and its specificity was identified by Western blotting. The rosette inhibition test (RIT) was used to detect its biological activity. Results: EPF cDNA was efficiently amplified from total RNA of HeLa cells. The EPF cDNA was inserted into the expression vector pGEX-5X-1 with the correct reading frame and induced by IPTG to express EPF recombinant protein efficiently. Western blotting showed that it has specific binding with anti-EPF antibody, RIT test results showed that the target protein has good biological characteristics of EPF. CONCLUSION: The prokaryotic expression vector of EPF was successfully constructed and purified to obtain the recombinant protein with biological characteristics, which lays the foundation for the further study of EPF protein.