人类可溶性TNFRⅠ-GFP融合蛋白重组体的构建、表达和特性分析

Construction,Expression and Characteristic Analysis of Recombinant Human TNFRⅠ-GFP Fusion Protein

目的 构建并表达 hs TNFR (hum an soluble tum or necrosis factorreceptor ) - GFP(green fluorescence pro-tein) - p ET2 8a融合蛋白 ,用以检测跨膜型 TNF- α(m TNF- α)。方法 用 PCR从 GFP- p KEN重组质粒中扩增 GFP的 c D-NA,将其插入 hs TNFR - p ET2 8a重组质粒中 hs TNFR 基因片段的下游 ,获得 hs TNFR - GFP- p ET2 8a重组体。转化大肠杆菌 BL- 2 1,用 IPTG诱导重组蛋白表达 ,经镍金属螯合层析柱纯化。用 MTT法检测重组融合蛋白对 s TNF- α细胞毒效应的抑制活性 ,并用重组融合蛋白直接对转染 TNF基因细胞表面的 m TNF- α进行定性检测。结果 hs TNFR -GFP重组融合蛋白可明显抑制 s TNF- α细胞毒效应 ,且呈剂量依赖性 ;该融合蛋白不但可与 NIH3T3细胞膜上鼠源性m TNF- α结合 ,而且也可与转基因 NIH3T3细胞表面人 m TNF- α结合。结论 hs TNFR - GFP重组融合蛋白具有 TN-FR的生物学活性 ,同时保留 GFP的发光特性 ,可用来检测细胞表达 m TNF-

Objective A recombinant human soluble tumor necrosis factor receptorⅠ(hsTNFRⅠ) green fluorescence protein (GFP) pET28a was constructed and hsTNFRⅠ GFP fusion protein was expressed by E.coli in order to establish an alternative method for detection of mTNF α. Methods The cDNA of GFP was amplified by PCR from GFP pKEN recombinant plasmid and was inserted into the downstream of hsTNFRⅠ gene fragment which is contained in plamid pET. The recombinant hsTNFRⅠ GFP pET28a was then transformed into E.coli BL 21. The expression of recombinant fusion protein hsTNFRⅠ GFP was induced by IPTG and the purification of the fusion protein was performed by nickel chelation chromatography. The inhibitory effect of hsTNFRⅠ GFP on cytotoxicity of sTNF α was detected by MTT and mTNF α expressed on the cell surface of TNF α gene transfected cell line NIH3T3 was qualitatively determined with hsTNFRⅠ GFP fusion protein. Results The hsTNFRⅠ GFP fusion protein could markedly suppress the cytotoxicity of sTNF α in a dose dependent manner. The hsTNFRⅠ GFP fusion protein could bind not only with murine TNF α expressed on NIH3T3, but also with human TNF α on transgenic NIH3T3 cell line. Conclusion The hsTNFRⅠ GFP fusion protein had both TNF α binding activities as native TNFR and the irradiance characteristic of green fluorescence. The fusion protein could be used for mTNF α detection.