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首页> 中文期刊> 《生物技术通讯》 >GFE-1多肽与重组人肿瘤坏死因子α融合蛋白的构建及其体外活性和体内分布研究

GFE-1多肽与重组人肿瘤坏死因子α融合蛋白的构建及其体外活性和体内分布研究

         
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目的：构建 GFE-1多肽与重组人肿瘤坏死子α(rmhTNF-α)融合蛋白(GFE-1-rmhTNF),研究该融合蛋白的体外活性和体内分布。方法：利用基工程方法,将人工合成的编码 GFE-1的寡核苷酸片段连接在 rmhTNF-α序列的3'端,转入大肠杆菌中诱导表达,采用 Q-Sepharose FF 离子层析柱和 SP-Sepharose FF 阳离子层析柱纯化蛋白,SDS-PAGE 和 Western 印迹鉴定,测定该融合蛋白的体外活性,观察其在小鼠体内的分布情况。结果：构建了融合蛋白 GFE-1-rmhTNF,并在大肠杆菌中获得高效表达。体外活性实验显示,GFE-1-rmhTNF 对 L929细胞有明显的杀伤活性；体内分布实验证实,GFE-1-rmhTNF 在小鼠肺组织的富集高肝肾组织。结论：构建了融合蛋白 GFE-1-rmhTNF,可显著杀伤 L929细胞并特异性富集小鼠肺组织。%Objective: To construct GFE-1-rmhTNF fusion protein and study its bioactivity in vitro and distribu⁃tion in vivo. Methods: The synthetic GFE-1 oligonucleotide fragment was linked to the 3' terminal of the se⁃quence encoding rmhTNF-α and expressed in E.coli DH5α. The fusion protein was purified by ion-exchange chro⁃matography and identified by SDS-PAGE and Western blot. Bioactivity in vitro and distribution in vivo of the fu⁃sion protein were further analysed. Results: GFE-1-rmhTNF was constructed and expressed in E.coli efficiently. In vitro, its significant cytotoxic activity in L929 cells was observed. In vivo, GFE-1-rmhTNF was dramatically en⁃riched in the lung tissue of mice than the liver and kidney tissue. Conclusion: We successfully constructed GFE-1-rmhTNF fusion protein. It could kill L929 cells efficiently and specifically enriched in the lung tissue of mice.

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