人肿瘤坏死因子受体-Fc融合蛋白的表达及生物活性分析  

Expression of human tumor necrosis factor receptor-Fc fusion protein and characterization of its biological activity

作　　者：杜芝燕[1] 王芳[1] 朱静潆[1] 于继云[1] 王宇[1] 朱晓明[1] 张巍[1] 

机构地区：[1]军事医学科学院基础医学研究所转化医学研究室

出　　处：《细胞与分子免疫学杂志》2014年第3期225-228,共4页Chinese Journal of Cellular and Molecular Immunology

基　　金：北京市自然科学基金资助项目(7132145)

摘　　要：目的构建携带人肿瘤坏死因子受体(TNFR)胞外区融合人IgG Fc片段的重组腺相关病毒载体(pAAV/hTNFR-Fc),并对融合蛋白hTNFR-Fc的表达和生物学活性进行鉴定。方法构建hTNFR-Fc融合基因重组腺相关病毒(AAV)载体,体外转染人胚肾HEK293T细胞,收集转染上清,以Western blot法检测目的蛋白的表达,应用L929细胞测定重组表达产物对人和大鼠TNF-α细胞毒中和活性,采用ELISA比较重组表达产物对人和大鼠TNF-α的结合活性。结果成功构建重组表达载体pAAV/hTNFR-Fc,在转染细胞上清中检测到目的蛋白hTNFR-Fc的表达,重组表达产物可有效结合人TNF-α并中和其L929细胞毒活性,并对大鼠TNF-α具有一定程度的结合和中和活性。结论成功构建了hTNFR-Fc融合基因,验证了其在AAV载体上的分泌性表达,并对其生物活性进行了鉴定,为进一步病毒包装和大鼠关节炎动物实验研究奠定了基础。Objective To construct a recombinant adeno-associated virus vector carrying the fusion product of extracellular domain of human tumor necrosis factor receptor （hTNFR） with human IgG Fc fragment, and detect the expression and biological activity of hTNFR-Fc. Methods The recombinant hTNFR-Fc fusion gene was cloned into adeno-associated virus （AAV） vector, which was used to transfect human embryo kidney cells （HEK293T） in vitro. The expression of the target protein hTNFR-Fc in the supernatants of the transfected HEK293T cells was identified by Western blot analysis. TNF-α inhibition assay in vitro was performed using TNF-α-sensitive mouse fibrosarcoma 1_929 cells to determine the neutralizing capacity of hTNFR-Fc against human or rat TNF-α The binding activity of hTNFR-Fc to human or rat TNF-a was identified by ELISA. Results The recombinant plasmid pAAV/hTNFR-Fc was constructed and the target protein of hTNFR-Fc was expressed in the supernatants of the transfected HEK293T cells. It could effectively bind to human TNF-α and neutralize its cytotoxicity to 1_929 cells. The hTNFR-Fc protein also exerted the binding and neutralization activities to rat TNF-α to some extent. Conclusion The study constructed hTNFR-Fc fusion gene and verified its secretory expression in AAV vector, as well as identified its biological activities, which lay the foundations for future viral package and animal experiments.

关 键 词：hTNFR—Fc 重组腺相关病毒载体 表达 活性 

分 类 号：R392.11[医药卫生—免疫学] R446.61[医药卫生—基础医学]