人肿瘤坏死因子受体（P55）-IgG Fc融合蛋白在真核细胞中的克隆与表达
[Cloning and expression of human TNFR (P55)-IgG Fc fusion protein in eukaryotic cells].
作者信息
An N, Xu C, Zhang Z
机构信息
State Key Laboratory of Molecular Virology and Genetic Engineering, Institute of Virology, Chinese Academy of Preventive Medicine, Beijing 100052, China.
出版信息
Zhonghua Shi Yan He Lin Chuang Bing Du Xue Za Zhi. 2001 Jun;15(2):116-7.
OBJECTIVE
To express soluble biologically active TNF receptor protein in eukaryotic cells, which can inhibit cytotoxic activity of TNF.
METHODS
PCR amplified the extra-cellular region of TNF receptor P55 and IgG Fc gene. Then the two were linked through an oligomer encoding a thrombin-sensitive peptide linker and cloned into eukaryotic expression vector pcDNA3.1 (+). The eukaryotic expression plasmid pcDNA3. 1/TI was then transfected to the mammalian cell line COS7 and BHK. Using the G418 system, BHK cell clones were selected and can continuously secrete biological protein in large amount.
RESULTS
The expressed protein was fused with IgG Fc and secreted into the cell culture supernatant. It has good antigenicity and binding ability to TNF. It can also inhibit the cytotoxic activity of TNF on L929 cell.
CONCLUSION
The TNFR-IgG Fc fusion protein expressed in eukaryotic cells has biological activities of human TNF receptor P55.
目的
在真核细胞中表达可抑制肿瘤坏死因子（TNF）细胞毒活性的可溶性生物活性TNF受体蛋白。
方法
采用聚合酶链反应（PCR）扩增TNF受体P55胞外区基因和IgG Fc基因，二者通过编码凝血酶敏感肽接头的寡聚核苷酸连接，克隆入真核表达载体pcDNA3.1（+）。将真核表达质粒pcDNA3.1/TI转染至哺乳动物细胞系COS7和BHK。利用G418系统筛选出能持续大量分泌生物蛋白的BHK细胞克隆。
结果
表达的蛋白与IgG Fc融合并分泌至细胞培养上清中。它具有良好的抗原性和与TNF的结合能力，还能抑制TNF对L929细胞的细胞毒活性。
结论
在真核细胞中表达的TNFR-IgG Fc融合蛋白具有人TNF受体P55的生物活性。
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