重组人肿瘤坏死因子α抑制肽-抗炎酸性尾巴融合蛋白的生物学活性检测

目的：评价原核表达、纯化的6×His-硫氧还蛋白（TRX）一人肿瘤坏死因子α（TNFα）抑制肽-C端抗炎酸性尾巴融合蛋白的生物学功能。方法：在大肠杆菌中分别表达带His标签的TRX对照蛋白及TRX蛋白融合的人TNFα抑制肽-抗炎酸性尾巴融合蛋白，并对2种蛋白进行N^2＋金属螯合层析纯化，采用MTT法检测纯化后的蛋白及化学合成多肽抑制TNFα标准品对L929细胞的细胞毒活性。结果：与对照蛋白相比，融合蛋白人TNFα抑制肽-C端抗炎酸性尾巴及合成多肽均能拮抗TNFα标准品对L929细胞的细胞毒作用。结论：融合蛋白人TNFα抑制肽-C端抗炎酸性尾巴及合成肽均能有效拮抗TNFα的生物学作用，为今后发展抑制TNFα为主的抗炎生物药物奠定了基础。

Detection of Biological Activity of Recombinant Human Tumor Necrosis Factor α Inhibitory Peptide Fused with Anti-Inflammatory Acidic Tail

Objective： To evaluate the biological activity of the purified recombinant fusion protein of N-termi- nal 6xHis tag-TRX fused human tumor necrosis factor α（TNFα） inhibitory peptide with a C terminal anti-inflam- matory acidic tail expressed in Escharia coli. Methods： The 6xHis tagged TRX control protein and TRX fused human TNFα inhibitory peptide with an anti-inflamatory acidic peptide tail were expressed in genetically engineered E.coli and then purified by nickel metal chelated affinity chromatography. The biological activity of the purified proteins and the chemically systhesized control peptide in inhibiting TNFα cytotoxic activity on the L929 cells was evaluated by MTT assay. Results： The TRX-human TNFα inhibitory peptide-anti-inflamatory acidic peptide tail fusion protein and the synthesized peptide can antagonize the eytotoxic activity of the TNFα standard on L929 cells. Conclusion： The recombinant fusion protein and the inhibitory peptide can antagonize the bioactivity of TNFα effectively, laiding the foundation for the development of anti-inflammatory biomedicine based on antagonist of TNFct in the future.