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目的 :在利用原核表达系统表达重组人突变体 4 71TNF α蛋白的基础上 ,对该蛋白进行初步纯化及生物学活性检测。方法 :利用最佳发酵和表达条件 ,诱导基因重组的人突变体4 71TNF α工程菌表达目的蛋白。收集菌体 ,经超声破碎 ,分离人突变体 4 71TNF α的包涵体 ,并观察变性剂及蛋白浓度对蛋白折叠的影响。采用MTT比色法检测并比较人野生型及突变体 4 71TNF α的生物学活性。结果 :在适当的变性与复性条件下 ,已成功地将突变体 4 71TNF α折叠并聚合形成具有生物学活性的三聚体。突变体 4 71TNF α对L92 9的细胞毒活性高于野生型TNF α的 15倍。结论 :原核表达系统中表达的人突变体 4 71TNF α经复性处理后具有显著的生物学活性 ,为进一步进行动物实验及临床研究奠定了基础
OBJECTIVE: To purify and test the biological activity of recombinant protein 471TNFα using prokaryotic expression system. Methods: The target gene was expressed in human mutant 4 71TNF α engineered bacteria with the best fermentation and expression conditions. The bacterial cells were harvested and disrupted by sonication. Inclusion bodies from human mutant 71TNFα were isolated and the effects of denaturant and protein concentration on protein folding were observed. MTT colorimetric assay was used to detect and compare the biological activity of human wild type and mutant 4 71TNFα. Results: Mutant 4 71TNF α has been successfully folded and polymerized to form a biologically active trimer under appropriate conditions of denaturation and renaturation. The cytotoxic activity of mutant 4 71TNFα on L92 9 was 15-fold higher than that of wild-type TNFα. CONCLUSION: The recombinant human 4T1TNF α gene expressed in prokaryotic expression system has significant biological activity after refolding, which lays the foundation for further animal experiments and clinical studies