人重组TNF-α突变体471蛋白的初步纯化及生物学活性

Study on the preliminary purification and bioactivity of recombinant human TNF-α mutein 471

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 。

AIM: To purify preliminary recombinant human TNF α mutein 471 and detect its bioactivity on the basis of the TNF α mutein 471 expressed in prokaryotic express system. METHODS: The expression of recombinant human TNF α mutein 471 in engineering bacteria strains E.coil was induced under the condition of optimal fermentation and expression. After cultured E.coil cells were collected and broken by using an ultrasonic disintegrator, the TNF α mutein 471 existed in the form of inclusion body was extracted and purified, and then the effects of denaturation and protein concentration on protein folding were examined. The bioactivities of wild type TNF α and the TNF α mutein 471 were detected by MTT colorimetry. RESULTS: The TNF α mutein 471 was folded and polymerized successfully to from a trimer with bioactivity under the condition of proper denaturation and renaturation. The cytotoxic activity of the TNF α mutein 471 to the L929 cells was 15 times as much as wild type TNF α. CONCLUSION: The TNF α mutein 471 expressed in prokaryotic expression system possesses significantly bioactivity after renaturation, which lays the foundation for further animal experiment and clinical experimental researches.