重组人TNF-α在大肠杆菌表达、纯化及生物学活性鉴定

Expression and purification of recombinant human tumor necrosis Factor α in Escherichia coli

[目的]获得人TNF-α蛋白,并鉴定其生物学活性。[方法]通过对人源TNF-α序列进行密码子优化构建了p Cold-TNFα原核表达载体,完成了TNF-α重组蛋白的表达,建立了两步分离纯化方案,并利用Western Blot和细胞刺激实验检测了重组人TNF-α蛋白抗原性和生物学活性。[结果]利用大肠杆菌成功表达重组人TNF-α蛋白,经酶切纯化后的人TNF-α蛋白不含标签序列,纯度达95%,收率约70%,并具有良好的生物学活性。[结论]获得具有生物学活性的人TNF-α蛋白,为进一步的中和抗体研究奠定了基础。

[Objective]To obtain the protein human TNF-α and analyze biological activity of this protein. [Methods] The p Cold-TNFα prokaryotic expression vector was constructed by codon optimization of human TNF-α sequence,and the expression of fusion protein TNF-α was completed. A two-step purification protocol was established. The experiments Western Blot and cell stimulation were performed on the recombinant protein TNF-α to detect the antigenicity and biological activity.[Results]The recombinant human TNF-α was successfully expressed in E. coli. The digested and purified protein human TNF-α without the tag sequence had a purity of 95%,a yield of about 70%,a good biological activity. [Conclusion]The protein human TNF-α with biological activity was obtained,which laid the foundation for the further studies on neutralizing antibodies.