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

作　　者：邓学天 毛海洲[2] 刘丹丹 于淼 于双营 张守涛 Xuetian Deng;Haizhou Mao;Dandan Liu;Miao Yu;Shuangying Yu;Shoutao Zhang(School of Life Science,Zhengzhou University,Zhengzhou 450001,China;Department of Gastroenterology,No.155 Hispital of PLA,Kaifeng 475003,China)

机构地区：[1]郑州大学生命科学学院,河南郑州450001 [2]解放军第155医院消化内科,河南开封475003

出　　处：《生物技术》2018年第4期335-340,312,共7页Biotechnology

基　　金：国家"重大新药创制"科技重大专项("纤溶/抑制血小板聚集双功能溶栓药物研究";No.2012ZX09102301)

摘　　要：[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.

关 键 词：人肿瘤坏死因子Α 原核表达 亲和纯化 

分 类 号：Q784[生物学—分子生物学] Q786