人源TNFα的原核表达及活性测定

doi:10.13523/j.cb.20140801

中国生物工程杂志

2014, Vol. 34

Issue (8): 1-6 DOI: 10.13523/j.cb.20140801

研究报告

人源TNFα的原核表达及活性测定

李翠琳, 张帆, 陈丹扬, 王昊, 郭强, 杜军

中山大学药学院微生物与生化制药实验室广州 510006

Study of Prokaryotic Expression and Biological Activity of Homo sapiens Kras Protein

LI Cui-lin, ZHANG Fan, CHEN Dan-yang, WANG Hao, GUO Qiang, DU Jun

Department of Microbial and Biochemical Pharmacy, School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China

全文: PDF(657 KB) HTML

摘要：

目的：利用SUMO标签构建人TNFα原核表达载体，通过表达及纯化获得重组蛋白，为深入研究和利用人TNFα奠定基础。方法：利用PCR技术，从质粒pET32a-hTNFα中扩增出人TNFα成熟肽编码序列，并在其上游添加SUMO标签，与原核表达载体pET28a连接，构建表达质粒pET28a-SUMO-hTNFα。在BL21（DE3）工程菌中表达融合蛋白，经Ni-NTA 纯化体系纯化，切除SUMO标签，纯化获得hTNFα成熟蛋白。CCK-8法检测TNFα对L929细胞的细胞毒性，以测定TNFα的生物学活性。结果：成功构建pET28a-SUMO-hTNFα原核表达质粒，酶切鉴定和测序分析与预期结果完全一致。在BL21（DE3）工程菌中实现了融合蛋白的可溶性表达。经纯化、水解酶切除标签及再次纯化获得hTNFα成熟肽。CCK-8法检测得所制备的TNFα蛋白ED50约为12.8 μg/ml。结论：成功构建原核表达载体pET28a-SUMO-hTNFα，经表达、纯化、酶切及再纯化，获得有生物活性的hTNFα蛋白，为深入研究和利用hTNFα奠定基础。

关键词： TNFα SUMO; 原核表达; 生物学活性

Abstract:

Objective: To construct a prokaryotic expression plasmid of human TNFα with SUMO,obtain recombinant protein by expression and purification,which is expected to provide basis for the further research and utilization of TNFα.Methods: The gene encoding mature TNFα protein was amplified from plasmid pET32a-hTNFα by PCR and cloned into pET28a with SUMO tag on the upstream.The fusion protein SUMO-hTNFα was expressed in BL21(DE3) engineering bacteria,then was purified by Ni-NTA Resin purification system.The purified production was digested by SUMO protease. One more purification in order to obtain mature TNFα protein.The biological activity was measured in a cytotoxicity assay using L929 cells by CCK-8 test. Results: The prokaryotic expression plasmid pET28a-SUMO-hTNFα was successfully constructed.The results identified by enzyme digestion and nucleotide sequencing corresponded exactly well as expected. The fusion protein SUMO-hTNFα expressed in a soluble form in BL21(DE3).Fusion protein was purified by Ni-NTA Resin purification system. Mature hTNFα protein was obtain after digestion by Sumo protease and purification. ED50 of recombinant hTNFα is 12.8μg/ml. Conclusion: The prokaryotic expression plasmid pET28a-SUMO-hTNFα was successfully constructed. TNFα protein was expressed and obtained by digestion and purification. These established a foundation of further research and utilization of hTNFα.

Key words: TNFα SUMO Prokaryotic expression Biological activity

收稿日期: 2014-04-08 出版日期: 2014-08-25

ZTFLH:

Q789

基金资助:

国家重点基础研究发展计划（2011CB935800）、国家自然科学基金（81272311，81071712）资助项目

通讯作者: 杜军，E-mail：dujun_tg@163.com E-mail: dujun_tg@163.com

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引用本文:

李翠琳, 张帆, 陈丹扬, 王昊, 郭强, 杜军. 人源TNFα的原核表达及活性测定[J]. 中国生物工程杂志, 2014, 34(8): 1-6.

LI Cui-lin, ZHANG Fan, CHEN Dan-yang, WANG Hao, GUO Qiang, DU Jun. Study of Prokaryotic Expression and Biological Activity of Homo sapiens Kras Protein. China Biotechnology, 2014, 34(8): 1-6.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20140801 或 https://manu60.magtech.com.cn/biotech/CN/Y2014/V34/I8/1

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