人白血病抑制因子在原核细胞中的表达与纯化

doi:10.13523/j.cb.20170902

中国生物工程杂志

2017, Vol. 37

Issue (9): 7-14 DOI: 10.13523/j.cb.20170902

研究报告

人白血病抑制因子在原核细胞中的表达与纯化

张鹤铭^1,2, 蔡楚凡^1,2, 刘杨^1,2, 甘龙站^1,2, 焦雪苗³, 田永强^1,2

1. 四川大学轻纺与食品学院生物质与皮革工程系成都 610065;
2. 四川大学皮革化学与工程教育部重点实验室成都 610065;
3. 河南大学植物逆境生物学重点实验室开封 475001

Soluble Expression of Human Leukemia Inhibitory Factor in Prokaryotic Cells and Its Purification

ZHANG He-ming^1,2, CAI Chu-fan^1,2, LIU Yang^1,2, GAN Long-zhan^1,2, JIAO Xue-miao³, TIAN Yong-qiang^1,2

1. Department of Biomaterials and Leather Engineering, College of Textiles and Food Science, Sichuan University, Chengdu 610065, China;
2. Key laboratory of Leather Chemistry and engineering(Sichuan University), Ministry of Education, Chengdu 610065, China;
3. Key Laboratory of Plant Stress Biology, Henan University, Kaifeng 475001, China

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摘要： 研究证明人白血病抑制因子（hLIF）是一种对多种不同类型的细胞和组织具有重要功能的细胞因子，其独特的生物学特性使其被广泛应用。介绍了自制的具有生物活性的hLIF，将hLIF基因克隆到pET32a中，并利用硫氧还蛋白（Trx）作为融合配体，在大肠杆菌中成功表达可溶性融合蛋白Trx-hLIF。亲和层析纯化后利用SDS-PAGE和Western blot对纯化结果进行检验。利用肠激酶（EK酶）切割融合蛋白，释放hLIF，随后通过简单的阳离子交换得到纯度高达98.1％的hLIF 4.75mg。小鼠M1髓系白血病细胞增殖分析测定纯化的rhLIF的功能与hLIF具有相似的生物活性，EC₅₀为5ng/ml，对应的比活性为0.5×107 IU/mg。

关键词： 简单纯化; 白血病抑制因子; 表达; 诱导

Abstract: Numerous studies have demonstrated that leukemia inhibitory factor (LIF) is one of the most important polyfunctional cytokine, hLIF is a pleiotropic cytokine with multiple effects on different types of cells and tissues, its unique biological characteristics make it widely used. The homemade hLIF which is biologically active is viesented, hLIF gene was cloned into pET32a, and it successfully expressed the soluble recombinant protein in E. coli using the thioredoxin (Trx) protein as a fusion partner. After purification based on membrane adsorber technology, the fusion protein was cleaved using EK protease. Released, soluble hLIF was subsequently purified by cation exchange, The samples analysis by SDS-PAGE and Western blot. This procedure yields up to 4.75mg rhLIF with 98.1% purity. Functional analysis of the purified rhLIF by murine myeloblastic leukemia M1 cell proliferation assay demonstrates biological activity that is similar and comparable to that of hLIF, and with the EC₅₀ of 6ng/ml and the corresponding specific activity of 0.5×10⁷ IU/mg.

Key words: Induction Soluble expression Leukemia inhibitory factor Simple purification

收稿日期: 2017-04-11 出版日期: 2017-09-25

ZTFLH:

Q819

基金资助: 中央高校基本科研业务费专项资金（SCU2015D008）、四川应用基础研究计划项目（2014JY0199）资助项目

通讯作者: 田永强 E-mail: yqtian@scu.edu.cn

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引用本文:

张鹤铭, 蔡楚凡, 刘杨, 甘龙站, 焦雪苗, 田永强. 人白血病抑制因子在原核细胞中的表达与纯化[J]. 中国生物工程杂志, 2017, 37(9): 7-14.

ZHANG He-ming, CAI Chu-fan, LIU Yang, GAN Long-zhan, JIAO Xue-miao, TIAN Yong-qiang. Soluble Expression of Human Leukemia Inhibitory Factor in Prokaryotic Cells and Its Purification. China Biotechnology, 2017, 37(9): 7-14.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20170902 或 https://manu60.magtech.com.cn/biotech/CN/Y2017/V37/I9/7

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