人β<sub>2</sub>-glycoprotein I及其第V结构域蛋白的原核表达、纯化和活性鉴定

中国生物工程杂志

2011, Vol. 31

Issue (11): 11-17

研究报告

人β₂-glycoprotein I及其第V结构域蛋白的原核表达、纯化和活性鉴定

王贺, 迟彦, 王仁军, 李文哲, 马艳华, 李敬达, 刘庆平

大连大学生命科学与技术学院生物有机化学重点实验室大连 116622

Expression, Purification and Activity Identification of Human β₂-Glycoprotein I and Domain V

WANG He, CHI Yan, WANG Ren-jun, LI Wen-zhe, MA Yan-hua, LI Jing-da, LIU Qing-ping

Key Laboratory of Bio-organic Chemistry, College of Life Science and Technology, Dalian University, Dalian 116622, China

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摘要：

目的:旨在重组表达人源β₂-GPI及其第V结构域基因,探讨后者在抗自身免疫性动脉粥样硬化实验中的干预作用。方法:以实验室保存的CTA727-1重组质粒为模板克隆β₂-GPI及其第V结构域基因,构建pET32-β₂-GPI和pET32-DV重组表达载体,分别转化至大肠杆菌Rosetta-gami。经IPTG诱导表达,镍离子亲和层析柱纯化重组蛋白,并采用Western blot、HPTLC和ELISA方法验证了重组蛋白的活性与功能。结果:β₂-GPI及其第V结构域目的基因被分别克隆至原核表达载体pET-32a-c(+),诱导出具备CL和oxLig-1结合活性的β₂-GPI 及第V结构域融合蛋白,ELISA实验显示第V结构域融合蛋白可抑制oxLig-1/(r)β₂-GPI/Antibody免疫复合物的形成。结论:成功构建了pET32-β₂-GPI及pET32-DV表达载体,获得高水平表达且具备活性的β₂-GPI 和第V结构域重组蛋白,为研究治疗动脉粥样硬化等疾病的多肽药物奠定了基础。

关键词： β₂-GPI; β₂-GPI及第V结构域; 原核表达; 动脉粥样硬化

Abstract:

Objective: To express and purify the recombinant proteins of human β₂-glycoprotein I (β₂-GPI) and β₂-GPI Domain V in E.coli. Methods: The plasmid CTA727-1 kept by our laboratory which contained the target DNA of β₂-GPI and Domain V was used as template. The recombinant plasmids pET32-β₂-GPI and pET32-DV were constructed and transformed into E.coli Rosetta-gami cells respectively. The recombinant proteins were induced by Isopropyl β-D-1-thiogalactopyranoside (IPTG) and purified by HisTrap affinity column and then identified by Western blot, high-performance thin-layer chromatography immunostaining assay (HPTLC) and enzyme-linked immunosorbent assay (ELISA). Results: The target DNA fragments of β₂-GPI and Domain V were inserted into prokaryotic expression vector pET-32a-c(+) respectively. The recombinant proteins were successfully expressed in E.coli. Furthermore, HPTLC showed that the binding activities of the recombinant proteins of β₂-GPI and Domain V to CL and 7-ketochorestery-9-caboxynonanoate (oxLig-1) were similar to that of human β₂-GPI. ELISA analysis illustrated the recombinant protein of Domain V competitively inhibits the formation of oxLig-1/(r) β₂-GPI/Antibody immune complex. Conclusion: The high level expression of β₂-GPI and Domain V were induced in E.coli expressing system and the purified proteins with activity were obtained which could lay the foundation for further research on functions of making drugs against arteriosclerosis.

Key words: β₂-GPI β₂-GPI Domain V Prokaryotic expression Arteriosclerosis

收稿日期: 2011-07-06 出版日期: 2011-11-25

ZTFLH:

Q786

基金资助:

国家自然科学基金资助项目(30371380,30571733)

通讯作者: 刘庆平 E-mail: qingpingliu40@hotmail.com

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引用本文:

王贺, 迟彦, 王仁军, 李文哲, 马艳华, 李敬达, 刘庆平. 人β₂-glycoprotein I及其第V结构域蛋白的原核表达、纯化和活性鉴定[J]. 中国生物工程杂志, 2011, 31(11): 11-17.

WANG He, CHI Yan, WANG Ren-jun, LI Wen-zhe, MA Yan-hua, LI Jing-da, LIU Qing-ping. Expression, Purification and Activity Identification of Human β₂-Glycoprotein I and Domain V. China Biotechnology, 2011, 31(11): 11-17.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2011/V31/I11/11

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