重组人源性脂联素球状结构的表达、纯化与活性检测

中国生物工程杂志

2013, Vol. 33

Issue (6): 68-73

技术与方法

重组人源性脂联素球状结构的表达、纯化与活性检测

张立剑¹, 孙璐¹, 郭云萍¹, 李冬冬¹, 王增禄², 刘毅¹, 陶凌¹

1. 第四军医大学西京医院西安 710032;
2. 第四军医大学药学系生物技术中心西安 710032

Expression, Purification of Human gAd in E.coli and Identification of Its Biological Activity

ZHANG Li-jian¹, SUN Lu¹, GUO Yun-ping¹, LI Dong-dong¹, WANG Zeng-lu², LIU Yi¹, TAO Ling¹

1. Department of Cardiology, Xijing Hospital, The Fourth Military Medical University, Xi’an 710032, China;
2. Biotechnology Center, School of Pharmacy, The Fourth Military Medical University, Xi’an 710032, China

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摘要：

目的:利用基因工程方法构建人源性脂联素球状结构(gAd) 基因的高效原核表达体系,并对重组蛋白进行诱导表达、纯化、鉴定及活性检测。方法:从正常人脂肪组织里面提取总RNA,反转录合成cDNA,经PCR扩增、酶切后连入pET-22b(+)载体构建重组质粒pET-22b(+)-gAd,重组质粒转化大肠杆菌BL21(DE3)感受态细胞。经诱导剂诱导后目的蛋白以包涵体形式产生,采用强碱促溶包涵体并用丙酮沉淀蛋白的方法进行复性和纯化,得到高纯度的人源性gAd。运用SDS-PAGE、Western blotting对重组蛋白进行鉴定,通过对蛋白激酶(AMPK)的磷酸化水平和对小鼠的心肌缺血再灌注损伤的保护作用来检测纯化蛋白的生物学活性。结果:成功构建了原核表达载体pET-22b(+)-gAd,实现了人源性gAd在原核细胞中的表达,并对形成的包涵体变性、复性和纯化,纯化出的蛋白经过SDS-PAGE 和Western分析证实为gAd;通过对AMPK的磷酸化水平的检测和对小鼠的心肌缺血再灌注损伤的保护作用证明纯化出的gAd具有高生物学活性。结论:成功构建、表达和纯化了无标签、高生物学活性的人源性脂联素球状结构(gAd),为其进一步的理论研究、生产开发奠定了基础。

关键词： 重组人脂联素球状结构; 表达纯化; 心脏保护

Abstract:

Objective: To construct the prokaryotic expression vector of human gAd gene and obtain the no-tagged recombinant human gAd protein. Methods: Total RNA was extracted from human adipose tissue. The gAd cDNA was obtained with RT-PCR technique and subcloned into a prokaryotic exprssion vector pET-22b(+) to generate pET-22b(+)-gAd. The expression of no-tagged recombinant human gAd protein was induced by IPTG in E.coli BL21(DE3). The gAd protein produced as inclusion bodies at an elevated level. The inclusion bodies were solubilized by alkaline shock, and then refolded and purified after acetone precipitation. SDS-PAGE and Western blot were used for identification of the purified gAd. The abilities of gAd to induce the phosphorylation of AMPK in HUVECs and to protect hearts after myocardial ischemia/reperfusion in mice were used for its biological activity assay. Results: The human gAd coding sequence was correctly cloned into pET-22b(+) vector. After expression and purification, the recombinant human gAd significantly induced the phosphorylation of AMPK in HUVECs and protected hearts from myocardial ischemia/reperfusion injury. Conclusion: The no-tagged recombinant human gAd was successfully expressed and purified in prokaryotic expression system with high biological activity.

Key words: Human gAd Expression and purification Cardioprotection

收稿日期: 2012-11-22 出版日期: 2013-06-25

ZTFLH:

Q786

基金资助:

国家重大新药创制(2009ZX09103-673); 国家自然科学基金(81170186、81100136);新世纪优秀人才支持计划(2011SXJ01)资助项目

通讯作者: 陶凌 E-mail: lingtao2006@gmail.com

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引用本文:

张立剑, 孙璐, 郭云萍, 李冬冬, 王增禄, 刘毅, 张英起, 陶凌. 重组人源性脂联素球状结构的表达、纯化与活性检测[J]. 中国生物工程杂志, 2013, 33(6): 68-73.

ZHANG Li-jian, SUN Lu, GUO Yun-ping, LI Dong-dong, WANG Zeng-lu, LIU Yi, ZHANG Ying-qi, TAO Ling. Expression, Purification of Human gAd in E.coli and Identification of Its Biological Activity. China Biotechnology, 2013, 33(6): 68-73.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2013/V33/I6/68

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