人HPPCn重组蛋白可溶性表达及其增殖活性检测

doi:10.13523/j.cb.20151203

中国生物工程杂志

2015, Vol. 35

Issue (12): 15-20 DOI: 10.13523/j.cb.20151203

研究报告

人HPPCn重组蛋白可溶性表达及其增殖活性检测

路青山¹, 乔媛媛², 李金凤³, 王运良³, 王姗姗³, 史成和², 杨霄鹏¹, 张达矜²

1. 郑州大学第二附属医院郑州 450014;
2. 中国人民解放军海军总医院基础医学研究中心北京 100048;
3. 中国人民解放军第148医院淄博 255300

Soluble Expression of Human HPPCn Recombinant Protein and Detection of Its Proliferation Activity

LU Qing-shan¹, QIAO Yuan-yuan², LI Jin-feng³, WANG Yun-liang³, WANG Shan-shan³, SHI Cheng-he², YANG Xiao-peng¹, ZHANG Da-jin²

1. Department of Neurology, The Second Affiliated Hospital of Zhengzhou University, Zhengzhou 450001, China;
2. Center for Basic Medical Sciences, Navy General Hospital of Chinese PLA, Beijing 100048, China;
3. The 148th Hospital of Chinese PLA, Zibo 255300, China

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摘要：

HPPCn是一种新的肝细胞刺激因子,获得较高纯度的具有生物活性的HPPCn蛋白对研究其生物学功能具有重要意义。HPPCn在重组质粒PET-24a(+)/HPPCn中,诱导表达产物主要以包涵体形式为主。为优化表达条件,运用冷休克表达载体pColdⅡ和无缝克隆技术获得重组质粒pColdⅡ/HPPCn,分别对诱导温度、时间、分子伴侣等诱导条件进行筛选,镍离子柱亲和层析纯化,Western blot分析目的蛋白特异性,不同浓度人HPPCn重组蛋白(0、10、100ng/ml)刺激SMMC7721细胞,免疫细胞化学方法测定Brdu掺入、PCNA表达变化。目的蛋白在培养温度为16℃,终浓度为0.1mmol/L的IPTG过夜诱导时为可溶性表达,最终得到人重组HPPCn蛋白纯度为94.88%,人HPPCn重组蛋白可刺激SMMC7721细胞Brdu掺入增加、PCNA表达水平上调,其刺激作用具有量效关系。通过冷休克表达系统获得可溶性表达的人重组HPPCn蛋白,其具有促进SMMC7721细胞增殖的生物学功能,为深入研究HPPCn的作用机制提供了技术条件。

关键词： HPPCn; 冷休克表达系统; ANP32; 可溶性表达

Abstract:

HPPCn is a new hepatocyte stimulating factor, and it is of great significance to obtain high purity of HPPCn for studying the biological function of HPPCn. The expression of HPPCn in the recombinant plasmid PET-24a(+)/HPPCn was mainly in the form of inclusion body. Cold-shock expression vector pColdⅡand seamless cloning technology were used to obtain recombinant plasmid pColdⅡ/HPPCn. The induction conditions, such as temperature, time and molecular chaperones, were optimized respectively. The expression product was subsequently purified by nickel-ion column. The specificity of the recombinant protein was analyzed by Western blotting. SMMC7721 cells were stimulated by human HPPCn recombinant protein at the concentrations of 0, 10 and 100ng/ml, respectively. The contents of PCNA and Brdu were determined by immunocytochemistry. Human HPPCn recombinant protein could be solubly expressed in the new system. The purity of recombinant HPPCn protein was 94.88%. Stimulated at different concentrations of HPPCn, Brdu incorporation and the expression levels of PCNA increased in a dose-dependent manner. The human HPPCn recombinant protein was induced to soluble expression in the cold-shock expression system, and the product could promote the proliferation of SMMC7721 cells. It may serve as the foundation for further studies on the mechanism of HPPCn.

Key words: HPPCn Cold-shock expression system Soluble expression ANP32

收稿日期: 2015-07-21 出版日期: 2015-12-22

ZTFLH:

Q786

基金资助:

国家自然科学基金面上项目(81472350,31071256)资助项目

通讯作者: 杨霄鹏, 张达矜 E-mail: dajinzhang@sina.com;yaxipe39@126.com

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引用本文:

路青山, 乔媛媛, 李金凤, 王运良, 王姗姗, 史成和, 杨霄鹏, 张达矜. 人HPPCn重组蛋白可溶性表达及其增殖活性检测[J]. 中国生物工程杂志, 2015, 35(12): 15-20.

LU Qing-shan, QIAO Yuan-yuan, LI Jin-feng, WANG Yun-liang, WANG Shan-shan, SHI Cheng-he, YANG Xiao-peng, ZHANG Da-jin. Soluble Expression of Human HPPCn Recombinant Protein and Detection of Its Proliferation Activity. China Biotechnology, 2015, 35(12): 15-20.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20151203 或 https://manu60.magtech.com.cn/biotech/CN/Y2015/V35/I12/15

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