人HMGB1基因原核表达载体的构建及其在大肠杆菌中的表达和纯化

山东大学学报(医学版) ›› 2010, Vol. 48 ›› Issue (4): 40-44.

人HMGB1基因原核表达载体的构建及其在大肠杆菌中的表达和纯化

崔彬1，2，王来城2，朱敏2，焦玉莲2，辛玮2，马春燕2，张捷2，赵跃然2

1. 山东省医学科学院基础医学研究所, 济南 250062；
2. 山东大学附属省立医院中心实验室, 济南 250021

收稿日期:2009-12-09 出版日期:2010-04-16 发布日期:2010-04-16
通讯作者: 赵跃然(1963- )，博导，主要从事免疫学及分子生物学研究。 Email:yrzhao@sdu.edu.com
作者简介:崔彬(1979- )，女，技师，硕士研究生，主要从事分子免疫学的研究。 Email:shytcb@126.com
基金资助:
山东省科技攻关计划资助项目（2009GG10002008）

Construction of recombinant prokaryotic expression plasmid of human high mobility group box1 and its expression and purification

CUI Bin1，2, WANG Laicheng2, ZHU Min2, JIAO Yulian2, XIN Wei2,MA Chunyan2, ZHANG Jie2, ZHAO Yueran2

1. Institute of Basic Medicine, Shandong Academy of Medical Sciences, Jinan 250062, China;
2. Central Laboratory, Provincial Hospital Affiliated to Shandong University, Jinan 250021, China

Received:2009-12-09 Online:2010-04-16 Published:2010-04-16

摘要/Abstract

摘要：

目的构建人高迁移率族蛋白B1（HMGB1）基因的原核表达载体，观察表达水平并纯化重组蛋白。方法根据GenBank中人HMGB1基因序列，用OptimumGeneTM行密码子优化并合成目的基因，经PCR扩增，NdeⅠ和XhoⅠ双酶切，插入原核表达载体pQE-T7-2的相应位点，构建原核表达质粒pQE-T7-2/HMGB1。经菌落PCR、酶谱分析及序列分析鉴定重组质粒，转化大肠杆菌BL21(DE3)，异丙基-β-D-硫代半乳糖苷(IPTG)诱导重组蛋白表达，采用SDS-PAGE和Western blot印迹法鉴定重组蛋白，NiNTA树脂亲和纯化目的蛋白。结果成功构建人HMGB1克隆载体和表达载体；表达的目的蛋白约占菌体总蛋白的20%以上，Western blot法显示，重组蛋白能与抗HMGB1抗体和抗His抗体特异结合，亲和层析纯化后纯度达90%以上。结论成功构建高效稳定的重组人HMGB1原核表达载体，获得纯化人HMGB1蛋白，为进一步研究HMGB1的功能奠定基础。

关键词: 高迁移率族蛋白质类；大肠杆菌；原核细胞；纯化

Abstract:

Objective To construct the prokaryotic expression plasmid of human high mobility group box1 (hHMGB1) gene, and to express and purify the His-tagged hHMGB1 fusion protein. Methods According to the sequence reported in GenBank, we optimized codons by OptimumGeneTM and synthesized the human HMGB1 gene. To construct prokaryotic expression plasmid pQE-T7-2/HMGB1, the DNA fragment obtained from PCR was digested with NdeⅠ and XhoⅠ, and then inserted into pQE-T7-2 that was cut with the same enzymes. The recombinants were screened by colony PCR, restriction mapping and sequencing. The recombinant E.coli BL21 (DE3) was induced for expression with isopropyl-beta-D-thiogalactopyranoside (IPTG), and the recombinant protein was identified by SDS-PAGE and Western blotting. The recombinant human HMGB1 was further purified by Ni-NTA Agarose. Results The prokaryotic expression plasmid pQE-T7-2/HMGB1 was successfully constructed. The expressed recombinant protein accounted for about 20% of the total bacterial proteins. The recombinant protein could specifically react with antiHMGB1 antibody and anti-His antibody. The purity of recombinant human HMGB1 separated with affinity chromatography was more than 90%. Conclusion The recombinant prokaryotic expression vector of human HMGB1 which was successfully constructed may lay the foundation for the further study of the function of human HMGB1.

Key words: High mobility group proteins； Escherichia coli； Prokaryotic cells； Purification

中图分类号:

R392.11

崔彬1，2，王来城2，朱敏2，焦玉莲2，辛玮2，马春燕2，张捷2，赵跃然2. 人HMGB1基因原核表达载体的构建及其在大肠杆菌中的表达和纯化[J]. 山东大学学报(医学版), 2010, 48(4): 40-44.

CUI Bin1，2, WANG Laicheng2, ZHU Min2, JIAO Yulian2, XIN Wei2,MA Chunyan2, ZHANG Jie2, ZHAO Yueran2. Construction of recombinant prokaryotic expression plasmid of human high mobility group box1 and its expression and purification[J]. JOURNAL OF SHANDONG UNIVERSITY (HEALTH SCIENCES), 2010, 48(4): 40-44.

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