隐源性肝炎相关新基因CHBP2的原核表达及蛋白纯化
隐源性肝炎;CHBP2;原核表达;蛋白纯化,李锟,叶进,肖凡,李国力,洪源,魏红山,通讯作者:,ExpressionandpurificationofnewgeneCHBP2associatedwithcryptogenichepatitisi
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李锟, 叶进, 华中科技大学同济医学院附属协和医院消化内科 湖北省武汉市 430022
肖凡, 李国力, 洪源, 魏红山, 北京地坛医院传染病研究室 北京市 100011
李锟, 华中科技大学同济医学院2004级硕士研究生, 主治医师, 主要从事肝病的分子生物学及临床研究.
通讯作者: 叶进, 430022, 湖北省武汉市, 华中科技大学同济医学院附属协和医院消化内科. yejin8688@sina.com
电话: 027-85726381
收稿日期: 2007-03-04 修回日期: 2007-07-18
Expression and purification of new gene CHBP2 associated with cryptogenic hepatitis in prokaryote cells
Kun Li, Jin Ye, Fan Xiao, Guo-Li Li, Yuan Hong, Hong-Shan Wei
Kun Li, Jin Ye, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, Hubei Province, China
Fan Xiao, Guo-Li Li, Yuan Hong, Hong-Shang Wei, Institute of Infectious Diseases, Ditan Hospital, Beijing 100011, China
Correspondence to: Dr. Jin Ye, Department of Digestive Disease, Union Hospital of Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, Hubei Province, China. yejin8688@sina.com
Received: 2007-03-04 Revised: 2007-07-18
Abstract
AIM: To construct a prokaryotic cell expression vector for a new gene, CHBP2, associated with cryptogenic hepatitis, and to abundantly express and purify CHBP2 protein.
METHODS: The open reading frame (ORF) of CHBP2 was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR), in which an mRNA template was extracted from Huh7 cells and ligated into the pGEM-T cloning vector. After sequencing, the correct DNA fragment was inserted into inducible Escherichia coli BL21. After the CHBP2 protein was induced with Isopropyl-beta-d-thiogalactopyranoside (IPTG), it was analyzed by SDS-PAGE and western blotting. Expressed bacteria were atomized by ultrasound and analyzed with SDS-PAGE. The expressed product was purified and renatured by Ni+ affinity column chromatography.
RESULTS: The prokaryotic cell expression vector pET-32a(+)-CHBP2 was successfully constructed and CHBP2 protein was abundantly expressed ......
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