隐源性肝炎相关新基因CHBP2的原核表达及蛋白纯化  

Expression and purification of new gene CHBP2 associated with cryptogenic hepatitis in prokaryote cells

作　　者：李锟[1] 叶进[1] 肖凡[2] 李国力[2] 洪源[2] 魏红山[2] 

机构地区：[1]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 [2]北京地坛医院传染病研究室,北京市100011

出　　处：《世界华人消化杂志》2007年第22期2394-2398,共5页World Chinese Journal of Digestology

摘　　要：目的:构建隐源性肝炎相关新基因CHBP2原核表达载体,在大肠杆菌中进行表达,并纯化CHBP2融合蛋白。方法:应用逆转录聚合酶链反应(RT-PCR),以提取的Huh7细胞mRNA为模板,扩增获得CHBP2基因片段,连接到pGEM-T载体,测序正确后插入至原核表达载体pET-32a(+)中,构建原核表达载体pET-32a(+)-CHBP2,转化大肠杆菌BL21,以异丙基-β-D-硫代半乳糖苷(IPTG)诱导,获得CHBP2融合蛋白的可诱导性表达,通过SDS-PAGE电泳、Western blot免疫印迹分析证实蛋白表达的特异性,超声破碎表达细菌,SDS-PAGE分析,利用镍离子亲和柱对表达蛋白进行纯化及柱上复性。结果:成功构建原核表达载体pET-32a(+)- CHBP2,并将CHBP2融合蛋白成功表达,通过Western blot免疫印迹,证实了蛋白表达的特异性。SDS-PAGE分析表明其为包涵体表达,并对蛋白成功进行了纯化和复性,获得了表达蛋白的纯品结论:成功表达、纯化CHBP2蛋白,为研究CHBP2蛋白的生物学功能打下了基础,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 vec- tor. 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 ex- pressed. The protein＇s specificity was verified by western blotting. Protein production was mainly in the inclusion body, as shown by SDS-PAGE analysis, and was purified and renatured by Ni^＋ affinity column chromatography. Purified protein was successfully obtained. CONCLUSION： Expression and purification of CHBP2 protein will be useful for studying the biological functions of CHBP2.

关 键 词：隐源性肝炎 CHBP2 原核表达 蛋白纯化 

分 类 号：R346[医药卫生—基础医学]