HCBP12融合蛋白的表达、纯化及多抗制备

中国临床药理学与治疗学 ›› 2008, Vol. 13 ›› Issue (4): 425-430.

HCBP12融合蛋白的表达、纯化及多抗制备

李越^1,3, 王琦^2,3, 林原¹, 成军³, 李康³, 李华¹

¹大连医科大学药学院药理教研室,大连116044,辽宁;
²大连医科大学医学微生物学教研室,大连116044,辽宁;
³北京地坛医院传染病研究所,北京100011

收稿日期:2007-12-27 修回日期:2008-02-01 出版日期:2008-04-26 发布日期:2020-10-12
通讯作者: 李华,女,博士,研究方向:心血管药理学及分子药理学。Tel:0411-86110406 E-mail:lihuadl@hotmail.com
作者简介:李越,女,硕士研究生,研究方向:生化药理学及分子药理学。Tel:010-64200407 E-mail:liyuedl04@126.com

Expression and purification of HCBP12 fusion protein and preparation of polyclonal antibody against HCBP12

LI Yue^1,3, WANG Qi^2,3, LIN Yuan¹, CHENG Jun³, LI Kang³, LI Hua¹

¹Department of Pharmacology , Dalian Medical University , Dalian 116044, Liaoning, China;
²Department of Medi-cal Microbiology, Dalian Medical University , Dalian 116044 , Liaoning, China ;
³Institute of Infectious Diseases, Beijing Ditan Hospital, Beijing 100011 , China

Received:2007-12-27 Revised:2008-02-01 Online:2008-04-26 Published:2020-10-12

摘要/Abstract

摘要： 目的: 构建丙型肝炎病毒核心抗原结合蛋白12( HCV Core binding protein, HCBP12) 原核表达载体, 在大肠埃希菌中进行表达、纯化HCBP12 融合蛋白并制备兔抗HCBP12 多克隆抗体。方法: 应用逆转录聚合酶链反应( RT-PCR) , 以提取的HepG2 细胞mRNA 为模板, 扩增获得 HCBP12 基因片段, 插入至原核表达载体 pET-32a( +) 中, 构建原核表达载体pET-32a(+)-HCBP12, 转化大肠埃希菌BL21, 以异丙基硫代β-D-半乳糖苷( IPTG)诱导, 获得HCBP12 融合蛋白的可诱导性表达, 并通过SDS-PAGE 电泳、Western Blot 免疫印迹分析和证实融合蛋白表达的特异性。利用Ni+亲和柱对表达蛋白进行纯化及柱上复性。纯化蛋白免疫新西兰兔, 获得抗HCBP12 多克隆抗体。以纯化HCBP12 为抗原, 利用 Western blot 和ELISA 法对多克隆抗体进行特异性和效价检测。结果: HCBP12 融合蛋白表达成功。SDS-PAGE 分析表明其为包涵体表达。成功获得了融合蛋白纯品及兔抗HCBP12 多克隆抗体。ELISA 法表明多克隆抗体效价>1：512000, Western blot 检测证明多克隆抗体的特异性良好。结论: 成功表达、纯化HCBP12 融合蛋白, 并获得高特异性、高效价兔抗HCBP12 多克隆抗体, 为研究HCBP12 蛋白的生物学功能及丙肝的临床治疗提供了重要的实验工具。

关键词: HCBP12, 融合蛋白, 蛋白纯化, 多克隆抗体

Abstract: AIM: To express and purify HCV core binding protein ( HCBP12) with prokaryotic cell ex-pressive vector and to prepare HCBP12 specific rabbit polyclonal antibody .METHODS: The DNA segment of HCBP12 was amplified by RT-PCR with mRNA tem-plate from HepG2 cells and inserted into inducible pro-karyotic expressive vector pET-32a(+) .The recombi-nant plasmid, pET-32a(+)-HCBP12, was trans-formed into the competent E.coli BL21 .The inducible HCBP12 fusion protein was analyzed by SDS-PAGE and conformed by Western blot .Then the inducible HCBP12 protein w as purified and renatured by Ni⁺ affirity column chromatography. The purified HCBP12 ploteinwas used to immunize New Zea land rabbits for preparing polyclonal antibody. The specificity and potency of polyclonal antibody was evaluated by Western blot and ELISA .RESULTS: The HCBP12 fusion pro-tein was highly expressed and purified .The polyclonal antibody against HCBP12 was obtained successfully with the titer >1:512000 and confirmed specifically with Western blot .CONCLUSION: The successful ex-pression and purification of HCBP12 fusion protein and the preparation of specific anti-HCBP12 polyclonal an-tibody will be valuable for the study on the biological function of HCBP12 and clinical therapy of Hepatitis C .

Key words: HCBP12, fusion protein, protein pu-rification, polyclonal antibody

中图分类号:

李越, 王琦, 林原, 成军, 李康, 李华. HCBP12融合蛋白的表达、纯化及多抗制备[J]. 中国临床药理学与治疗学, 2008, 13(4): 425-430.

LI Yue, WANG Qi, LIN Yuan, CHENG Jun, LI Kang, LI Hua. Expression and purification of HCBP12 fusion protein and preparation of polyclonal antibody against HCBP12[J]. Chinese Journal of Clinical Pharmacology and Therapeutics, 2008, 13(4): 425-430.

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