生物技术通报 ›› 2013, Vol. 0 ›› Issue (11): 130-135.

• 研究报告 • 上一篇    下一篇

人血白蛋白的原核表达及应用

李向茸1,3, 冯若飞1,2, 谢晶莹3, 田伟3, 杨妍梅3, 王丹3, 马忠仁2   

  1. 1.西北民族大学生物工程与技术国家民委重点实验室,兰州 730030;2.甘肃省动物细胞工程技术研究中心,兰州 730030;3.西北民族大学生命科学与工程学院,兰州 730030
  • 收稿日期:2013-05-12 出版日期:2013-11-14 发布日期:2013-11-14
  • 作者简介:李向茸,女,硕士研究生,研究方向:生物制品的研发与质量控制;E-mail:491753586@qq.com

Prokaryotic Expression and Application of Recombinant Human Serum Albumin

Li Xiangrong1,3 Feng Ruofei1,2, Xie Jingying3, Tian Wei3, Yang Yanmei3, Wang Dan3, Ma Zhongren2   

  1. (1. The Key Bio-engineering and Technology Laboratory of Nationality Commission,Northwest University for Nationalities,Lanzhou 730030;2. Gansu Engineering Research Center for Animal Cell,Lanzhou 730030;3. Life Science and Engineering College of Northwest University for Nationalities,Lanzhou 730030)
  • Received:2013-05-12 Published:2013-11-14 Online:2013-11-14

摘要: 旨在构建人血白蛋白(HSA)基因的原核表达载体pGEX-4T-1-rHSA,诱导表达后纯化重组HSA蛋白,制备多克隆抗体并对其进行鉴定。根据GenBank发表的HSA(NM_000477.5)的核苷酸序列设计合成1对特异性引物,以人胚肺二倍体细胞的总RNA为模板,利用RT-PCR扩增HSA基因,将其克隆至pGEX-4T-1载体,构建重组原核表达质粒pGEX-4T-1-rHSA,经PCR、酶切及测序鉴定正确后转化大肠杆菌BL21(DE3),进行IPTG诱导表达,表达产物经变性、复性、亲和层析纯化后作为免疫原制备多克隆抗体,应用间接ELISA和Western-blot方法对所得抗体进行检测。结果表明,重组蛋白主要以包涵体的形式存在,分子质量为92 kD;重组蛋白具有良好的反应原性和免疫原性;应用其制备的抗血清效价达到1:100 000;纯化的多克隆抗体与血源人血清白蛋白和重组人血白蛋白均能产生特异性结合,具有良好的生物学活性。

关键词: 人血清白蛋白, 原核表达, 包涵体, 多克隆抗体

Abstract: This study was aimed to express and purify the protein of pGEX-4T-1-rHSA in prokaryocytes and prepare anti-HSA polyclonal antibody. The HSA gene was amplified from human embryo lung diploid total RNA by RT-PCR, with primers based on the published HSA(NM_00047.5)sequence in GenBank, then it was cloned into prokaryotic expression vector pGEX-4T-1 to construct a recombinant expression vector pGEX-4T-1-rHSA. The pGEX-4T-1-rHSA was transformed into E. coli BL21(DE3). The purified expressed protein was injected into a rabbit and the polyclonal antibody was then prepared, which were identified by the indirect ELISA and Western-blot analysis. Results showed that the recombinant protein mainly exists in the inclusion body, about 92 kD and it has good reactogenicity and immunogenicity. The ELISA titer of the antiserum was approximately 1:100 000. Western-blot analysis revealed that the purified polyclonal antibody against pGEX-4T-1-rHSA had a specific affinity for natural human serum albumin and the expressed protein.

Key words: Human serum albumin, Prokaryotic expression, Inclusion body, Polyclonal antibody