人血白蛋白的原核表达及应用

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

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.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 Xiangrong^1，3 Feng Ruofei^1，2, Xie Jingying³, Tian Wei³, Yang Yanmei³, Wang Dan³, Ma Zhongren²

(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/Abstract

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

李向茸, 冯若飞, 谢晶莹, 田伟, 杨妍梅, 王丹, 马忠仁. 人血白蛋白的原核表达及应用[J]. 生物技术通报, 2013, 0(11): 130-135.

Li Xiangrong, Feng Ruofei, Xie Jingying, Tian Wei, Yang Yanmei, Wang Dan, Ma Zhongren. Prokaryotic Expression and Application of Recombinant Human Serum Albumin[J]. Biotechnology Bulletin, 2013, 0(11): 130-135.

参考文献

[1] Lawn RW, Adelman J, Bock SC, et al. The sequence of human serum albumin cDNA and its expression in E.coli[J]. Nucleic Acids Res, 1981, 9(22)：6103-6114.
[2] Goodey AR, Park M, Sleep D, et al. High purity albumin and method of producing[P]. US：5728553, 1998.
[3] Carter DC, He XM, Munsoon SH, et al. Three-dimensional structure of Human serum albumin[J]. Nature, 1989, 244(4909)：1195-1198.
[4] 郭美锦, 储炬, 杭海峰, 等.重组人血清白蛋白表达研究进展[J].生物工程进展, 2000, 20(5)：39-42.
[5] Kobayashi K, Nakamura N, Sumi A, et al. The development of recombinant human serum albumin[J]. Ther Aphe, 1998, 2(4)：257-262.
[6] Meloun B, Moravek L, Kostka V. Complete amino acid sequence of human serum albumin[J]. FEBS Lett, 1975, 58(1)：134-137.
[7] Saunders CW, Schmidt BJ, Mallonee RL, et al. Secretion of human serum albumin from Bacillus subtilis[J]. J Bacteriol, 1987, 169(7)：2917-2925.
[8] Kalman M, Cserpan I, Bajszar G, et al. Synthesis of a gene of human serum albumin and its expression in Saccharomyces cerevisiae[J]. Nucleic Acid Res, 1990, 18(20)：6075-6081.
[9] Quirk AV, Geisow MJ, Woodrow JR, et al. Production of recombinant human serum albumin from Saccharomyces cerevisiae[J]. Bio-technology Appl Biochem, 1989, 11(3)：273-287.
[10] Okabayashi K, Nakagawa Y, Hayasuke N, et al. Secretory express-ion of the human serum albumin gene in the yeast Saccharomyces cerevisiae[J]. J Biochem, 1991, 110(l)：103-110.
[11] Sijmons PC, Dekker BM, Schrammeijer B, et al. Production of correctly processed human serum albumin in transgenic plants[J]. Biotechnology, 1990, 8(3)：217-221.
[12] Shani M, Barash I, Nathan M, et al. Expression of human serum albumin in the milk of transgenic mice[J]. Transgenic Res, 1992, 1(5)：195-208.
[13] Ilan N, Barash I, Faerman A, et al. Dual regulation of Beta-lactoglobulin/human albumin gene expression by extracellular matrix in mammary cells from transgenic mice[J]. Exp Cell Res, 1996, 224(1)：28-38.
[14] Pieper FR, Wit IC, Pronk AC, et al. Efficient generation of functional transgenes by homologous recombination in murine zygotes[J]. Nucleic Acids Res, 1992, 20(6)：1259-1264.
[15] Ohtani W, Masaki A, Ikeda Y, et al. Structure of recombinant human serum albumin from Pichia pastoris[J]. Yakugaku Zasshi, 1997, 117(4)：220-232.
[16] 仇晴川, 杨代常.分子医药农业研究进展[J].武汉植物学研究, 2009, 27(1)：83-89.
[17] Ohtani W, Ohda T, Sumi A, et al. Analysis of Pichia pastoris components in recombinant human serum albumin by immunological assays and by HPLC with pulsed amperometric detection[J]. Anal Chem, 1998, 70(2)：425-429.
[18] Ohtani W, Nawa Y, Takeshima K, et al. Physicochemical and immunochemical properties of recombinant human serum albumin from Pichia pastoris[J]. Anal Biochem, 1998, 256(1)：56-62.
[19] 黄琛, 佘菲菲, 陈月秀, 等.幽门螺杆菌CagA蛋白肽段的基因克隆、表达及抗原性检测[J].福建医科大学学报, 2003, 37(1)：60-63.
[20] 张春艳, 李树浓, 宁波, 等.人CD154-GST融合蛋白基因在大肠杆菌中的表达[J].中国病理生理杂志, 2000, 16(8)：673-677.