一种融合抗体ScFv-Fc通用表达载体的构建

中国生物工程杂志

2011, Vol. 31

Issue (8): 110-117

技术与方法

一种融合抗体ScFv-Fc通用表达载体的构建

王丁丁¹, 苏曼曼², 胡丽莉¹, 袁丽颖⁴, 孙艳¹, 汪炬³, 颜炜群²

1. 广东药学院生命科学与生物制药学院广州 510006;
2. 吉林大学药学院长春 130021;
3. 暨南大学生命科学技术学院广州 510632;
4. 吉林大学附属第三幼儿园长春 130021

A Vector System for the Production of Single-chain Fv-Fc Fusions in Pichia pastoris

WANG Ding-ding¹, SU Man-man², HU Li-li¹, YUAN Li-ying⁴, SUN Yan¹, WANG Ju³, YAN Wei-qun²

1. Institute of Life Science and Biological Pharmacy, Guangdong Pharmaceutical University, Guangzhou 510006, China;
2. College of Pharmaceutical, Jilin University, Changchun 130021, China;
3. College of Life Science and Technology, Jinan University, Guangzhou 510632, China;
4. The 3rd Kindergarten Affiliated to Jilin University, Changchun 130021, China

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摘要：

为了构建一个可供自由替换的ScFv区,表达人小分子融合抗体ScFv-Fc的通用载体,利用RT-PCR技术扩增人抗体IgG1的Fc片段克隆至毕赤酵母表达载体pPICZα,将一段人工合成的互补寡核苷酸链插入重组载体pPICZα/Fc中Fc区的上游,引入2个可供小分子抗体ScFv-Fc的ScFv区自由替换的限制性酶切位点。分别扩增人抗狂犬病毒以及抗乙型肝炎表面抗原的ScFv片段,克隆至已构建的通用载体pPICZα/Fc,在毕赤酵母中诱导表达。进一步在1L条件下对活性抗体进行发酵,并利用protein A亲和层析柱进行纯化。应用酵母基因组PCR、ELISA、Western blotting、活性检测等试验对此小分子抗体的表达进行生物学及免疫学分析。结果表明具有狂犬病毒抗原结合活性以及乙肝表面抗原结合活性的人源抗体分子均获得成功表达,1L发酵条件下表达量达到20~30mg/L, protein A亲和层析纯化后纯度＞95%。研究构建了可用于功能性抗体分子ScFv-Fc筛选和表达的通用载体并对其发酵、纯化条件进行了摸索,为重组抗体分子诊断、治疗试剂的开发以及抗体的人源化奠定了物质基础。

关键词： 融合抗体; ScFv-Fc; 通用载体; 表达; 毕赤酵母

Abstract:

Recombinant antibodies, especially ScFv fragments, can be applied as detection reagents and even substitute for some reagents used in immunoassays such as antibody-enzyme conjugates. For ScFv fragments, a universal system available is necessary. A vector system was constructed based on pPICZα/Fc, in which the hinge, CH2 and CH3 domains (Fc fragment) of human IgG1 and His-tag were cloned into the Pichia expression vector pPICZα. Two fragments of ScFv were introduced into pPICZα/Fc, which can bind HBsAg and rabies virus antigen, to yield the expression cassette pPICZα/ScFv-Fc. Following fermentation in a 1-liter reactor, the fusions were expressed at high levels in the methylotrophic yeast Pichia pastoris, secreted as dimeric forms in the culture, and purified by protein A column chromatography. The expression yield can reach 20~30mg/L of culture medium. The ScFv-Fc fusion proteins retain the biological binding ability of the parent ScFv. Furthermore, the successful expression and maintenance of the binding activity verify the efficacy of the vector system for use as detection reagents in vitro, by reacting with the specific antigens and being readily detected using general anti-human IgG antibodies.

Key words: Fusion protein ScFv-Fc Vector system Expression Pichia pastoris

收稿日期: 2011-06-10 出版日期: 2011-08-25

ZTFLH:

Q81

基金资助:

国家自然科学基金青年基金资助项目(81000923)。

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引用本文:

王丁丁, 苏曼曼, 胡丽莉, 袁丽颖, 孙艳, 汪炬, 颜炜群. 一种融合抗体ScFv-Fc通用表达载体的构建[J]. 中国生物工程杂志, 2011, 31(8): 110-117.

WANG Ding-ding, SU Man-man, HU Li-li, YUAN Li-ying, SUN Yan, WANG Ju, YAN Wei-qun. A Vector System for the Production of Single-chain Fv-Fc Fusions in Pichia pastoris. China Biotechnology, 2011, 31(8): 110-117.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2011/V31/I8/110

[1] Skerra A, Pluckthun A. Assembly of a functional immunoglobulin Fv fragment in Escherichia coli. Science,1988,240:1038-1041.

[2] Better M, Chang C P, Robinson R R, et al. Escherichia coli secretion of an active chimeric antibody fragment. Science,1988,2(40):1041-1043.

[3] Desplancq D, Rinaldi A, Stoessed A, et al. Single-chain Fv fragment antibodies selected from an intrabody library as effective mono-or bivalent reagents for in vitro proten detection. J immunol Methods, 2011, (in print).

[4] Sakamoto S, Taura F, Tsuchihashi R, et al. Expression, purification, and characterization of anti-plumbagin single-chain variable fragment antibody in Sf9 insect cell. Hybridoma (Larchmt), 2010,29(6):481-488.

[5] Sandrine M, Ahmed E M, Ole V, et al. A multi-Fc-species system for recombinant antibody production. BMC Biotechnology, 2009, 9, Article ID 14.

[6] Yan W Q, Wang D D. Recombinant antibody against rabies antigen. China, 200710055307.1.

[7] Sambrook J, Russell D. Molecular Cloning: A Laboratory Manual, 2001. 663-667.

[8] Bradford M M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal Biochem, 1976,72:248-254.

[9] Stish B J, Chen H, Shu Y, et al. Increasing anticarcinoma activity of an anti-erbB2 recombinant immunotoxin by the addition of an anti-EpCAM sFv. Clin Cancer Res, 2007,13(10):3058-3067.

[10] Hamada T S, Suzuki K, Akahori Y. Antibody-dependent cell-mediated cytotoxicity is induced by a single-chain Fv-protein III fusion in the presence of a rabbit anti-protein III polyclonal antibody. Immunol Lett, 2011,136(1):44-48.

[11] Niculescu-/duvaz I, Springer C J. Antibody-dircted enzyme prodrug therapy (ADEPT): a review. Adv Drup Deliv Rev, 1997,26:151-172.

[12] Chamow S M, Ashkenazi A. Immunoadhesins: principles and app1ication. TIBTECH, 1996, 14: 52-60.

[13] Fischer R, Drossard J, Emans N, et al. Towards molecular farming in the future: Pichia pastoris-based production of single-chain antibody fragments. Biotechnol Appl Biochem, 1999, 30:117-120.

[14] Shapiro R I, Wen D, Levesque M, et al. Expression of Sonic hedgehog-Fc fusion protein in Pichia pastoris. Identification and control of post-translational, chemical, and proteolytic modifications. Protein Expr Purif, 2003,29(2):272-283.

[15] Cregg J M, Vedvick T S, Raschke W C. Advances in the expression of foreign genes in Pichia pastoris. Biotechnology, 1993,11:905-910.

[16] Jafari R, Sundstrom B E, Holm P. Optimization of production of the anti-keratin 8 single-chain Fv TS1-218 in Pichia pastoris using design of experiments. Microb Cell Fact, 2011,10(1):34-41.

[17] Gurkan C, Symeonides S N, Ellar D J. High-level production in Pichia pastoris of an anti-p185HER-2 single-chain antibody fragment using an alternative secretion expression vector. Biotechnol Appl Biochem, 2004,39(1):115-122.

[18] Schoonooghe S, Kaigorodov V, Zawisza M, et al. Efficient production of human bivalent and trivalent anti-MUC1 Fab-scFv antibodies in Pichia pastoris. BMC Biotechnol, 2009,9:70-84.

[19] Yamawaki S, Matsumoto T, Ohnishi Y, et al. Production of single-chain variable fragment antibody (scFv) in fed-batch and continuous culture of Pichia pastoris by two different methanol feeding methods. J Biosci Bioeng, 2007,104(5):403-407.

[20] Jafari R, Holm P, Piercecchi M, et al. Construction of divalent anti-keratin 8 single-chain antibodies (sc(Fv)(2)), expression in Pichia pastoris and their reactivity with multicellular tumor spheroids. J Immunol Methods, 2011,364(1-2):65-76.

[21] Damasceno L M, Pla I, Chang H J, et al. An optimized fermentation process for high-level production of a single-chain Fv antibody fragment in Pichia pastoris. Protein Expr Purif, 2004, 37(1):18-26.

[22] Freyre F M, Vazquez J E, Ayala M, et al. Very high expression of an anti-carcinoembryonic antigen single chain Fv antibody fragment in the yeast Pichia pastoris. J Biotechnol, 2000,76(2-3):157-163.

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