抗FGF2人鼠嵌合抗体在HEK293T细胞中的表达优化

doi:10.13523/j.cb.20140503

中国生物工程杂志

2014, Vol. 34

Issue (5): 14-22 DOI: 10.13523/j.cb.20140503

研究报告

抗FGF2人鼠嵌合抗体在HEK293T细胞中的表达优化

王金胜¹, 姜浩武², 张晋霞¹, 潘磊¹, 赵凤芝³, 于云飞¹, 蔡亚雄¹, 邓宁¹

1 暨南大学生科院生物医学转化研究院广东省分子免疫与抗体工程研究中心广州 510632;
2 中国医学科学院/北京协和医学院基础医学研究所北京 100005;
3 暨南大学第一临床医学院肿瘤科广州 510632

Optimized Expression of a Mouse-human Chimeric Antibody Production in HEK 293T Cells Against Human FGF2

WANG Jin-sheng¹, JIANG Hao-wu², ZHANG Jin-xia¹, PAN Lei¹, ZHAO Feng-zhi³, YU Yun-fei¹, CAI Ya-xiong¹, DENG Ning¹

1 Guangdong Province Key Laboratory of Molecular Immunology and Antibody Engineering，Biomedicine Translational Institute in Jinan University, Guangzhou 510632, China;
2 Institute of Basic Medical Sciences Chinese Academy of Medical Sciences, School of Basic Medicine Peking Union Medical College, Beijing 100005, China;
3 Department of Oncology Department, First Affiliated Hospital of Jinan University, Guangzhou 510632, China

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摘要：

目的：FGF2是肿瘤血管新生过程中最重要的因子之一，因此通过制备抗FGF2人鼠嵌合抗体中和其发挥作用，以达到抑制肿瘤生长的目的。方法：利用分泌抗FGF2抗体的杂交瘤细胞株IgG9B9和人B淋巴细胞，分别克隆抗体轻链可变区V_L、重链可变区V_H和人重链恒定区C_H基因，从pComb3λ载体中扩增出人λ 链恒定区C_L基因，通过重叠PCR，将V_L，V_H和C_L，C_H片段分别连接形成嵌合抗体的轻链L和重链H，将L/H链以单独构建或串联于同一载体的方式，构建抗FGF2嵌合抗体表达载体，并通过调控元件WPRE优化载体、共转染促生长因子aFGF以及调整表达温度等方式提高嵌合抗体在真核细胞中的表达。结果：成功构建了优化表达载体PLexm-WPRE、PLexm-aFGF；L、H链基因也成功构建，并以L、H或L-F2A-H（2A连接肽将L和H连接起来）的方式分别成功连接到PLexm，PLexm-WPRE载体中。转染细胞上清的ELISA鉴定结果表明，L/H链单独构建要比串联构建的方式具有更高的表达水平，WPRE能有效促进抗体的表达而aFGF并不能促进其表达，与31、37℃相比，33℃时抗体的表达量最高，同时嵌合抗体表现出了很好的结合活性及中和活性，竞争IC50=6.25μg/ml。通过亲和层析获得了高纯度的抗FGF2嵌合抗体。结论：在33℃下，人鼠嵌合抗体基因在WPRE存在下表达量最高，且与抗原FGF2有很好的结合活性及中和活性，为临床应用奠定了基础。

关键词： 碱性成纤维生长因子; 嵌合抗体; HEK293T细胞; 真核表达载体

Abstract:

FGF2 is one of the most important factors in tumor angiogenesis. Blocking FGF-2/FGFR activity with antibodies could be proposed as a potential therapeutic strategy, especially in the treatment of tumors. A mouse anti-FGF2 monoclonal antibody (mAb), designated as 9B9, has previously been produced and characterized by our laboratory. We report here the construction and the optimized expression of mouse-human chimeric antibody derived from the mAb. cDNAs encoding variable regions of heavy and light chains were prepared from 9B9 cells by polymerase chain reaction, and introduced to eukaryotic expression vectorsp Lexm containing cDNA for human gamma1 constant regions, respectively. Cotransfection of the vectors into HEK 293T cells resulted in production of antibody and detected by western blots of reduced and non-reduced SDS-PAGE. The chimeric antibody has a special binding activity to FGF2 and can inhibit the binding of FGFR1βⅢc to immobilized FGF2, (IC50=6.25μg/ml) in ELISA. On the other hand, we optimized the recombinant plasmids through woodchuck hepatitis virus post-transcriptional regulation element (WPRE) and acidic Fibroblast Growth Factor (aFGF) to improve chimeric antibody yelid. The results suggest that WPRE enhanced expression of the recombinant antibody, while aFGF can not effectively promote the expression of chimeric antibody, and with the increase of aFGF, antibody expression quantity reduces instead. Furthermore, recombinant antibody yield was enhanced 12- to 18-fold compared to levels at 37 ℃ by exposure of cells to mild hypothermia (31℃ to 33℃) after transfection. This simple and efficient approach of antibody production is expected to provide a sufficient amount of antibody for screening experiments.

Key words: FGF-2 Chimeric antibody HEK293T cell Eukaryotic expression vector Optimization

收稿日期: 2014-03-18 出版日期: 2014-05-25

ZTFLH:

Q789

基金资助:

中央高校基本科研业务费专项资金（21612112）资助项目

通讯作者: 邓宁 E-mail: tdengn@jnu.edu.cn

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王金胜, 姜浩武, 张晋霞, 潘磊, 赵凤芝, 于云飞, 蔡亚雄, 邓宁. 抗FGF2人鼠嵌合抗体在HEK293T细胞中的表达优化[J]. 中国生物工程杂志, 2014, 34(5): 14-22.

WANG Jin-sheng, JIANG Hao-wu, ZHANG Jin-xia, PAN Lei, ZHAO Feng-zhi, YU Yun-fei, CAI Ya-xiong, DENG Ning. Optimized Expression of a Mouse-human Chimeric Antibody Production in HEK 293T Cells Against Human FGF2. China Biotechnology, 2014, 34(5): 14-22.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20140503 或 https://manu60.magtech.com.cn/biotech/CN/Y2014/V34/I5/14

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