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| 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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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.
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Received: 18 March 2014
Published: 25 May 2014
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