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| Effect of Chimeric Intron on the Expression of Anti-bFGF Antibody Genes in 293T Cells |
| Antibody Engineering Center of Ji’nan University, Guangzhou 510632, China |
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Abstract Objective: To construct eukaryotic expression vectors containing genes of chimeric intron and anti-bFGF antibody and express in 293T cells for exploring the effect of chimeric intron on antibody expression. Methods: Genes of chimeric intron and Fd、κ chain were amplified from vectors pCl-neo and pComb3-Fab25 respectively, then inserted in different combinations into expression vector pIgG containing human immunoglobulin IgG1 Fc region. Recombinant plasmids were transfected into 293T cells by liposome polyethyleneimine(PEI). Transfection result was observed by fluorescence microscope and antibody expression was analyzed by sandwich ELISA and Western blot. Results: The results of sequencing and restriction enzyme digestion analysis showed that intron and antibody genes were successfully inserted into expression vector pIgG. Fluorescence observations indicated that plasmids were transfected into 293T cells and antibody in culture supernatant was detected by Western blot. Antibody concentration of pIgG-Fd-κ、pIgG-Fd-intron-κ、pIgG-Fd-κ-intron and pIgG-Fd-intron-κ-intron was 1.21mg/L、0.468mg/L、7.39mg/L、0.601mg/L respectively. Conclusion: Recombinant expression vectors containing chimeric intron and antibody genes were constructed and expressed in 293T cells successfully and the chimeric intron may conduct important action. Antibody expression can be improved by inserting chimeric intron into 5’ upstream of κ chain, but depressed into 5’ upstream of Fd.
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Received: 18 December 2009
Published: 25 March 2010
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