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Optimization of Transient Expression Conditions of Anti-bFGF Antibody Gene in 293T Cell

Author: GongYiPing
Tutor: DengNing；WangHong
School: Jinan University
Course: Biochemistry and Molecular Biology
Keywords: bFGF antibody expression condition optimization chimeric intron
CLC: Q78
Type: Master's thesis
Year: 2010
Downloads: 77
Quote: 0
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Abstract


Aim:To construct eukaryotic expression vectors of human anti-bFGF antibody, optimize the soluble expression conditions of anti-bFGF antibody in 293T cells.Methods:Gene fragments of Fd、κchain were amplified from vectors pComb3-Fab respectively, and then were inserted into expression vectors pIgG、pIRES、pN(A+B) and pA(X+A). The recombinant plasmids were transfected into 293T cells by liposome polyethyleneimine (PEI) and the different transfection conditions were optimized by means of vector pEGFP. Antibody expression was analyzed by indirect ELISA. Decreasing temperature, inserting chimeric intron and adding deacetylase inhibitors were used to improve antibody expression in 293T cells.Results:The results of sequencing and restriction enzyme analysis showed that the antibody genes were successfully inserted into expression vectors pIgG、pIRES and pN(A+B)、pA(X+A). The GFP expression level showed the best expression condition:PEI/DNA ratio was 4:1, medium serum concentration was 2%, plasmid amount was 1μg, cells was treated with 25μmol/L chloroquine, time of transfection complex and cells was 6h.The result of antibody concentration detected by sandwich ELISA showed that vector pIgG showed the strongest expression ability and antibody concentration was 2.1mg/L. Antibody level arised from 2.1mg/L to 12.3mg/L by inserting chimeric intron at upstream ofκchain 5’terminal, decreasing expression temperature(33℃) and adding valproic acid(4mmol/L) to medium.Conclusions:Recombinant expression vectors were constructed, the antibody gene was expressed in 293T cells and antibody concentration was 12.3mg/L.

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