融合蛋白基因与抗体基因电转染CHO-S细胞的条件摸索优化

Optimization of Electrotransfection Conditions of Genes for Fusion Protein and Antibody to CHO-S Cells

旨在对编码融合蛋白和抗体的重组质粒转染CHO-S细胞的转染条件进行摸索优化,提高外源基因的转染效率,从而增加外源蛋白的表达。应用Amaxa Nucleofector-II电转仪,从电转染程序、质粒用量及细胞用量三方面着手,最终发现编码融合蛋白的重组质粒的最佳电转条件为:电转程序U-030,质粒用量20μg/孔,细胞用量1×10~7个/孔;编码抗体的重组质粒的最佳电转条件为:电转程序U-024,质粒用量15μg/孔,细胞用量1×10~7个/孔;实验结果进一步显示抗体重组质粒电转染CHO-S细胞的效果要优于融合蛋白重组质粒,利用高通量细胞筛选仪Clone Pix对转染后重组细胞的阳性克隆数进行统计,证实了该抗体重组质粒的转染效率更高。这为以后的抗体及融合蛋白重组质粒电转染CHO-S细胞提供了一定的数据支持,同时提示不同类型的细胞及外源基因,都需要设计相应的电转染实验进行条件优化,进而为获得高产稳定的生产用细胞株奠定基础。

The objective of this work is to optimize the conditions while recombinant plasmids encoding Fc-fusion protein and antibody transfect into CHO-S cells for improving the transfection efficiency and subsequently increasing the expression of exogenous proteins.The target genes were transfected into the CHO-S cells with Amaxa Nucleofector-II and the transfection conditions were screened from the 3 perspectives of electrotransfection programs,plasmid dosage and cell dosage.The optimized conditions for the Fc-fusion protein were found to be electroporation program U-030,plasmid dosage 20μg/well,and cell dosage 1×10^7 cells/well;while the optimized conditions for the antibody were electroporation program U-024,plasmid dosage 15μg/well,and cell dosage 1×10^7 cells/well.It was found that the transfection efficiency for the gene of the antibody was higher than that for the gene of Fc-fusion protein,which was also demonstrated by the ratio of positive clone analyzed with Clone Pix.This study provides data on electrotransfection of genes for antibody and Fc-fusion protein to CHO-S cells,also indicating that the importance of optimizing the electroporation conditions for different cell lines and target genes for obtaining stable and highyield producing cell lines.