不同质粒载体对GFP单基因瞬时转染ExpiCHO-S细胞表达量的影响

Effects of Vectors on Transient Expression of GFP in CHO Cells

【目的】ExpiCHO细胞瞬时表达能够快速有效地生产重组蛋白,为筛选有效的重组蛋白节省了大量的时间,细胞系的选择、表达载体的选择、转染试剂的选择等是制约ExpiCHO细胞瞬时表达蛋白的重要因素,本研究旨在筛选出在CHO细胞中瞬时表达量较高的载体。【方法】以GFP基因作为目的基因,用不同的真核表达载体构建GFP重组质粒,瞬时转染ExpiCHO-S细胞。转染后4 d观察不同载体表达GFP的荧光数量和强度;转染后8 d对细胞进行裂解,收获裂解上清,用SDS-PAGE对不同载体的表达量进行鉴定。同时用His Trap FF亲和层析柱对裂解上清中的GFP蛋白进行纯化,通过Western Blot比较不同载体GFP蛋白的表达量。【结果】pCDNA3.1-GFP、pCDNA3.4-GFP载体表达的荧光数量最多,pCIneo-GFP载体表达的荧光强度最强,pCMVHA载体表达的荧光数量最少,荧光强度最弱。SDS-PAGE和Western Blot结果均表明pCDNA3.1-GFP、pCDNA3.4-GFP、pCIneo-GFP重组质粒的蛋白表达量高于pCHO-GFP、pCMVHA-GFP重组质粒的蛋白表达量。【结论】筛选出表达量较高的真核表达载体pCDNA3.1、pCDNA3.4、pCIneo,为后续重组蛋白的瞬时表达载体选择提供依据。

【Objective】Appropriate cell line,expression vector,and transfection reagent were selected to enhance the transient expression of GFP recombinant protein for quick and efficient production of the proteins in CHO cells.【Methods】Targeting GFP,various eukaryotic vectors were employed to carry the gene protein for transfection into ExpiCHO-S cells.Four days after transfection,number and intensity of expression fluorescence on the recombinant plasmids by different vectors were recorded.Four more days later,the cells were lysed,and the lysate supernatant harvested to verify the gene expressions by SDS-PAGE.Meanwhile,GFP recombinant plasmids in the supernatant were purified on a His Trap FF affinity chromatography column to quantify the protein expressions by western blot.【Results】The recombinant plasmids,pCDNA3.1-GFP and pCDNA3.4-GFP had the expression fluorescence the greatest in number,pCIneo-GFP the highest on intensity,while pCMVHA the fewest in number and the lowest on intensity.SDS-PAGE and western blot showed the expressions of the first 3 recombinant plasmids were higher than those of pCHO-GFP or pCMVHA-GFP.【Conclusion】The GFP recombinant plasmids constructed with the eukaryotic vectors,pCDNA3.1,pCDNA3.4,and pCIneo,exhibited the greatest expressions and were considered the choice vectors for future studies requiring transient expression.