重组人红细胞生成素二聚体工程细胞的建立

Construction of Engineering Cell Strain of Recombinant Human Dimeric Erythropoietin

目的建立重组人红细胞生成素二聚体工程细胞株。方法用重组人红细胞生成素二聚体表达载体转染COS-7细胞,通过dot blot进行检定。然后对CHO-dhfr-细胞进行稳定转染,再经过稀释克隆和MTX的加压扩增,筛选二聚体工程细胞株并进行传代及无血清培养基培养、目的蛋白纯化及检测。结果瞬时转染70h细胞有目的蛋白表达。稳定转染后选取表达量最高的细胞株为工程细胞株。该细胞株连续传15代及经无血清培养基培养,目的蛋白表达量均无明显下降。所表达的目的蛋白相对分子质量为70000,并具有高度特异性。结论已获得稳定高效表达重组人红细胞生成素二聚体的工程细胞株。

Objective To establish the engineering cell strain of recombinant human dimeric erythoropoietin. Methods Transiently transfect COS-7 cells with expression vector pBT-2s of recombinant human dimeric erythropoietin and identify by dot blot. Perform stable transfection test on the expression vector pBT-2s with CHO-dhfr^- cells, and screen the engineering cell strain of recombinant human dimeric erythropoietin by dilution cloning and MTX pressure screening for subculture and culture in serum-free medium. Purify the expressed product and identify by SDS-PAGE and Western blot. Results Target protein was expressed in the CHO-dhfr^- cells 70 h after transient transfection. After stable transfection, the cell strain with the highest expression of target protein was served as engineering cell strain. The expression of target protein showed no significant decrease in the engineering cell strain subcuhured for 15 passages or cultured in serum-free medium. The expressed protein with a relative molecular weight of 70 000 showed high specificity. Conclusion An engineering cell strain was established by transfection of CHO-dhfr^- ,dilution cloning and MTX pressure screening,and may be used for stable and high expression of human dimeric erythropoietin.