分泌表达bFGF的中国仓鼠卵巢(CHO)细胞的构建及其特性分析

CHO细胞在无血清或无蛋白培养条件下培养通常会遇到贴壁能力差,细胞活力差等问题。通过构建分泌型bFGF基因,克隆到pIRESneo3表达载体上,转染CHO细胞,通过MTT法间接检测细胞培养上清中bFGF表达,并在无蛋白培养基中观察细胞的生长。结果显示转染的CHO细胞表达bFGF,且分泌的bFGF有生物活性;转染的CHO细胞在无蛋白培养基中较未转染的CHO细胞的贴壁能力和活力强。成功改造了CHO细胞,为CHO细胞在无血清或无蛋白条件下大规模培养提供了基础。

Reconstruction and Characterization of CHO Cells Transfected bFGF Gene

Lacking of various celluar growth factors, Chinese hamster ovary(CHO) cells can not grow normally, with poor adhesion and weak activity in serum-free or protein-free medium. Some researches about mediums have been done. A strategy was used, and transfected CHO cells with basic fibroblast growth factor (bFGF) gene could secrete bFGF into medium and have an autocine or paracrine regulation for CHO cells. A bFGF nucleotides, of which nine residues from the amino-terminus of wild-type FGF-2 were replaced with eight amino acid residues of signal peptide of FGF-4, was cloned into pIRESneo3 vector, and then transfected into CHO cells. The positive CHO clones were screened with G418 and the culture medium was assayed for the mitogenic activity of bFGF by MTT method. The behavior of the positive CHO cells cultured in protein-free medium were observed. The results showed that the positive CHO cells could secrete bFGF with mitogenic activity, and could grow normally in protein-free medium, with good adhesion and normal bioactivity, while control CHO cells could not. A foundation for the large-scale culture of CHO cells in serum-free or protein-free medium was laid.