Zhang Ganliang, Zhang Haifeng, Nian Weihong, Sun Jiuru
Genor Biopharma Co. Ltd., Shanghai 201203, China.
Sheng Wu Gong Cheng Xue Bao. 2011 Jun;27(6):935-42.
We established a stable Chinese hamster ovary (CHO-S) cell line for recombinant human VEGF165-expressing. We co-transfected GS-expression vector and rhVEGF165 expression plasmid into CHO-S cells, and selected the highest VEGF165-expressing clone as the working cell line to express VEGF165 protein. After 7-day fed-batch culture in a 5 L bioreactor and 3 steps chromatographic purification, we got the rhVEGF165 protein for series of binding and biological activity examination. The production was over 50 mg/L. The purified rhVEGF165 protein was functionally active with a half-maximal Human Umbilical Vein Endothelial Cells (HUVEC) growth-enhancing effect concentration of 1.94 ng/mL. It was slightly better than commercially available Escherichia coli expressing rhVEGF165. So we expressed successfully rhVEGF165 protein in high-level and obtained the fully active rhVEGF165 protein in large quantity.
我们建立了一个稳定的用于表达重组人VEGF165的中国仓鼠卵巢(CHO-S)细胞系。我们将GS表达载体和rhVEGF165表达质粒共转染到CHO-S细胞中,并选择VEGF165表达量最高的克隆作为工作细胞系来表达VEGF165蛋白。在5L生物反应器中进行7天的补料分批培养并经过三步色谱纯化后,我们得到了用于一系列结合和生物活性检测的rhVEGF165蛋白。产量超过50mg/L。纯化后的rhVEGF165蛋白具有功能活性,其对人脐静脉内皮细胞(HUVEC)生长增强作用的半数有效浓度为1.94ng/mL。其效果略优于市售的大肠杆菌表达的rhVEGF165。因此,我们成功地高水平表达了rhVEGF165蛋白,并大量获得了具有完全活性的rhVEGF165蛋白。