rhVEGF165在中国仓鼠卵巢细胞中的稳定表达、纯化及其生物学活性

Stable expression of rhVEGF165 in Chinese hamster ovary cells

摘要:

利用哺乳动物细胞表达系统，稳定表达和纯化高生物学活性的人重组血管内皮生长因子 (VEGF165) 蛋白。将VEGF165克隆于表达载体pCDNA4.0，与T-GS载体共同转染CHO-S (中国仓鼠卵巢细胞) 细胞，MSX (Methionine sulphoximine) 加压筛选高表达细胞株，5 L发酵罐培养，细胞培养上清液通过三步纯化得到rhVEGF165蛋白，通过Western blotting、Biacore和人脐静脉内皮细胞增殖实验等对表达蛋白的特异性、亲和力及生物学活性等进行检测。所建立的细胞

关键词:血管内皮生长因子 (VEGF165)，中国仓鼠卵巢细胞，重组蛋白稳定表达，生物反应器，rhVEGF165蛋白纯化

Abstract:

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.

Key words:rhVEGF165, Chinese hamster ovary (CHO) cell, stable expression, bioreactor process, rhVEGF165 purification