人源DKK1真核悬浮分泌表达系统的建立及其特异性抗体的制备和鉴定

Production of human DKK1 protein from an eukaryotic secreted expression system and the preparation and identification of its specific antibody

目的:利用哺乳动物细胞悬浮培养表达系统分泌表达人源Dickkopf-1(DKK1)蛋白,纯化蛋白并制备特异性抗体。方法:构建DKK1真核表达载体pCMVcStrep-DKK1,并借助脂质体将载体瞬时转染入Free-Style293-F细胞(无血清培养),ELISA和蛋白印迹法检测DKK1蛋白表达量。利用亲和层析法纯化DKK1蛋白,Wnt信号通路荧光素酶报告系统检测其生理活性。以纯化的DKK1蛋白免疫BALB/c小鼠获得相应抗体,并初步鉴定其抗原特异性。结果:DKK1蛋白分泌表达在Free-Style293-F细胞培养液中,蛋白浓度在转染后第5天达最高(20mg/L)。所纯化的DKK1蛋白能够抑制Wnt3a蛋白诱导的报告基因的表达。抗DKK1小鼠抗体能特异性识别细胞内源性DKK1蛋白。结论:建立了在哺乳动物细胞悬浮培养系统中高效分泌表达人源DKK1重组蛋白的表达系统,并获得相应的特异性抗体,为其下一步结构与功能研究及在肿瘤中的应用奠定了实验基础。

Objective： To obtain and purify the human Dickkopf-1 （DKK1） protein expressed from an eukaryotic secreted expression system, and to prepare mouse antibody against human DKK1 protein. Methods： The DNA sequence of human DKK1 gene was amplified by PCR and subcloned into an eukaryotic expression vector pCMVcStrep. The recombined plasmid was transiently transfected by lipofectin regeant into Free-Style 293-F cells, which were grown in a serum-free suspension culture. The expression of secreted DKK1 protein was measured by ELISA and Western blotting. DKK1 protein was purified by affinity chromatography, and the biological activity was detected by its capability to inhibit Wnt-activated luciferase activity. Then the BALB/c mice were immunized with the purified DKK1 protein for antibody production, and the specificity of the antibodies was determined by ELISA and Western blotting. Results： DKK1 protein was expressed and secreted in the culture medium of Free-Style 293-F cells, and its peak yield （20 mg/L） was obtained 5 days after transfection. The purified DKK1 protein was able to inhibit Wnt3a protein-induced reporter activity in a dose-dependent effect. The mouse specific antibody against human DKK1 protein could bind specifically to endogenous DKK1 protein. Conclusion： High quantity of DKK1 protein can be obtained from the suspension expression system, and the mouse specific antibody against human DKK1 protein can be successfully prepared.