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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 purify protein and prepare specific antibody by using mammalian cell suspension culture expression system to secrete and express human Dickkopf-1 (DKK1) protein. METHODS: DKK1 eukaryotic expression vector pCMVcStrep-DKK1 was constructed and transfected into Free-Style293-F cells by lipofectamine 2000 without serum. The expression of DKK1 protein was detected by ELISA and Western blotting. The DKK1 protein was purified by affinity chromatography and the Wnt signaling pathway luciferase reporter system was used to detect its physiological activity. BALB / c mice were immunized with the purified DKK1 protein to obtain the corresponding antibodies, and the antigen specificity was initially identified. RESULTS: DKK1 protein was secreted in Free-Style293-F cell culture medium and the protein concentration was highest (20mg / L) on the 5th day after transfection. The purified DKK1 protein can inhibit the Wnt3a protein-induced reporter gene expression. Anti-DKK1 mouse antibodies specifically recognize cellular endogenous DKK1 protein. CONCLUSION: A highly efficient expression system of human DKK1 recombinant protein in mammalian cell suspension culture system was established, and the corresponding specific antibodies were obtained, which laid the experimental foundation for the further study of its structure and function and its application in tumors .