Objective To clone,express, purify and verify human Dickkopfl (DKK1). Methods The cervical cancer cells from cervical cancer tissue were cultured, and mRNA was harvested from the fifth generation. The DKK1 gene was acquired via RT-PCR and cloned in pCMV-HA2 plasmid containing the HA tag to construct the recombinant plasmid pCMV-HA2/DKKl. The recombi-nant plasmid was identified by restriction enzyme cutting and sequence analysis. The recombined plasmid was transiently transfected by lipofectin regent into Free-Style 293-F cells, which were grown in a serum-free suspension culture. The expression of DKK1 protein was measured by Western blotting. Results The product of RT-PCR was coincide with what we preconceived. Restriction enzyme EcoR I cutting and sequence analysis proved the recombinant plasmid to be pCMV-HA2/DKKl. The product of expression was verified properly via Western Blot with special anti-DKKl antibody. Conclusion The successful cloning of DKK1 gene and expression of DKK1 protein lay a basis for studying the effects on the biologic activity of tumorigenesis.%目的 构建人DKK1基因的真核表达质粒,表达、纯化、鉴定其表达产物.方法 自宫颈癌组织提取癌细胞,抽提mRNA,RT-PCR获得人DKK1基因片断,构建重组质粒pCMV-HA2/DKK1,EcoRⅠ酶切、测序鉴定重组质粒;借助脂质体将载体瞬时转染入Free-Style 293-F细胞(无血清培养)蛋白印迹法检测DKK1蛋白.结果 RT-PCR获得片断与预期结果相符,酶切鉴定、测序鉴定证实pCMV-HA2/DKK1重组质粒构建成功;DKK1蛋白分泌表达在Free-Style 293-F细胞培养液中,Western Blot鉴定表达产物为DKK1蛋白.结论 成功构建了人DKK1的真核表达质粒,并对该基因的真核表达产物进行鉴定,为下一步的DKK1蛋白对肿瘤发生中的生物学活性研究奠定基础.
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