【摘要】 目的:构建带Flag标签的酪蛋白激酶1α(CK1α)激酶区截短突变体CK1α(1-285aa)的真核表达载体,获得其表达产物,并验证该激酶区与已知相互作用蛋白Myc-Cdc25A之间的相互作用。方法:用PCR技术从人全长CK1α真核表达载体中扩增CK1α(1-285aa),将其克隆到Flag载体中,将重组质粒转染人胚肾293T细胞,Western印迹检测其在人293T细胞中的表达情况,免疫共沉淀分别检测Flag-CK1α全长、Flag-CK1α(1-285aa)与Myc-Cdc25A的相互作用。结果:双酶切和基因测序鉴定显示Flag-CK1α(1-285aa)真核表达载体克隆构建成功;Western印迹结果表明Flag-CK1α(1-285aa)转染人胚肾细胞293T细胞后获得表达;免疫共沉淀结果显示Flag-CK1α全长、Flag-CK1α(1-285aa)与Myc-Cdc25A在蛋白质水平上具有相互作用,证实其具有生物学活性。结论:构建了Flag-CK1α(1-285aa)的真核表达载体,为进一步探讨Flag-CK1α激酶区对细胞信号通路的调节作用奠定了实验基础。更多还原

【Abstract】 Objective: To express casein kinase 1α(CK1α) kinase domain(1- 285aa) interacted with Myc-Cdc25 A in 293 T cells. Methods: Human CK1α(1-285aa) coding region amplified from the full-length CK1α ex-pression vector by PCR was inserted into the Flag vector. The recombinant plasmid Flag-CK1α(1-285aa) transfect-ed into human 293 T cell lines was investigated and examined by Western blotting. In addition, immunoprecipita-tion assay was applied to determine the interaction between Flag-CK1α full length or kinase domain and Myc-Cdc25 A. Results: Amplication of CK1α(1-285aa) and its cloning into pc DNA3.0 vector were identified by doubleenzyme digestion and gene sequencing. The expression of Flag- CK1α(1- 285aa) in human 293 T cell lines wasdemonstrated by Western blotting assay. Immunoprecipitation analysis indicated that Flag-CK1α full length and ki-nase domain(1-285aa) both interacted with Cdc25 A protein in line with their reported function. Conclusion: Theeukaryotic expression vector of CK1α kinase domain is obtained, which will contribute to research of CK1α mediat-ed cell signal pathway.更多还原