人酪蛋白激酶1激酶区真核表达载体的构建及鉴定  

Construction and Identification of the Kinase Domain of Human Casein Kinase 1α Eukaryotic Expression Vector

作　　者：尤文叶 褚春雨 徐小洁[3] 梁迎春[3] 洪甜[3] 黄俊[3] 韩笑 叶棋浓[3] 杨俊兰[1] 李瑛[1] 

机构地区：[1]中国人民解放军总医院,北京100853 [2]河北省文安县医院普内科,河北文安065800 [3]军事医学科学院生物工程研究所,北京100850

出　　处：《生物技术通讯》2016年第6期808-811,共4页Letters in Biotechnology

基　　金：吴阶平医学基金(wjp-lc-11037);国家自然科学基金(31100604);北京市科技新星计划(Z141102001814055);北京市自然科学基金(7132155)

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。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.

关 键 词：酪蛋白激酶1α 真核表达 CDC25A 

分 类 号：Q78[生物学—分子生物学]