人1型酪蛋白激酶的真核表达及其在肿瘤细胞中的定位  

Eukaryotic Expression of Casein Kinase 1 and its Localization in Tumor Cells

作　　者：冀全博[1,2] 徐小洁[1] 张强[2] 康磊[3] 李玲[1] 黄蓉[2] 周丽英[1] 王荣福[3] 王岩[2] 叶棋浓[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100850 [2]解放军总医院骨科,北京100853 [3]北京大学第一医院核医学科,北京100034

出　　处：《生物技术通讯》2014年第4期451-454,共4页Letters in Biotechnology

基　　金：国家自然科学基金(31100604;81101387;81101065)

摘　　要：目的:构建带FLAG标签的人1型酪蛋白激酶(CK1)基因的真核表达载体,获得其表达产物,并研究该激酶在骨肉瘤U2OS、乳腺癌ZR-75-1、肝癌HepG2等多种肿瘤细胞中的表达及定位情况。方法:应用PCR技术从人乳腺文库中扩增人CK1基因的全长编码区,将其克隆到带FLAG标签的pCMV-Tag2B载体中;将重组质粒转染骨肉瘤U2OS细胞、乳腺癌ZR-75-1细胞、肝癌HepG2细胞,以SDS-PAGE和Western印迹鉴定表达情况;细胞免疫荧光观察FLAG-CK1质粒在骨肉瘤U2OS细胞、乳腺癌ZR-75-1细胞、肝癌HepG2细胞中的细胞定位。结果:双酶切和测序结果显示FLAG-CK1真核表达质粒构建成功;SDS-PAGE和Western印迹结果表明,FLAG-CK1转染骨肉瘤U2OS细胞、乳腺癌ZR-75-1细胞、肝癌HepG2细胞后成功表达;细胞免疫荧光实验显示,CK1在骨肉瘤U2OS细胞、乳腺癌ZR-75-1细胞、肝癌HepG2细胞的胞核和胞质中均有分布,且胞核信号强于胞质。结论:构建了CK1的真核表达载体,且FLAG-CK1能在不同肿瘤细胞系的细胞核和细胞质中表达,为进一步研究CK1对细胞的调控奠定了实验基础。Objective： To construct the eukaryotic expression vector of human casein kinase 1（CK1） labeled with FLAG tag, obtain the expressed product, and measure its cellular localization in osteosarcoma U2OS, breast cancer ZR-75-1, hepatocellular carcinoma HepG2 cells. Methods： Human CK1 coding gene region, amplified from mammary cDNA library by PCR, was inserted into the pCMV-Tag2B vector. The resulting recombinant plas-mid pCMV-Tag2B-CK1 was transfected into U2OS, ZR-75-1 and HepG2 cells and cell lysates were examzined by SDS-PAGE and Western blotting. In addition, immunofluorescence was applied to determine the cellular local-ization. Results： The CK1 was successfully amplified by PCR and cloned into pCMV-Tag2B, as evidenced by dou-ble enzyme digestion and gene sequencing. U2OS, ZR-75-1 and HepG2 cell lines transfected by the recombinant plasmid pCMV-Tag2B-CK1 successfully expressed the protein, according to SDS-PAGE and Western blotting. Im-munofluorescence indicated that CK1 protein was expressed in all three cell lines and localized in both the nucle-us and cytoplasm, predominantly in the nucleus. Conclusion： The CK1 eukaryotic expression vector was successful-ly obtained, and the CK1 protein could be expressed in both the nucleus and cytoplasm in different tumor cell lines, which lays foundation for further research on cell regulation by CK1.

关 键 词：人1型酪蛋白激酶 真核表达 细胞定位 

分 类 号：Q78[生物学—分子生物学]