人1型酪蛋白激酶的真核表达及其在肿瘤细胞中的定位

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。

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

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.