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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 a eukaryotic expression vector of FLAG-tagged human type 1 casein kinase (CK1) gene and to obtain its expression product, and to study the expression of CK1 gene in osteosarcoma U2OS, breast cancer ZR-75-1 and HepG2 Tumor cells in the expression and location. METHODS: The full-length coding region of human CK1 gene was amplified from human breast cDNA library by PCR and cloned into pCMV-Tag2B vector with FLAG tag. The recombinant plasmids were transfected into U2OS osteosarcoma cells and breast cancer cell line ZR-75 The expression of FLAG-CK1 in osteosarcoma U2OS cells, breast cancer ZR-75-1 cells and HepG2 hepatocellular carcinoma cells were observed by immunofluorescence staining. Results: Double-digestion and sequencing results showed that FLAG-CK1 eukaryotic expression plasmid was successfully constructed. The results of SDS-PAGE and Western blotting showed that FLAG-CK1 transfected into osteosarcoma U2OS cells, breast cancer ZR-75-1 cells and HepG2 cells Immunocytochemistry showed that CK1 was distributed in the nucleus and cytoplasm of osteosarcoma U2OS cells, breast cancer ZR-75-1 cells and HepG2 hepatoma cells, and the nuclear signal was stronger than the cytoplasm. CONCLUSION: The eukaryotic expression vector of CK1 was constructed and FLAG-CK1 could be expressed in the nucleus and cytoplasm of different tumor cell lines, which laid the experimental foundation for further study on the regulation of CK1 on cells.