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目的:构建带Myc标签的CCAAT增强子结合蛋白β(C/EBPβ)的真核表达载体,并检测其对骨肉瘤细胞增殖的影响。方法:应用PCR技术从人乳腺文库中扩增C/EBPβ全长编码区基因,并克隆到pXJ-40载体中;将重组质粒转染人胚胎肾293T细胞,采用SDS-PAGE和Western印迹鉴定蛋白表达;另将重组C/EBPβ质粒转染入骨肉瘤细胞系U20S,生长实验检测其对肿瘤细胞增殖的影响。结果:基因测序和双酶切鉴定表明,Myc-C/EBPβ真核表达质粒构建成功;SDS-PAGE和Western印迹结果显示,Myc-C/EBPβ转染人胚胎肾293T细胞后获得表达;生长实验证明C/EBPβ具有抑制骨肉瘤细胞增殖的功能。结论:构建了Myc-C/EBPβ的真核表达载体,为全面了解C/EBPβ的生物学功能及调控机理奠定了基础。
OBJECTIVE: To construct eukaryotic expression vector of Myc tagged CCAAT enhancer binding protein β (C / EBPβ), and to detect its effect on the proliferation of osteosarcoma cells. METHODS: The full-length coding region of C / EBPβ gene was amplified from human breast cDNA library by PCR and cloned into pXJ-40 vector. The recombinant plasmid was transfected into human embryonic kidney 293T cells and identified by SDS-PAGE and Western blot The recombinant plasmid was transfected into human osteosarcoma cell line U20S. The growth of the cell line was detected by MTT assay. Results: The sequencing of Myc-C / EBPβ eukaryotic expression plasmid was successfully constructed. The results of SDS-PAGE and Western blotting showed that Myc-C / EBPβ was transfected into human embryonic kidney 293T cells. It is demonstrated that C / EBPβ has the function of inhibiting osteosarcoma cell proliferation. Conclusion: The eukaryotic expression vector Myc-C / EBPβ was constructed, which laid the foundation for understanding the biological function and regulation mechanism of C / EBPβ.