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目的:构建HPC2不同截短功能片段的真核表达载体,并检测各片段在骨肉瘤U2OS细胞中的表达。方法:以质粒pcDNA3.2-V5/HPC2为模板,分别设计各片段PCR引物进行扩增,EcoR I酶切鉴定PCR产物,正确的PCR产物进行连接后转化到DH5α感受态细胞中并涂板筛选,从而得到带有V5标签的不同截短动能片段的表达载体,提取质粒并转染U2OS细胞,Western blot法鉴定目标蛋白的表达。结果:测序及酶切的结果均表明HPC2不同功能片段的真核表达载体构建成功,并能够在U2OS细胞中表达。结论:成功构建了HPC2不同功能域片段的真核表达载体。
Objective: To construct eukaryotic expression vectors with different truncated functional fragments of HPC2 and to detect the expression of each fragment in osteosarcoma U2OS cells. METHODS: The plasmid pcDNA3.2-V5/HPC2 was used as a template to design the PCR primers of each fragment for amplification. The PCR products were identified by EcoR I digestion. The correct PCR products were ligated and transformed into DH5a competent cells and plated. The expression vector with different truncated kinetic fragments with V5 tag was obtained. The plasmid was extracted and transfected into U2OS cells. The expression of target protein was identified by Western blot. Results: The results of sequencing and digestion showed that the eukaryotic expression vector of different functional fragments of HPC2 was successfully constructed and expressed in U2OS cells. Conclusion: The eukaryotic expression vector of different functional domain fragments of HPC2 was successfully constructed.