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目的构建带FLAG标签的过氧化物还原酶3(peroxiredoxin3,PRDX3,PRX3)的真核表达载体,并对其在人舌癌SCC15细胞中的定位进行检测。方法以乳腺文库为模板,PCR扩增带有FLAG标签的PRDX3基因片段,并将扩增产物插入到PCDH空载体中,构建成PCDH-FLAG-PRDX3,质粒测序正确后瞬时转染入人胚肾293T细胞,Western免疫印迹检测克隆载体的表达情况,细胞免疫荧光实验检测PRDX3的亚细胞定位。结果双酶切和测序结果表明,PCDH-FLAG-PRDX3真核表达载体构建成功,转染293T细胞后成功表达了FLAG-PRDX3融合蛋白;细胞免疫荧光显示FLAG-PRDX3蛋白定位于舌癌SCC15细胞的细胞质中,不存在于细胞核内。结论成功构建了带FLAG标签的PRDX3真核表达载体,并检测其定位于舌癌SCC15细胞质中,为进一步明确PRDX3在舌癌发生发展中的功能及分子机制奠定基础。
Objective To construct eukaryotic expression vector of FLAG tagged peroxiredoxin3 (PRDX3, PRX3) and detect its localization in human tongue cancer SCC15 cells. METHODS: The FLAG-tagged PRDX3 gene fragment was amplified by PCR using the breast cDNA library as a template. The amplified product was inserted into PCDH empty vector to construct PCDH-FLAG-PRDX3. The plasmid was transfected into human embryonic kidney 293T cells and Western blotting were used to detect the expression of cloned vector. The subcellular localization of PRDX3 was detected by immunofluorescence assay. Results The results of double enzyme digestion and sequencing showed that the eukaryotic expression vector pcDH-FLAG-PRDX3 was constructed successfully and the FLAG-PRDX3 fusion protein was successfully expressed after transfected into 293T cells. Immunofluorescence showed FLAG-PRDX3 protein localized in tongue cancer SCC15 cells Cytoplasm, does not exist in the nucleus. Conclusion The FLAG-tagged PRDX3 eukaryotic expression vector was successfully constructed and localized in the cytoplasm of SCC15 tongue cancer cells, which laid the foundation for further clarifying the function and molecular mechanism of PRDX3 in the genesis and progression of tongue carcinoma.