PRDX3真核表达载体构建及亚细胞定位

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。

Construction and identification of PRDX3 eukaryotic expression vector

Objective To construct the eukaryotic expression vector of PRDX3 labeled with FLAG tag and to study its localization in human tongue cancer cell line SCC15.Methods PRDX3 gene was obtained from the breast library by PCR and cloned into PCDH vector to construct PCDH-FLAG-PRDX3.The plasmid was transiently transfected into 293T cells and the expression was detected by Western blot.Subcellular localization was detected by cellular immunofluorescence.Results The result of double digestion and sequencing showed that PCDH-FLAG-PRDX3 eukaryotic expression vector was constructed.The expression of FLAG-PRDX3 in human 293T cells was positively confirmed by Western blotting.In human tongue cancer cell line SCC15, the result of cellular immunofluorescence showed FLAG-PRDX3 was located in the cytoplasm rather than in the nucleus.Conclusion PRDX3 eukaryotic expression vector labeled with FLAG tag is constructed successfully, which is located in cytoplasm in human SCC15 cells.Construction and identification of PRDX3 could shed light on the function and mechanism of PRDX3 in tongue cancer.