硫氧还蛋白和氧化/还原因子的融合荧光蛋白载体的构建及在293T细胞中的分布

Construction of thioredoxin-DsRed and APE/ref-1-EGFP fusion protein vectors and their localization in 293T cells

目的构建硫氧还蛋白和氧化/还原因子的融合荧光蛋白真核表达载体,并在293T细胞中得到表达和定位。方法以RT-PCR方法从PC12细胞中克隆氧化/还原因子(APE/ref-1)的cDNA,然后亚克隆构建APE/ref-1-GFP融合真核表达载体。以PCR方法将质粒pQE30-TRX上的硫氧还蛋白cDNA亚克隆到pDsred1-1质粒上,然后将硫氧还蛋白-DsRed融合基因序列亚克隆到pCMV5质粒上,构建硫氧还蛋白-DsRed融合真核表达载体.通过磷酸钙转染293T细胞,以荧光显微镜分析融合蛋白的表达及其亚细胞定位。结果成功构建了硫氧还蛋白-DsRed和APE/ref-1-GFP融合表达载体,并在293T细胞中得到表达,APE/ref-1-GFP融合蛋白定位在293T细胞核内;硫氧还蛋白-DsRed融合蛋白定位在293T细胞质和细胞核内。结论为进一步研究硫氧还蛋白和APE/ref-1之间的动态相互作用奠定基础。

Objective To construct thioredoxin and APE/ref-1 fusion protein expression vector and to investigate their subcellular localization of the fusion proteins in 293Tcells. Methods The APE/ref-1 cDNA was cloned by RT-PCR from PC12 cell. APE/ref-1-EGFP fusion protein expression vector was constructed through subcloning. Thioredoxin cDNA was subcloned into pDSredl-1 from pQE30-TRX plasmid by PCR, and thioredoxin-DsRed fusion protein expression vector was constructed in pCMV5 plasmid. The expression and subcellular localization of the fusion proteins in 293T cells transfected with the vectors by calcium phosphate was analyzed by fluorescent microscopy. Results The results demonstrated that thioredoxin DsRed and APE/ref-1-EGFP fusion protein expression vectors were successfully constructed and expressed in 293T cells. APE/ref-1-EGFP fusion protein was located only in nucleus, and thioredoxin-DsRed fusion protein was located in cytoplasm as well as nucleus. Conclusion This study has set up a solid base for further to study on the dynamic interaction between thioredoxin and APE/ref-1 fusion proteins.