摘要
目的构建增强型绿色荧光蛋白（EGFP）和人转铁蛋白受体（TfR）双融合报告基因表达载体，并进行功能鉴定，为活体光学／磁共振双模式成像提供实验基础。方法将TfR基因克隆到pEGFP．C1载体，构建重组pEGFP-C1-TfR质粒。pEGFP-C1-TfR质粒转染293T细胞48h后，通过荧光显微镜观察EGFP的表达情况；通过RT-PCR检测TfR的表达情况；通过激光共聚焦扫描显微镜检测EGFP-TfR融合蛋白的亚细胞定位；通过Tf探针摄取和竞争实验检测EGFP-TfR融合蛋白的功能。结果测序结果显示，EGFP．TfR基因序列正确，无突变或缺失。重组质粒转染293T细胞后，荧光显微镜下观察到EGFP的表达；RT-PCR结果显示TfR高效表达；EGFP-TfR融合蛋白主要位于细胞膜，并能够特异介导Tf的内吞。结论EGFP-TfR双融合报告基因表达载体构建成功，并且能够高效表达发挥各自的功能，可以用于后续的活体光学／磁共振双模式成像。
Objective To construct dual fusion reporter gene expression vector containing enhanced green fluorescence protein （EGFP） and human transferrin receptor （TfR）, and validate the reconstructed plasmid, which will provide experimental foundation for in vivo dual-modality optical/Magnetic Resonance （MR） imaging. Methods Clone TfR into the pEGFP-C1 vector to construct pEGFP-C1-TfR plasmid, pEGFP-C1-TfR plasmid was transfected into 293T ceils for 48 h, then investigate EGFP expression under a fluorescence microscope; detect TfR expression through PT-PCR ; inspect the subcellular location of EGFP-TfR fusion protein through Confocal Scanning Laser Microscopy; evaluate the function of EGFP-TfR fusion protein through Tf probe uptake and competition assays. Results DNA sequencing analysis confirmed that EGFP-TfR gene sequence was correct, and there was no mutation and deletion. After transfecting the reconstructed plasmid into 293T cells, fluorescence microscope observation and RT-PCR results demonstrated that EGFP and TfR were expressed efficiently. EGFP-TfR fusion protein was located predominantly in the cellular membrane, and could specifically mediate internalization of Tf. Conclusion EGFP- TfR dual fusion reporter gene expression vector has been successfully constructed, and could be expressed efficiently with functional features. Thus, the expression vector could be applied for in vivo dual-modality optical/Magnetic Resonance （MR） imaging.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2011年第47期3363-3366,共4页
National Medical Journal of China
基金
国家自然科学基金（81071206,81070349,81101096,81172193）
NSFC-广东联合基金（U1032002）
部属（管）医院2010-2012年度临床学科重点项目（164）
关键词
磁共振成像
基因
报告
重组融合蛋白质类
Magnetic resonance imaging
Genes,reporter
Recombinant fusion proteins
