F10基因与绿色荧光蛋白融合载体的构建及亚细胞定位

目的：构建F10基因与绿色荧光蛋白的融合蛋白表达载体，进而观察F10基因编码蛋白在细胞内的定位情况。方法：运用RT—PCR技术从葡萄胎组织中分离F10基因的完整ORF，构建pEGFP-N2／F10和pEGFP—C2／F10的融合表达载体，以1Apofect AMINE2000为介导剂，将重组载体转染入人乳腺癌细胞MCF-7，并在荧光显微镜和共聚焦显微镜下观察表达的融合蛋白。结果：显微镜下观察到，GFP／F10融合蛋白呈不规则的颗粒状、环状分布于细胞核，或细胞浆，或者在核浆中均有分布，而且在细胞浆中主要分布在细胞器中。而对照的pEGFP蛋白均匀分布在整个细胞浆和细胞核。结论：F10蛋白在细胞的不同周期分布不同，或在细胞核，或在细胞胞浆中，或者在核浆中均有分布。

Construction and Subcellular Localization of F10-incorporating GFP Fusion Protein Vector

Objective:To construct the F10-incorporating GFP fusion protein vector,and observe the subcellular fraction of its encoding protein.Methods: the complete coding sequence of F10 gene was amplified from hydatidiform mole tissues by RT-PCR method,and then cloned into pEGFP-N2 and pEGFP-C2 on special direction.After these recombinant plasmids were transfected into human breast cancer (MCF-7) cells by lipofectimane 2000 mediation,the expression of F10 gene and its subcellular fraction were observed by fluorescent microscope and laser confocal microscope.Results: detected by the microscope,GFP/F10 fusion protein is localized in nucleus or cytoplasm,or both.In cytoplasm,the protein is localized in cell organelles.As control,GFP is distributing in the whole cell.Conclusion:With different cell cycle,F10 protein has different distribution,localizesd in nucleus or cytopasm,or both of them.