原钙黏蛋白7b与绿色荧光蛋白融合基因表达载体的构建及其在MCF10A细胞中的表达定位

Vector Construction for PCDH7b-GFP Fusion Protein and its Subcellular Localization in MCF10A Cells

目的:构建原钙黏蛋白7b(PCDH7b)与绿色荧光蛋白(GFP)的融合基因表达载体pQCXIP-PCDH7b-EGFP,并检测PCDH7b-GFP融合蛋白在细胞中的表达定位。方法:以MDA-MB-436细胞基因组DNA为模板,PCR扩增人PCDH7b基因,克隆入逆转录病毒载体pQCXIP-EGFP-N1构建pQCXIP-PCDH7b-EGFP,病毒包装后感染人乳腺上皮细胞系MCF10A,免疫荧光染色检测其与膜蛋白E钙黏蛋白的相对表达定位。结果:构建获得逆转录病毒载体pQCXIP-PCDH7b-EGFP,融合蛋白PCDH7b-GFP定位于细胞浆和细胞膜,与E钙黏蛋白有共定位。结论:PCDH7b-GFP融合蛋白的表达,为后续活细胞成像分析PCDH7b在细胞内的动态分布奠定了基础。

Objective：To construct retroviral expression vector pQCXIP-PCDH7b-EGFP for PCDH7b-GFP fusion protein,and examine the subcellular localization of PCDH7b-GFP fusion protein.Methods：Human proto-cadherin 7b（PCDH7b） gene was first amplified by PCR with the template of genomic DNA form MDA-MB-436 cells,and then cloned into retroviral expression vector pQCXIP-EGFP-N1 to make pQCXIP-PCDH7b-EGFP,from which retrovirus was made and used to infect MCF10A,an immortal cell line from human breast epithelium.Afterwards,immune-fluorescence staining was performed to examine the subcellular localization of PCDH7b-GFP fusion protein together with E-cadherin.Results：Retroviral expression vector for pQCXIP-PCDH7b-EGFP was successfully made,and PCDH7b-GFP fusion protein was found in cytoplasm and on cell membrane,and partially colocalized with E-cadherin.Conclusion：Expression of PCDH7b-GFP fusion protein will be helpful to explore dynamic distribution of PCDH7b protein by live cell microscopy.