小鼠3D3／LYRIC基因重组质粒构建及蛋白表达和定位

Construction of Fusion Plasmid of Mouse 3D3/LYRIC Gene and Identification of Its Recombinant Protein Expression and Localization

目的构建小鼠3D3/LYRIC真核表达载体并证实融合蛋白在细胞内的表达及定位。方法提取小鼠乳腺上皮细胞系C127的mRNA,反转录为cDNA。PCR扩增m3D3/LYRIC全长编码基因,亚克隆至pEGFP-C1表达载体中。将构建的重组质粒测序并转染到腺样囊性癌细胞系ACC-2中,提取细胞蛋白进行Western blot检测。利用免疫荧光显微镜观察pEGFP-m3D3/LYRIC在腺样囊性癌ACC-2细胞内的定位。结果 m3D3/LYRIC全长基因序列克隆到真核表达载体pEGFP-C1中,酶切鉴定片段大小为1 740 bp。Western blot检测到融合蛋白GFP-m3D3/LYRIC表达,分子量约为91 kDa。pEGFP-C1-m3D3/LYRIC主要在细胞质内定位,在细胞核内未见表达。结论成功构建了m3D3/LYRIC全长基因真核表达载体,pEGFP-m3D3/LYRIC蛋白主要定位于细胞质内。

Objective To construct eukaryotic expression plasmid of mouse 3D3/LYRIC gene and identify its recombinant protein expression and localization.Methods Total mRNA isolated from mouse mammary epithelial C127 cells was reversely transcribed into cDNA.The m3D3/LYRIC coding sequence was amplified by polymerase chain reaction（PCR） method and was subcloned into pEGFP-C1 expression vectors.After the target region was sequenced,the plasmids were transfected into ACC-2 cells,which were established from adenoid cystic carcinoma cells of the salivary gland（ACC-2 cells）.Expression of the recombinant plasmids in ACC-2 cells was detected by Western blot.The localization of pEGFP-m3D3/LYRIC in ACC-2 cells was observed by laser scanning confocal microscopy.Results m3D3/LYRIC has been constructed into eukaryotic expressing vector pEGFP-C1 successfully.The length of the fragment was 1 740 bp,identified by restriction enzymes digestion.The expression of GFP-m3D3/LYRIC fusion protein was detected by Western blot,with a molecular weight of 91 kDa.The pEGFP-m3D3/LYRIC protein was mainly localized in the cytoplasma without any expression in the nucleus.Conclusion The recombinant plasmids were successfully cloned into eukaryotic expressing vector m3D3/LYRIC.The pEGFP-m3D3/LYRIC fusion protein was mainly expressed in the cytoplasma.