目的：运用基因克隆技术构建人源β1，4-半乳糖转移酶Ⅱ（β4GalTⅡ）真核表达质粒，并将其转染到 COS7细胞中，观察蛋白在细胞内的定位，同时转染到 HEK 293T 细胞中检测其表达。方法分别设计带 FLAG 标签和 GFP 标签的β4GalTⅡ基因的特异性引物，以含人β4GalTⅡ全长 cDNA序列为模板，定向构建到 pcDNA3．1（＋）及 pCDGFP 真核表达载体中，构建重组表达质粒。通过酶切和测序鉴定阳性重组质粒，利用 Lipofectamine 2000分别转染 COS7、HEK 293T细胞，免疫荧光显微镜观察重组蛋白在细胞内的定位，West-ern blot 法鉴定重组蛋白的表达。结果成功构建 pcD-NA3．1-β4GalTⅡ-FLAG 和 pCDGFP-β4GalTⅡ重组表达载体，定位实验表明：β4GalT Ⅱ-FLAG 和 GFP-β4GalT Ⅱ二者在COS7细胞中均主要定位在细胞核周围，并有明显的块状染色，胞核中未见明显分布；Western blot 法检测显示重组β4GalTⅡ蛋白在 HEK 293T 细胞中稳定表达。结论获得了人β4GalTⅡ的真核表达质粒，并能在 COS7和 HEK 293T 细胞中表达，为进一步研究β4GalTⅡ的功能及其与其他蛋白的相互作用奠定了一定基础。%Objective To construct the eukaryotic expression plasmids tagged FLAG or GFP of β4GalTⅡgene by PCR,then the plasmids were transfected into COS7 cells respectively to investigate the localization of recombinant proteins.They were also transfected into HEK 293T cells to define their expression.Methods Specific primers with FLAG or GFP tag were designed for β4GalTⅡgene.The gene encoding β4GalTⅡwas amplified directly by PCR using cDNA fragment as template.Then the amplified products were ligated into the eukaryotic expression vec-tors pcDNA3.1 (+)and pCDGFP to construct recombinant plasmids.Both of the plasmids were confirmed by re-striction enzyme digestion and DNA sequencing.The plasmids were transfected into COS7 and HEK 293T cells re-spectively via Lipofectamine 2000 Reagent.The localization and expression of the recombinant proteins in cells were examined by the fluorescence microscopy or Western blot.Results The pcDNA3.1-β4GalTⅡ-FLAG and pCDGFP-β4GalTⅡrecombinant expression vectors were successfully constructed and expressed in eukaryotic cells. The results which observed by the fluorescence microscope demonstrated that both β4GalTⅡ-FLAG and GFP-β4GalTⅡ proteins were predominantly detected in the perinuclear spot and showed obvious signs of massive dying, and they were not distributed in the nucleus evidently.Western blot identified that both of the β4GalTⅡtagged FLAG and GFP could express stably in HEK 293T cells.Conclusion Construction of eukaryotic expression re-combinant plasmids of β4GalTⅡgene provides some basis for further function studies of β4GalTⅡin cells and their interaction with other proteins.
展开▼
