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目的:构建磷脂酰肌醇蛋白聚糖3(GPC3)真核表达载体pEGFP-GPC3,并在小鼠树突状细胞(DC)表达。方法:将质粒pCMV-SPORT6-GPC3和载体pEGFP-N1分别进行双酶切获得目的基因GPC3片段和空载体,回收纯化后用T4连接酶将两者连接并转化E.coli DH5α菌株,用EcoR I、BamH I双酶切鉴定后通过脂质体法转染到DC中,然后进行荧光检测和Western blot分析。结果:构建的真核表达载体pEGFP-GPC3,转染DC后,荧光显微镜下可见转染的DC中有EGFP-GPC3融合蛋白的表达,Western blot分析发现有相对分子质量(Mr)大小约为67 000的蛋白条带。结论:成功地构建了真核表达载体pEGFP-GPC3并在转染DC后能在其中表达,为进一步研究GPC3基因的功能提供实验基础。
OBJECTIVE: To construct the eukaryotic expression vector pEGFP-GPC3 of glypican 3 (GPC3) and express it in dendritic cells (DCs) of mice. Methods: Plasmid pCMV-SPORT6-GPC3 and vector pEGFP-N1 were digested with restriction endonucleases to obtain the target gene GPC3 fragment and empty vector. After purification, they were ligated with T4 ligase and transformed into E. coli DH5α strain. , BamH I double restriction enzyme digestion after transfection into DC, and then fluorescence detection and Western blot analysis. Results: The eukaryotic expression vector pEGFP-GPC3 was constructed. After transfected with DC, EGFP-GPC3 fusion protein was expressed in transfected DCs by fluorescence microscopy. Western blot analysis showed that the relative molecular mass (Mr) was about 67 000 protein band. CONCLUSION: The eukaryotic expression vector pEGFP-GPC3 was successfully constructed and expressed in DCs after transfection, which provided the experimental basis for further study on the function of GPC3 genes.