真核表达载体pEGFP-GPC3的构建及表达

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(M,)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.

Construction and expression of eukaryotic expression vector for pEGFP-GPC3

AIM: To construct and expression of eukaryotic expression vector of glypican-3(GPC3) pEGFP-GPC3 in mouse dendritic cells(DCs).METHODS: The target gene fragment of GPC3 and empty vector were obtained from plasmid pCMV-SPORT6-GPC3 and vector pEGFP-N1 digested by double enzymes.GPC3 and empty vector were linked by ligase T4,and then transfected into E.coli DH5α.Identification with EcoR I,BamH I digestion,the recombinant eukaryotic expression vector pEGFP-GPC3 was transfected into DCs by LipofectamineTM2000.Fluorescent microscopy and Western blot were used to detected the expression of GPC3.RESULTS: The expression of EGFP-GPC3 fusion protein was detected in DCs,and GPC3 fusion protein of 67 kDa was detected by Western blotting.CONCLUSION: The eukaryotic expression vector pEGFP-of GPC3 was constructed succesfully and transfected effectively into DCs.