Kras突变基因真核表达载体的构建及在不同肝细胞株中的表达

目的：构建携带突变Kras基因，以增强型绿色荧光蛋白（EGFP）为报告基因的重组真核表达载体，并导入两种不同的肝细胞株中表达。方法：PCR扩增突变Kras目的基因，将该全长基因定向克隆至真核表达载体pEGFP-N1上，构建重组质粒载体。并利用脂质体转染人肝癌细胞株Huh7．5和鸡肝癌细胞株LMH，在活细胞状态下用荧光显微镜直接观察Kras-EGFP融合蛋白在细胞中的表达；用WesternBlotting方法验证Kras蛋白水平的表达。结果：酶切和测序证实pEGFP—N1-Kras重组质粒构建正确，将EGFP报告基因融合在突变的Kras基因的3’端；在Huh7．5和LMH中均观察到了绿色荧光，转染率分别为19％和53％；WesternBlott—ing也检测到融合蛋白的表达。结论：通过基因克隆方法成功构建了pEGFP—N1-Kras重组质粒载体，并且在Huh7．5和LMH中均稳定表达，为下一步筛选针对突变Kras基因的靶向药物奠定了基础。

Construction of pEGFP-N 1-Kras Expression Vector and Its Expression in Different Liver Cell Lines＊

Objective： To construct pEGFP-N1 eukaryotic expression vector by using enhanced green fluorescence protein as reporter gene and transfeeting two different liver cell lines. Methods： The mutated Kras gene was amplified by PCR technique and inserted into pEGFP-N1 vector. The human hepatoma cell line （Huh7.5） and chicken hepatoma cell line （LMH） was transfected with the recombinant plasmid by means of lipidosome. The Kras-EGFP fused protein was viewed directly with fluorensce microscope, and the expression of Kras was detected by Westem Blotting. Results： The recombinant plasmid was formed correctly by the analyses of enzyme restriction and DNA sequencing, and the 3＇ end of mutatd Kras gene was fused with EGFP reporter gene. The green fluorescence could be seen in both Huh7.5 and LMH cell lines and the transfection rate was 19% and 53% respectively. The expression of fusion protein could be detected by Western Blotting. Conclusions： The expression vector of recombinant plasmid pEGFP-N1-Kras is successfully constructed and is effectively expressed after being transfected into Huh7.5 and LMH, which provides support for the study of the screening of mutated Kras gene targeted drugs.