pEGFP-N1-亚麻苦甙水解酶重组真核表达载体的构建及亚麻苦甙水解酶在SGC-7901细胞中的表达

Construction of the pEGFP-N1-linamarase eukaryotic expression vector and its expression in SGC-7901 cells

目的:构建携带亚麻苦甙水解酶(lis)基因的重组真核表达载体,以绿色荧光蛋白(GFP)为报告基因,导入SGC-7901细胞中表达。方法:提取木薯总RNA,PCR扩增lis目的基因,将该基因全长定向克隆至真核表达载体pEGFP-N1上,构建重组质粒载体。用电穿孔法转染体外培养的SGC-7901细胞,在活细胞状态下用荧光显微镜直接观察lis-EGFP融合蛋白在细胞中的表达。用PCR检测lis转录水平的表达,用Western blot法验证lis蛋白水平的表达。结果:酶切和测序证实pEGFP-N1-lis重组质粒构建正确,重组质粒载体在SGC-7901细胞中获得了表达,表达的融合蛋白具有lis和EGFP的双重活性。结论:通过基因克隆方法成功地构建了pEGFP-N1-lis重组质粒载体,并且在SGC-790细胞中稳定表达。

Objective： To construct a pEGFP-N1-linamarase eukaryotic expression vector by using the green fluo rescence protein （GFP） as the report gene and transfecting SGC-7901 cells. Methods： The cassava tissue linamarase （lis） genes were amplified with PCR technique and inserted into the pEGFP-N1 vector. The SGC-7901 cells were transfected with the formed plasmid by means of electrotransfer. The linamarase-EGFP fused protein was viewed directly with the fluorescence microscope and the expression of linamarase detected by Western-blot. Results： Plasmids were formed correctly and expressed in＇the SGC-7901 cells. The fused protein had the activities of both linamarase and EGFP. Conclusion： The pEGFP-NI-lis eukaryotic expression vector was successfully constructed by gene cloning, and it was stably expressed in the SGC-7901 cells.