质粒超螺旋比例对其细胞转染效率及表达的影响

The Effect of Proportion of Supercoiled Plasmid on Transfer Efficiency and Expression Level in Target Cells

检测质粒开环、闭环比例不同时对其细胞转染效率及表达的影响。构建携带人肝细胞生长因子基因的质粒(pcDNA3-HGF)及携带LacZ报告基因的质粒(pcDNA3-LacZ),用核酸纯化试剂盒提取开环、闭环比例不同的质粒,然后用LipofectAMINE介导它们分别转染NIH3T3细胞,采用X-Gal染色和ELISA法分别观察转染效率和表达活性。结果表明,用超螺旋比例分别为85.45%和48.44%的质粒pcDNA3-LacZ转染生长旺盛的NIH3T3细胞,48h检测转染效率分别为23.4%±3.8%和9.3%±2.5%,前者约是后者的2.5倍。用超螺旋比例分别为93.28%和40.53%的质粒pcDNA3-HGF转染1×106NIH3T3细胞,ELISA法检测48h细胞培养上清中HGF的表达量分别为46.5±6.3ng和25.6±4.2ng,前者是后者的1.8倍。初步认为质粒开环、闭环比例不同对其细胞转染效率及表达有一定影响,超螺旋比例高时其转染效率及表达量较高。

To detect the effect of different proportions of supercoiled plasmid on transfer efficiency and expression level in target cells. A plasmid pcDNA3-HGF carrying human hepatocyte growth faetor（HGF） gene and a plasmid pcDNA3-LacZ carrying LacZ reporter gene at first were constructed, then expanded and purified supercoiled plasmids of different proportions with DNA purification kit. The proportion of supercoiled plasmids was determined by a Gel image analysis system. Subsequently, transfection of the plasmids to fibroblast NIH3T3 cells was carried out in vitro by LipofectAMINE reagent. The transfer efficiency was observed by X-Gal staining and the expression level was quantitated by ELISA. The transfer efficiency of supercoiled plasmid pcDNA3-LacZ 85.45% and 48.44% proportions to NIH3T3 cells was 23.4%±3.8% and 9.3%±2.5%, respectively, at 48 h after transfection. The former is 2.5 times than the later. The amount of expressed HGF of supercoiled plasmid pcDNA3-HGF with 93.28% and 40.53% proportions in 1×10^6 NIH3T3 cells was 46.5±6.3 ng and 25.6±4.2 ng, respectively, insupernatant at 48 h after transfection. The former is 1.8 times than the later. These results demonstrate that proportion of supercoiled plasmid may affect its transfer efficiency and expression level in target cells. Higher proportion of supercoiled plasmid may lead to higher transfer efficiency and expression level.