重组人核糖核酸酶抑制因子基因的siRNA表达载体构建及B16细胞中基因沉默的鉴定

人核糖核酸酶抑制因子(Human ribonuclease inhibitor, hRI)是细胞质中的一种酸性糖蛋白,可以与血管生长因子(Angiogenin, Ang)紧密结合从而抑制血管形成.利用全基因组合成法合成针对人核糖核酸酶抑制因子基因(hri)的发夹shRNA序列,亚克隆到siRNA表达载体pKD;重组载体经酶切鉴定后,用脂质体法与报告基因绿色荧光蛋白重组融合的人核糖核酸酶抑制因子的逆转录病毒载体pLNCX-EGFP-C1-hri共转染到小鼠黑色素瘤细胞B16中,在荧光显微镜下检测干扰效果.用Image-Pro plus 4.5软件对绿色荧光照片半定量分析干扰效率.结果表明,荧光显微镜显示B16中表达的绿色荧光被干扰,荧光强度半定量分析干扰效率可达80%以上.成功重组构建了针对hri的siRNA表达载体.图5参9

Construction and Identification of siRNA Plasmid Targeting Recombinant hri in B16 Cells

To construct a siRNA plasmid targeting the gene of human ribonuclease inhibitor (hri) which is capable of inhibiting angiogenin expression in the mammalian cells and to observe its silencing effect on the rat melanoma cells (B16), a shRNA sequence targeting hri was chemically synthesized and subcloned into vector pKD. The vector was identified by the enzyme digested and then co-transfected into B16 cells carrying reporter plasmid of pLNCX-EGFP-C1-hri. The silencing effect of the siRNA plasmid in the B16 cells was visualized by fluorescent microscope. Restriction enzyme digestion proved that the construction of siRNA plasmid targeting hri was correct. The observation from fluorescent microscope showed that the potent of green fluorescent was obviously decreased in the co-transfected B16 cells compared with B16 cells carrying the plasmid of pLNCX-EGFP-C1-hri. The percentage of the green fluorescent was decreased by about 80% detected by the semi-quantitative analysis using Image-Pro plus 4.5 version of computer software. The results showed that the plasmid of siRNA targeting hri was successfully constructed and the expression of hri was effectively inhibited in the co-transfected B16 cells. Fig 5, Ref 9