shRNA抑制外源绿色荧光蛋白在肝癌细胞株中的表达

Inhibition of GFP expression in hepatocellular carcinoma cells by shRNA

目的 :构建绿色荧光蛋白 (GFP)的短发夹状RNA(shRNA) ,观察其在哺乳动物细胞中抑制外源GFP的表达。方法 :设计、合成含GFP编码基因片段 ,中间被 4个bp间隔的反向重复序列 ,与转录载体 pTZU6 +1连接 ,构建pshRNA—GFP重组质粒 ,与pEGFP -C1共转染肝癌细胞株HepG2和SMMC - 772 1,检测其对GFP表达的影响。结果 :pshRNA—GFP对共转染的 pEGFP -C1中的绿色荧光蛋白有明显的抑制作用 ,抑制率达 70 %～ 85 %。结论 :构建的 pshRNA—GFP重组质粒能有效地抑制GFP在哺乳动物细胞中的表达。

Objective:To construct the plasmid containing short hairpin RNA(shRNA) of green fluorescent protein(GFP) to suppress the expression of GFP.Methods:A 20bp reverse repeated motif of GFP target sequence with 4bp spacer were synthesized,and inserted into pTZU6+1 to generate the pshRNA—GFP plasmid;pshRNA—GFP and pEGFP-C1 or pTZU6+1 and pEGFP-C1 were cotransfected into mammalian cells to detect effect of GFP expression separately.Results:pshRNA—GFP suppressed the GFP expression by 70%～85% in mammalian cells.Conclusion:The result shows that the short hairpin RNA of GFP can efficiently suppress its expression in HepG2 and SMMC-7721.