靶向FTL基因的shRNA真核表达载体的构建及鉴定

Construction and Identification of shRNA Eudaryotic Expression Vectors for Ferritin Light Chain Gene

目的:为了构建铁蛋白轻链(ferritin light chain,FTL)的短发夹小干扰RNA表达载体,并提供进一步研究FTL功能的平台。方法:针对小鼠FTL基因的特异性位点设计并合成特异性长度约65bp的短发夹状小干涉RNA(shRNA)寡核苷酸序列,退火形成双链后将其插入真核表达载体pRNA-U6.1/neo的限制性内切酶HindⅢ和Bam HⅠ之间中,构建成FTL基因的干扰载体(FTL shRNA)。经测序鉴定正确后,用lipofectamineTM2000将FTL shRNA及其对照空载体pRNA-U6.1/neo分别转染到小鼠腹腔单核巨噬细胞(RAW264.7)中,提取蛋白后利用Western blot技术检测细胞中FTL蛋白的表达。结果:得到与预期目的片段大小相似的重组载体,测序结果显示所构建的小鼠FTL shRNA质粒序列正确。细胞内的FTL表达被FTL shRNA成功干扰,效率高达80%。结论:实验成功构建了小鼠FTL基因的短发夹小RNA干扰表达载体FTL shRNA。

Objective： To construct the specific short hairpin small interfering RNA（ shRNA） expression vector of ferritin light chain（ FTL）,and provide a platform for further studies of FTL. Method： The specific short hairpin shRNA sequences about 65 bp length were designed based on the mouse FTL gene- specific sites and inserted between the restriction endonuclease HindⅢ and Bam HⅠ recognition sites on the eukaryotic expression vector pRNA- U6. 1 / neo,and the shRNA eukaryotic expression plasmid of FTL（ FTL shRNA） was constructed. After sequence analysis,FTL shRNA and its empty vector pRNA- U6. 1 / neo were transfected into RAW264. 7 cells using lipofectamineTM2000. Extracting protein and then detected FTL expression by Western blot. Result： The recombinant vectors showed the expected length of the target fragments and the sequencing result was correct. The cellular FTL expression was downregulated in 80% by FTL shRNA successfully. Conclusion： The present study showed that the short hairpin RNA expression vector for FTL was constructed successfully.