摘要
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.
出处
《生物技术》
CAS
CSCD
北大核心
2014年第5期8-12,共5页
Biotechnology
基金
国家自然科学基金项目("胞质铁蛋白对炎症介质产生和释放的影响及其分子机制的研究"
No.31000632)
中国博士后科学基金项目("JNK1调节L-Ferritin泛素化降解的分子机理研究"
No.20090450922)
河北省自然科学基金项目("JNK活化导致铁蛋白表达降低的分子机理研究"
No.C2010000409)资助~~