膜联蛋白Ⅱ基因短发夹RNA表达载体的构建和鉴定  

Construction and identification of a vector expressing short hairpin RNA targeting annexinⅡgene

作　　者：刘亚伟[1] 戴兵[1] 梅长林[1] 付莉莉[1] 蔡厚安[1] 

机构地区：[1]第二军医大学长征医院肾内科解放军肾脏病研究所,上海200003

出　　处：《第二军医大学学报》2006年第11期1181-1185,共5页Academic Journal of Second Military Medical University

基　　金：国家自然科学基金重点项目(30330640);国家自然科学基金面上项目(30271523);国家自然科学基金(30576867)~~

摘　　要：目的:构建针对膜联蛋白(annexin)Ⅱ的短发夹RNA(shRNA)的表达载体,观察其对目的基因表达的影响。方法:根据GenBank中annexinⅡ基因已知序列设计了2条序列,即annexinⅡshRNA1、annexinⅡshRNA2,同时设计1条非特异性序列作为阴性对照。据此设计合成各自的寡核苷酸链,退火后连接人pGenesil1载体,转化扩增后进行序列测定。3种重组表达载体均转染HEK293细胞,通过实时荧光定量RT-PCR和Western印迹法分别在基因和蛋白水平上检测各自annexinⅡ的表达,以未转染细胞作为空白对照。结果:3种重组表达载体(pGenesil1-annexinⅡshRNA1、pGenesil1-annexinⅡshRNA2和pGenesil1-negative shRNA)经限制性酶切及测序分析证明基因插入正确。转染HEK293细胞后,转染pGenesil1-annexinⅡshRNA2组细胞annexinⅡ基因和蛋白表达明显低于转染pGenesil1-annexinⅡshRNA1、pGenesil1-negative shRNA以及空转染细胞(P<0.05),而后三者间没有明显差异。结论:成功构建了针对annexinⅡ的shRNA表达载体(pGenesil1-annexinⅡshR-NA2),转染细胞后可抑制annexinⅡ基因表达。Objective：To construct a vector encoding short hairpin RNA（shRNA） targeting annexin Ⅱ and to observe its influence on annexinⅡ expression. Methods： Annexin Ⅱ-targeted hairpin RNA1 and RNA2 were devised according to the GenBank annexin Ⅱ sequence. Meanwhile, a nonspecific sequence was taken as negative control. The oligonucleotide strands of DNA fragments encoding the above shRNAs were synthesized. After annealing of the complementary strands, the DNA fragments were cloned into human pGenesill plasmid followed by amplification in E. coli and sequencing. The 3 pGenesill constructs were then transfected into HEK293 cells and the expression of annexin Ⅱ was examined by real-time fluorescence quantitative RT-PCR and Western bolt. The non-transfected cells were taken as blank control. Results： Restrictive enzyme digestion and sequencing verified that the 3 recombinants（pGenesill-annexin Ⅱ shRNA1, pGenesill-annexin Ⅱ shRNA2, and pGenesill- negative shRNA） were correct. The annexin Ⅱ expression was significantly lower in HEK293 cells transfected with pGenesill-annexin Ⅱ shRNA2 compared with those transfected with pGenesill annexin Ⅱ shRNA1, pGenesill-negative shRNA, and blank control （P〈0.05） ; and there was no significant difference between the latter 3. Conclusion： We have successfully generated an annexin Ⅱ-targeted shRNA construct, which can significantly inhibit the expression of annexin Ⅱ.

关 键 词：膜联蛋白质类 RNA干扰 短发夹RNA 

分 类 号：Q786[生物学—分子生物学]