bcl-2 shRNA表达载体的构建及其在K562细胞中的干扰作用研究  

Construction of bcl-2 shRNA expression vector and study on the interfering effect of bcl-2 shRNA expression vector in K562 cells

作　　者：李君君[1] 颜家运[1] 刘劼[2] 黄丽芳[1] 

机构地区：[1]南华大学附属第一医院血液科,湖南衡阳421001 [2]南华大学病原微生物研究室,湖南衡阳421001

出　　处：《中国现代医学杂志》2008年第7期869-872,共4页China Journal of Modern Medicine

基　　金：湖南省卫生厅资助项目(No:B20050102)

摘　　要：目的构建bcl-2shRNA表达载体,观察其在人白血病K562细胞系中对bcl-2基因的干扰作用。方法设计合成有短发夹结构靶位bcl-2基因的65个碱基的寡核苷酸,连接到载体pSIREN-Shuttle(pSS),构建了bcl-2shRNA表达载体,得到重组质粒pSS-shbcl-2-1、pSS-shbcl-2-2、pSS-shbcl-2-3和一个阴性对照,将其稳定转染K562细胞,分别用RT-PCR、Western-blot法检测转染后K562细胞bcl-2 mRNA及蛋白质表达水平。结果成功构建了bcl-2shRNA的表达载体,并筛选到转染后K562细胞在pSS-shbcl-2-3组bcl-2基因的mRNA和蛋白质表达水平明显抑制。结论特异性的bcl-2shRNA可以干扰K562细胞中bcl-2基因的表达,针对bcl-2基因不同位点的不同shRNA也有干扰效果的差异。[Objective] To construct short hairpin RNA interfering （shRNA） expression vector of bcl-2, and detect the specific interfering effect of bcl-2 shRNA expression vector on human leukemia K562 cells. [Methods] Oligos of 65 base pairs for short hairpin RNA targeting for bcl-2 were designed and connected to the expression vector pSIREN-Shuttle（pSS） to construct the bcl-2 shRNA expression vectors. The recombinant plasmid pSS-sh- bcl-2-1, pSS-sh-bcl-2-2, pSS-sh-bcl-2-3 were generated and transfected into K562 cells. Expression of bcl-2 mRNA was assayed by RT-PCR, and the level of protein was determined by Western-blot. [Results] A specific bcl2 shRNA expression vector was successfully constructed.The third shRNA （pSS-sh-bcl2-3） was more effective in the suppression of bcl-2 with significant reduction of bcl-2 mRNA and the level of protein expression. [Conclusion] Specific bcl-2 shRNA could interfere the expression of Bcl-2 gene in K562 cells, shRNA matching different sites of bcl-2 gene exert different inhibitory effects.

关 键 词：RNA干扰 基因BCL-2 细胞系 K562 

分 类 号：R446.63[医药卫生—诊断学]