大鼠PER2基因RNAi慢病毒载体的构建及干扰效率的测定  

Construction of lentivial RNA interference vectors of rat PER2 gene and determination of its interference efficiency

作　　者：王翔凌[1,2] 孙宁玲[1] 马丽萍[3] 郭晓夏[1] 姜娟[1] 王鲁雁[1] 何湘君[3] 

机构地区：[1]北京大学人民医院心内科 [2]卫生部北京医院心内科,北京100730 [3]北京大学人民医院中心实验室,北京100044

出　　处：《西安交通大学学报（医学版）》2014年第1期16-21,共6页Journal of Xi’an Jiaotong University（Medical Sciences）

摘　　要：目的构建及筛选有效干扰大鼠血管平滑肌细胞PER2基因的shRNA慢病毒载体,并测定其干扰效率。方法设计大鼠PER2的siRNA靶序列,插入慢病毒载体质粒pGLV-U6-EGFP中,构建pLentivirus-per2-rat表达重组体并转染至A7r5大鼠胸主动脉血管平滑肌细胞内,利用Real-time PCR方法检测PER2基因的mRNA表达水平,筛选有效沉默钟基因PER2表达的RNAi慢病毒载体。结果经电泳、基因测序证实插入的目的序列正确,有PER2表达,成功构建了pLentivirus-per2-rat表达重组体;转染慢病毒载体pLentivirus-per2-rat至A7r5大鼠胸血管平滑肌细胞,测定转染效率达70%;Real-time PCR检测PER2基因的mRNA相对表达量,筛选出有效干扰大鼠血管平滑肌细胞钟基因Per2表达的siRNA片段,使PER2mRNA表达量下调约84%。结论成功构建了大鼠PER2的RNAi慢病毒载体,为进一步研究时钟基因的功能奠定了基础。Objective To construct the lentiviral RNA interference （RNAi） vectors of rat PER2 gene and evaluate the effects of silencing PER2 gene expression by siRNA. Methods The target sequence of siRNA- PER2 in rats was designed, and the complementary DNA containing both sense and antisense oligonucleotides was synthesized. After phosphorylation and annealing, the double-stranded DNA was cloned to pGLV-U6-EGFP to construct pLentivirus-per2-rat. Then the effects of RNAi in reducing gene expression were further confirmed by real-time PCR in transfected rat ATr5 aortic vessel smooth muscle cells expressing PER2. Results The rat lentivirus vectors expressing PER2-specific shRNA were synthesized, and the specificity of the designed target sequence was primarily identified by electrophoresis and gene sequence analysis. Rat ATr5 aortic vessel smooth muscle cells were successfully transfected with pLentivirus-per2-rat, and the transfection rate was 70%. The real- time PCR analysis confirmed that the level of PER2 mRNA was decreased significantly as compared with that in the negative control group, and suppressed by approximately 84% in ceils transfected with pLentivirus-per2-rat. Conclusion The successful construction of rat lentivirus vectors expressing PER2-specific shRNA may lay a foundation for further study on the clock gene＇s function.

关 键 词：PER2基因 RNA干扰 慢病毒载体 血管平滑肌细胞 时钟基因 

分 类 号：R349[医药卫生—基础医学]