泛素C端水解酶L1的RNA干扰载体的构建及效果评价

Construction and Evaluation of Interference Vector Targeting Ubiq uitin C Terminal Hydrolase L1 Gene

目的:获得抑制效果好的泛素C端水解酶L1(UCH-L1)基因小干扰RNA(siRNA)干扰载体。方法:根据Gen Bank中大鼠UCH-L1基因序列设计并合成4对siRNA寡核苷酸序列,将4对寡核苷酸序列退火成双链后分别插入siRNA表达载体pcDNA6.2-GW/EmGFP-miR中构建4个siRNA表达质粒,测序鉴定后将4个siRNA表达质粒分别转染HEK293细胞,利用Western印迹和qPCR方法检测干扰效果;将干扰效果最好的质粒包装成腺病毒,感染大鼠血管平滑肌细胞(VSMC),并采用TNFα干预诱导UCH-L1表达升高,Western印迹验证干扰效果。结果:测序分析证实4对siRNA寡核苷酸序列分别插入siRNA表达载体pcDNA6.2-GW/EmGFP-miR;qPCR检测与Western印迹均表明第3号siRNA表达载体对UCH-L1表达的抑制程度最高,将其包装成腺病毒并转染VSMC能显著抑制TNFα诱导的UCH-L1表达升高。结论:构建并筛选出干扰效果好的UCH-L1 siRNA干扰载体。

[Abstract] Objective： To obtain high effeelive siRNA interference w＇ctor of ubiquiliu C lernfinal hydrolase 1,1 （UCH-LI） gene. Methods： Four pairs of siRNA oligonue｜eotide seqttences were designed synthesized accord- lug to UCH-L1 gene sequence of rat in GenBank. and then were annealed into doubh-straud and inserted into the plasmid of peDNA6.2-GW/EmGFP-miR respectively to construct expression vectors. After I）NA sequencing. [bur siRNA expression vectors were transfected into HEK293 cells. The interf＇erenee effet＇t of Ihese leeted bv the method of Western bh）t and qPCR. Then the siRNA vector with the best into adenovirus. After infected with adenovirus, vascular smoolh nuts（.h, cells（VSMC） were treated ~ith TNFa to induce high UCH-LI expressions, and the interference effect of the adenovirus was verified by Results： Fm, r pairs of oligonueleotide sequences were successfidly inserted into the /EmGFP-miR and confirmed by sequencing a,mlysis. I1 was found /hat the No.3 siRNA expression vec- tor had the highest intelSerenee effect on UCH-LI expression when compared with other veclors. After packaged into adenovirus and Iransfeet into VSMC. No.3 siRNA expression vector also could significanll5 suppress high UCH-1A expression induced by TNF-a. Conclusion： UCH-L1 siRNA inlerference veelor with siguificant interfer- ence effect was constructed successfully.