摘要
目的构建人Period2(Per2)基因shRNA慢病毒。方法设计并合成3对特异性人Per2基因shRNA靶序列,构建到pENTRTM/U6(GV112)慢病毒载体中,包装慢病毒,用real-time PCR筛选取最佳片段。对感染的U343细胞,经嘌呤霉素初步筛选,Western blot鉴定Per2蛋白表达,得到Per2低表达的细胞。结果成功构建了具有Per2沉默效应的shRNA慢病毒。结论此次包装的慢病毒可以成功抑制Per2基因在胶质瘤U343细胞中的表达,并可用嘌呤霉素筛选低表达细胞株。
Objective To construct shRNA lentiviral vectors which can interfere expression of Period2( Per2) gene,and to establish glioma U343 cell lines with a low expression of Per2 gene. Methods Three pairs of specific human Per2 gene shRNA target sequence were designed and synthesized,which could link with pENTRTM / U6( GV112) lentiviral vector after annealing. Screening single colonies,then plasmid were extracted for PCR and sequencing. The plasmids were transfected into 293T cells,and the viral supernatant was collected to detect the titer of viral. Real-time PCR was used for selecting the best jamming effect of the lentivirus infection U343 cells,then getting cell lines with a low expression of Per2 by puromycin,Western blot was used for detecting the effects of interference. Results The results of PCR and DNA sequencing showed that knock-down of Per2 was successfully constructed with interference effects by shRNA lentiviral vectors; filter out the best sequence for Interference by real-time PCR,then the drug screening method was used for the determination of viral and the titer was 6. 0 × 108Tu·mL-1; Western blot validated shRNA lentiviral infection after glioma U343 cells Per2 protein expression levels were significantly decreased. Conclusion Lentiviral-mediated shRNA interference can be effectively suppressed Per2 gene in glioma U343 cells for further in-depth study and research Per2 association between glioma experimental basis.
出处
《宁夏医科大学学报》
2014年第3期252-255,359,共5页
Journal of Ningxia Medical University
基金
国家自然科学基金(81160313)
