靶向大鼠Nogo受体基因的小发夹RNA慢病毒载体的构建及病毒包装

Construction and packaging of lentiviral shRNA targeting rat Nogo-receptor gene

目的构建靶向大鼠Nogo受体(NgR)的小发夹RNA(shRNA)慢病毒载体并进行病毒颗粒包装及滴度测定。方法设计合成3对针对大鼠NgR及1对非特异性的寡核苷酸单链,退火后得到shNgR双链,克隆入慢病毒载体pGC-LV,行聚合酶链反应(PCR)并测序鉴定重组体;构建NgR过表达载体,双酶切并测序鉴定插入序列正确性;NgR过表达载体和各慢病毒shRNA干扰载体共转染293T细胞后,定量反转录(RT)-PCR及蛋白印迹法筛选有效的慢病毒shRNA干扰载体;重组NgR慢病毒shRNA干扰载体和病毒包装质粒共转染293T细胞,包装并收集病毒颗粒,进行病毒浓缩液滴度检测。结果 PCR及DNA测序结果均显示慢病毒载体pGC-LV-shNgR构建正确;双酶切及测序亦显示NgR过表达载体插入序列正确;定量RT-PCR及蛋白印迹法结果表明所构建的3个慢病毒载体pGC-LV-shNgR均可以有效干扰NgR的表达,其中pGC-LV-shNgR-A干扰效率最高;包装病毒颗粒后,检测病毒浓缩液的滴度为2×107 TU/mL。结论成功构建靶向大鼠Nogo受体的shRNA慢病毒载体并进行病毒颗粒包装,浓缩的病毒液可以用于后续研究。

Objective To construct shRNA lentivirus vector targeting at rat NgR and packaging of lentivirus granule.Methods Three pairs of oligonucleiotide single strands targeting at rat NgR and one pair of non-targeting strands were designed and sythesized.shNgR double-strands were got after annealing and were cloned into lentivirus vevtor pGC-LV.NgR overexpression vector was also constructed and identified by enzyme digestion and DNA sequencing.The effect of recombinant pGC-LV-shNgR vectors were screened by co-transfection with NgR overexpression vector into 293T cells before analysis by qPCR and Western blotting.Then the most effective pGC-LV-shNgR and virus packaging vectors were co-transfected into 293T cells.And the virus solution was collected,concentrated and subjected to titer determination.Results pGC-LV-shNgR vectors and NgR overexpression vector were all shown to be accurately constructed by DNA sequencing.qPCR and Western blotting showed that the three lentivirus vectors could effectively interfere NgR expression.After packaging and collection,the titer of the concentrated virus solution was detected to be 2×107 TU/mL.Conclusion shRNA lentivirus vector targeting at rat NgR was successfully constructed and packaged.The concentrated virus solution can be applied in future studies.