NgR特异性siRNA筛选及其表达载体构建

Establishment of an expression vector of NgR-specific siRNA

目的:将siRNA技术应用于NgR,筛选出高效的NgR(Nogo受体)特异性siRNA,并构建其表达载体。方法:2005-03/2006-03,在杭州新瑞佳生物制药有限公司、解放军第二军医大学神经生物教研室设计筛选NgR特异性siRNA,按照E1bashir等设计原则和siRNA的要求,设计、合成4对siRNA,转染体外培养大鼠原代皮层和海马细胞,筛选出基因高沉默效率的NgR特异性siRNA;2004-03/2005-03上海生工生物工程公司将获得的高沉默效率siRNA设计成带有BamHI、HindⅢ酶切粘性末端、终止识别序列和LOOP环的发卡式RNA,并将其克隆入载体pRNAT-U6.1/Neo,构建NgR特异性siRNA表达载体,然后进行酶切鉴定及基因测序。结果:①4对NgR特异siRNA中,siRNA199下调NgRmRNA的表达水平效率最高,转染72h效果最显著(96.04%)。NgR特异性siRNA199序列为:5’-CCGAAUCUCUUACGUGCCATT-3’。②将该序列的发卡式RNA成功克隆入载体pRNAT-U6.1/Neo,构建成NgR特异性siR-NA199表达载体,酶切鉴定及基因测序表明设计序列完全相符,目的基因序列准确无误。结论:NgR特异性siRNA199及其表达载体构建成功,为进一步合成病毒载体及观察NgR特异性RNA干扰抑制大鼠NgRmRNA表达对脊髓损伤的修复作用奠定了基础。

AIM： To find a greater specific siRNA suppressing Nogo Receptor （NgR） gene and establish its expression vector. NETHODS： According to the criteria of Elbashir et al and siRNA interference＇s rule, four pairs of NgR-specific siRNA were designed and synthesized by Hangzhou New Ruijia Biomedical Corporation and Staff Room of Neurology, Second Military Medieal University of Chinese PLA from March 2005 to March 2006, and then were transfected into rats＇ primary cortical and hippoeampal cells in vitro to find the NgR-specific siRNA of high gene silencing efficiency; And the siRNA of high gene silencing efficiency was designed from March 2004 to March 2005 by Shanghai Sangon Biological Engineering Co.,Ltd into a short hairpin RNA with BamH Ⅰ , Hind Ⅲ sticky end, terminated cognition sequence and a loop structure, followed by being cloned in pRNAT-U6.1/Neo to construct a expression vector of NgR-specific siRNA; At last the vector were evaluated by enzyme cutting and gene sequencing test. RESULTS： ①Among 4 pairs of NgR-specific siRNA, siRNA199 suppressed the expression of NgR mRNA mostly, and the effect was the most significant after 72 hours of transfection （96.04%）. The sequence of NgR- specific siRNA was 5＇-CCGAAUCUCUUACGUGCCATT-3＇. ②The short hairpin RNA was cloned successfully in pRNAT-U6.1/Neo vector to construct NgR-specific siRNA199 expression vector. The gene sequence proved by enzyme cutting and gene sequencing test was identical to that of targeting gene. CONCLUSION： NgR-specific siRNA199 can create an expression vector successfully, which will be used to generate viral vector and observe the repairing effect on spinal cord injury after suppressing NgR mRNA of rats by vector-based RNA interference.