巨细胞病毒IE2的红色靶基因与绿色荧光-慢病毒系统的建立及其对人神经干细胞的感染

An EGFP-Lentivirus and red target HCMV-IE2 gene system and its infection in human NSCs

目的 RNA干扰技术沉默连接红色荧光蛋白mCherry与靶基因巨细胞病毒IE2的表达,并初步研究慢病毒感染神经干细胞的可能性。方法以人巨细胞病毒IE2 mRNA编码序列作为干扰靶点,以慢病毒质粒UTG作为载体,构建靶向巨细胞病毒IE2的shRNA表达质粒,构建携带IE2的mCherry红色荧光蛋白的质粒作为验证shRNA有效性的靶点,并转染293FT细胞。通过红色荧光的表达及RT-PCR和Western Blot确定最佳沉默IE2序列后,将有效的UTG-IE2质粒与辅助质粒共转染293FT细胞,获取高滴度慢病毒。应用慢病毒感染人神经干细胞,观察神经干细胞被慢病毒感染的情况并检测神经干细胞的绿色荧光表达情况。结果成功构建IE2-shRNA慢病毒载体UTG-shRNA-IE2,RT-PCR及Western Blot结果显示慢病毒感染的293FT细胞IE2 mRNA及蛋白表达相对于对照组显著降低。构建的慢病毒可以成功感染神经干细胞。结论红色靶基因-绿色慢病毒系统具有简便的筛选性能;UTG慢病毒对神经干细胞具有较高感染效率,可实现慢病毒介导的RNA干扰在神经干细胞内的有效表达,并为相关研究提供技术支持。

Objective To effectively silence the mCherry combined HCMV IE2 target gene expression and a possibility in Lentivirus transduction NSCs was explored.Methods A mCherry plasmid carrying the whole backbone of IE2 gene was set as the target to shRNAs.Both the UTG and mCherry-IE2 plasmids were co-transfected into 293FT cells.The optimized IE2 sequence was determined by knocking out of mCherry color and confirmed by RT-PCR and Western Blot.The Lentivirus was added into human nerve stem cells（hMSCs） to observe the situation of transduction.Results A set of Lentiviral shRNA vectors targeting IE2 was constructed successfully and one optimized shRNA sequence was selected.The results showed IE2 mRNA and protein were decreased significantly.In addition,Lentivirus with shRNA can infect human MSCs.Conclusions The mCherry target-EGFP Lentivirus characterizes as simple and convenient tool in screening an optional shRNA.UTG Lentivirus shows a high efficiency in infecting NSCs as a vector to study knocking down gene expression in NSCs by HCMV.