干涉UBXN1慢病毒颗粒制备及稳定细胞系的建立

Packaging and Identification of Lentivirus Particles Interfering UBXN1 and Stable Cell Line Construction

构建并鉴定UBXN1-shRNA慢病毒表达载体,以便应用RNAi技术以及慢病毒感染系统建立稳定干涉细胞系并进一步研究UBXN1的功能。将携带不同特异性干涉序列的DNA片段插入PLKO.1载体中构建慢病毒表达载体,并制备慢病毒颗粒。将慢病毒颗粒感染U2OS细胞,建立稳定干涉细胞系,应用real-time PCR和western blot技术分别检测U2OS细胞中UBXN1 mRNA和蛋白质水平的表达差异。重组克隆经酶切证实shRNA正确插入慢病毒载体,DNA测序证实插入的序列正确,western blot检测证实设计的五条shRNA干扰序列有效的敲低U2OS细胞中内源性UBXN1的表达。成功制备UBXN1的慢病毒干涉颗粒,并建立UBXN1稳定下调的U2OS细胞系。

For studying the function of UBX domain-containing protein 1（UBXN1）,lentivirus expression vectors is constructed and set up stable knock-down cell line using RNAi technique and lentiviral system.Synthesized DNA segments is inserted including specific interfering sequences into PLKO.1 vector,and packaged them into lentivirus particles.Stable cell lines were established though infecting U2OS cells with lentivirus particles.Realtime PCR and western blot were used respectively to detect the level of UBXN1 mRNA and protein expression after lentivirus infection.The small hairpin RNA sequences were successfully inserted into PLKO.1 vector,and the sequences were identified by DNA sequencing.Further,western blot results validated that the five small hairpin RNAs effectively knockdown the level of endogenous UBXN1 in U2OS cell lines.It is conclused UBXN1 stably down-regulated cell lines though effective lentivirus particles infection is successfully constructed.