FLG基因RNAi慢病毒载体的构建与鉴定

Construction and Identification of RNAi Lentiviral Vectors Targeting FLG Gene

目的构建人类中间丝聚合蛋白(filaggrin,FLG)基因慢病毒载体及观察慢病毒表达载体介导的RNA干扰RNAi对人HaCat细胞FLG表达的影响。方法应用基因工程技术筛选出3条针对FLG基因的RNAi靶序列,分别与GV115载体连接,构建3个重组慢病毒表达载体FLG-vshRNA 10154,FLG-vshRNA 10155,FLG-vshRNA 10156;将连接产物转化到DH5α感受态细胞,经PCR筛选阳性克隆、测序鉴定,将FLG-vshRNA,pHelper 1.0,pHelper 2.0共转染293T细胞,包装产生慢病毒颗粒并测定病毒滴度,将包装产生的3种重组慢病毒分别感染HaCat细胞,实时定量PCR检测HaCat细胞FLG mRNA表达。根据筛选的结果,选取最有效的载体进行病毒的大量包装。结果慢病毒载体PCR和测序结果表明3对碱基成功插入到预计位点,序列完全一致。慢病毒载体感染HaCat细胞后,FLG基因mRNA的表达量与未感染慢病毒的细胞组及空载体感染组相比均明显下降,下降程度达70%以上(P<0.05)。3个慢病毒载体经包装产生的病毒滴度分别为1.2×109,1.5×109,1×109TU/mL。结论成功构建针对FLG基因的3个慢病毒载体FLG-vshRNA,体外感染HaCat细胞后可有效抑制FLG基因的表达。

Objective To construct expressing FLG siRNA plasmid vector and examine the effect of the letiviral vector-mediated RNA interference（RNAi） on the expression of human FLG gene in HaCaT cells. Methods Gene engineering technique was used to screen three RNAi sequencestargeting FLG gene （FLG-vshRNA 10154, FLG-vshRNA 10155 and FLG-vshRNA 10156）. The sequences were separately cloned into the GV115 vector to construct 3 lentiviral vectors, which were subsequently confirmed by PCR and DNA seuencing. The titer of lentiviruses was determined after 293T cells were co-transfected with FLG-vshRNA, pHelper 1.0 and pHelper 2. 0. The three kinds of reconbinant lentiviruses were used to infect HaCat cells and the expression of FLG mRNA were detected by real-time RT-PCR. Results PCR analysis and DNA sequencing confirmed that the three FLG-vshRNA sequences were successfully inserted into the letiviral vectors. The FLG expression in HaCat cells infected with lentiviral vectors was significantly inhibited at mRNA levels when compared with that in non-transfected and empty vector-transfected HaCat cells. The FLG mRNA expression was decreased by over 70% after FLG-vshRNA infection. The titer of the conentrated virus was 1.2×10^9, 1.5×10^9 and 1×10^9 TU/mL, respectively. Conclusion Three levtiviral RNAi vectors of FLG gene were successfully constructed, which could effectively inhibit the expression of FLG gene in HaCat cells in vitro.