Robo1 RNA干扰慢病毒载体的构建及其基因沉默效应

Construction of Lentivirus Vector of Robo1 siRNA and Its Silencing Effects

目的构建Roundabout(Robo1)siRNA慢病毒载体,并在SKOV3卵巢癌细胞中鉴定其转染及基因沉默效果。方法根据GenBank中基因信息,采用干扰序列设计软件设计靶点,制备Robo1DNA片段,在T4DNA连接酶作用下,将线性化的病毒载体GV118与DNA片段制备合成GV118-siRNA目的质粒,转化感受态细胞,对于长出的克隆应用菌落PCR鉴定,再对PCR鉴定阳性的克隆进行测序和对比分析,重组病毒质粒与另外2种辅助包装原件载体质粒通过LipofectamineTM2000共转染293T细胞,培养48h后,收集细胞培养上清液,将病毒浓缩后在293T细胞中测定病毒滴度;并检测病毒载体在工具细胞SKOV3卵巢癌细胞中的转染效率,实时荧光定量PCR(RT-PCR)和Western blot方法检测Robo1siRNA慢病毒对SKOV3中Robo1的干扰作用。结果成功构建Robo1siRNA慢病毒载体GV118-siR-NA,病毒滴度为2×109 TU/mL;用该病毒体外转染SKOV3卵巢癌细胞,当感染复数(MOI)为10时,感染效率大于90%;RT-PCR和Western blot方法检测Robo1基因沉默的效率分别为76.7%、56.0%。结论成功构建了Robo1siR-NA慢病毒载体GV118-siRNA,该病毒载体在体外转染SKOV3卵巢癌细胞的效率较高,且具有显著的基因沉默效果。

Objective To construct the lentivirus vector that can encode siRNA targeting Roundabout 1 （Robol） gene and examine the efficiency of its infection and gene silencing in the SKOV3 cells. Methods The target plasmid GV118-siRNA was prepared by connecting the linearized vector GVll8 and the DNA sequence by using T4 DNA convertase. The target plasmids were transformed into competent cells. The grown colonies were identified by colony PCR,and then the positive colonies were sequenced and aligned. Recombinant lentivirus vector plasmids and the other two auxiliary plasmids were co-transfected into 293T cells by LipofectamineTM2000, and the cell culture supernatant was collected 48 h later. The virus supernatant was concen- trated and tittered in 293T cells. The infection efficiency of the constructed virus was examined in SKOV3 cells and the gene si lencing efficiency was determined by using real-time quantitative PCR and Western blot. Results Robol siRNA was successfully cloned into lentivector GV118 ,and the titer of the concentrated virus was 2 × 10^9 TU/mL. The infection efficiency of the virus in SKOV3 cells was more than 90; at a MOI of 10. Robol was significantly knocked down by the recombinant lentivirus vector. The mRNA and protein expression levels of Robol were decreased by 76.7% and 56.0% respectively （P〈0.05）. Conclu- sion Lentivirus vector encoding siRNA targeting Robol is successfully constructed and the virus can effectively infect cells and knock down Robol expression.