慢病毒介导的大鼠肝BRL-3A细胞Tmub1基因沉默及稳定感染细胞系的建立

Lentivirus-mediated Tmub1 Silencing in BRL-3A and Establishment of RNAi Stable Cell Line

目的:构建Tmub1基因慢病毒干扰载体,建立稳定转染细胞系,检测大鼠肝BRL-3A细胞中Tmub1基因表达的干扰效果。方法:设计并构建4对针对大鼠Tmub1基因的特异性shRNA干扰质粒,酶切鉴定、DNA测序所得质粒。将由pRSV-Rev、pMDLg-pRRE、pMD2G和pll3.7干扰质粒组成的包装系统共转染293T细胞,产生慢病毒。所得慢病毒感染大鼠正常肝细胞BRL-3A,Western Blot检测不同靶点RNAi后Tmub1蛋白表达情况,确定有效靶点。针对有效靶点大量包装慢病毒。测定病毒滴度并以最适感染复数(multiplicity of infection,MOI)感染BRL-3A细胞后,G418抗生素筛选稳定感染细胞系BRL-3A/256。RT-PCR和Western Blot检测各组细胞Tmub1 mRNA和蛋白质的表达差异。结果:结果显示Tmub1 RNAi慢病毒载体构建成功,C0020Sh2-Hops-256干扰靶点RNAi效果最强。成功包装Tmub1基因RNAi慢病毒,测定病毒滴度为2.3×108TU/ml,对293T细胞的最适感染复数为60。成功建立Tmub1 RNAi慢病毒载体稳定感染细胞系BRL-3A/256,且在该细胞系中Tmub1 mRNA和蛋白质表达明显降低。结论:成功构建Tmub1 RNAi慢病毒载体,有效干扰BRL-3A细胞中Tmub1 mRNA和蛋白表达;成功筛选出Tmub1RNAi慢病毒稳定感染细胞系BRL-3A/256。

Objective： To construct a RNA interference（RNAi） lentivirus vector of Tmub1 gene,to establish a stable cell line with Tmub1 gene knockdowned,and to detect the silence effect of Tmub1 gene in rat hepatocyte cell line（BRL-3A） transfected with different RNAi vector.Methods： Four pairs of oligonucleotide sequences of the Tmub1 gene were designed and synthesized,and cloned into the Pll3.7 vector digested by Xho I and Hpa I,and then the vectors were confirmed by PCR and DNA sequencing.293T cells were cotrans-fected with pRSV-Rev,pMDLg-pRRE,pMD2G and pll3.7,to produce lentivirus.Carrying Tmub1 shRNA,BRL-3A cells were infected with lentivirus.RNA interference effect on Tmub1 expression in BRL-3A cells was detected with Western-Blot.The strongest interfer-ence plasmid was packaged to produce Tmub1 RNAi lentivirus LV256.Determine the virus titer and fittest multiplicity of infection（MOI）.After BRL-3A cells was infected,G418 antibiotic as used to screen out the stably infected cells line BRL-3A/256.The Tmub1 mRNA and protein expression in the BRL-3A/256 cells line were detected by RT-PCR and Western Blot.Results： The data demonstrated that the lentivirus RNAi vector of Tmub1 was constructed successfully.And the C0020 Sh2-Hops-256 target showed the best interference effects.The titer of virus was 2.3×108 TU/ml,and the fittest MOI in 293T was 60.Stably infected cells line BRL-3A/256 was successfully established and showed low expression of Tmub1 mRNA and protein.Conclusion： The lentivirus RNAi vector of Tmub1 is constructed successfully,and it had effect on the mRNA and protein expression of Tmub1.The stably transfected cells line BRL-3A/256 is estab-lished successfully.