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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，C0020 Sh2-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Tmub1 RNAi 慢病毒稳定感染细胞系BRL-3A/256。

英文摘要:

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 cotransfected 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 interference 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 established successfully.

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