靶向大鼠Smad3基因的siRNA筛选及其shRNA重组慢病毒的构建

摘要

Objective To design and select the siRNA targeting rat Smad3 gene and construct recombinant lentivirus expressing Smad3small hairpin RNA(shRNA).Methods Six pairs of siRNAs of rat smad3 gene named as siRNA 001-006 and an irrelevant siRNA were designed and synthesized.The siRNAs were transfected into the rat liver cell line BRL-3A, and the inhibitory effects on SMAD3protein expression were determined by Western Blot.According to the efficient siRNAs and irrelevant siRNA sequences, DNA sequences coding corresponding shRNAs were synthesized and inserted into plasmid plentilox3.7-shRNA(pLL3.7-shRNA).Then, the pLL3.7-shRNA, pRSV-rev, pMDLG/pRRE and VSV-G were transfected into 293FT cells together to package lentivirus.Infective vector titers were determined by GFP gene expression analysis using flow cytometry.Results Western Blot analysis demonstrated that siRNA001,siRNA005 and siRNA006 had high interfering efficiency, with the inhibitory rate being 83.36％, 86.99％ and 64.88％, respectively.Meanwhile, the irrelevant siRNA had no apparent effect.Enzyme digestion and DNA sequencing results showed that shRNA recombinant vector pLL3.7-shRNA were constructed successfully.Infective lentivirus have been produced.Conclusion Three siRNAs targeted to rat Smad3 gene have been selected.The recombinant lentivirus which express corresponding shRNA have been constructed successfully,laying foundations for further studying the influence on liver regeneration or liver fibrosis after Smad3 silence.%目的 设计并筛选针对大鼠Smad3基因的siRNA,构建靶向Smad3基因的shRNA重组慢病毒.方法 针对大鼠Smad3基因设计并合成6对siRNA(siRNA001～006)及1对无关对照siRNA,转染大鼠肝细胞株BRL-3A,应用Western Blot检测各siRNA对SMAD3蛋白表达的抑制作用,挑选抑制效率高的siRNA.依据所得的序列合成并克隆到pLL3.7载体中,与包装质粒pRSVrev、pMDLg-pRRE和VSV-G共转染293FT细胞获得靶向Smad3的慢病毒.通过流式细胞仪绿色荧光蛋白(GFP)荧光计数来检测病毒滴度.结果 Western Blot检测证实siRNA001、siRNA005、siRNA006对SMAD3蛋白表达的抑制作用明显,抑制率分别可达83.36%、86.99%及64.88%.而对照siRNA无明显作用.酶切和测序结果显示Smad3 shRNA及对照shRNA重组载体质粒pLL3.7shRNA构建成功.将构建的质粒进行慢病毒包装可产生有感染活性的慢病毒颗粒.结论 筛选出针对大鼠Smad3基因有明显抑制作用的3对siRNA,并成功构建表达相应shRNA的4种重组慢病毒,为研究调控Smad3的表达对肝再生或肝纤维化的影响提供了实验条件.