小鼠4-1BB特异性RNA干扰表达载体的构建及体外瞬时干扰效应的研究

目的 采用小片段RNA干扰(small interference RNA,siRNA)技术,通过构建系列小鼠4-1BB(m4-1BB)基因的特异性siRNA表达载体,体外观察对m4-1BB基因的瞬时沉默作用,筛选具有最佳干扰效果的siRNA表达载体.方法 设计并合成4条针对小鼠4-1BB全长特异性的siRNA寡核苷酸序列,插入真核表达载体(pENTRTM/U6 ),构建m4-1BB序列特异性siRNA 表达载体,根据靶位点分别命名为p336、394、498及763 siRNA,无关干扰质粒为pLacZ siRNA;通过体外m4-1BB真核表达质粒p4-1BB分别与各siRNA表达载体同时转染COS-7细胞,48h后通过RT-PCR和FACS(fluorescence-activated cell sorter,荧光活化细胞分选仪)观测COS-7细胞4-1BB mRNA及蛋白表达水平.结果经测序分析,各m4-1BB siRNA表达载体构建成功;其中p498 siRNA与p4-1BB双质粒转染COS-7 细胞,48h后可观察到细胞表面4-1BB分子明显下调,其胞内mRNA水平也显著降低,与无关干扰组比较,差异有统计学意义(P＜0.05).其余siRNA载体,几乎无干扰效应,与无关干扰组比较,差异无统计学意义(P＞0.05).结论我们所构建的p498 siRNA 真核表达载体,能明显干扰m4-1BB蛋白的瞬时表达,可望用于m4-1BB Lentivirus干扰表达系统的构建,进一步在小鼠原代T淋巴细胞内实现高效4-1BB表达沉默,以深入研究4-1BB对T淋巴细胞的生物学影响.

Construction and suppressive effect of siRNA expression vector targeting mouse 4-1BB in vitro

Objective To construct the small interfering RNA(siRNA) expression vector specific to mouse 4-1BB gene and observe its silencing effect in vitro and identify the one with high efficiency.Methods The pENTRTM/U6 siRNA expression vectors were constructed by gene recombination,then transfected into the cultured COS-7 cells.At 48h after transfection,the mRNA and protein level were separately detected by RT-PCR and FACS.Results The pENTRTM/U6 siRNA expression vectors were successfully constructed.And LipofectamineTM 2000 mediated gene transfection of p498 siRNA expression vector into COS-7 cells downregulated the mRNA and protein level of 4-1BB at 48h after transfection.Conclusion The pENTRTM/U6-p498 siRNA expression vector can specifically and efficiently inhibit 4-1BB gene expression,which may provide a foundation to construct 4-1BB Lentivirus and study 4-1BB gene function in mouse T lymphocytes.