Lenti-Toll样受体4-siRNA的构建及对大鼠肺泡巨噬细胞
NR8383的影响
张小艺;孟红艳;彭敏;司皓;马宏博
【期刊名称】《中国医药导报》 【年(卷),期】2015(000)029
【摘要】目的：构建 Toll样受体(TLR)4基因慢病毒表达载体介导的小干扰 RNA(siRNA),观察其对大鼠肺泡巨噬细胞NR8383 TLR4的沉默效应。方法设计4条针对TLR4基因的siRNA靶序列,经合成、退火、酶切,与线性载体GV115连接,转入细菌感受态细胞,经聚合酶链反应(PCR)筛选阳性克隆、测序鉴定。共转染293T细胞,包装产生慢病毒颗粒,分别感染NR8383细胞,实时定量PCR(RT-PCR)检测NR8383细胞TLR4 mRNA的表达。根据筛选结果,选取最有效的载体进行病毒大量包装。结果经测序验证,慢病毒载体构建成功并获得相应的慢病毒,NR8383细胞感染慢病毒后,TLR4基因mRNA表达明显下降,LV-TLR4-RNAi(Tgt-2)作用较明显,敲减效率达到65%。结论成功构建针对靶向大鼠TLR4基因的RNAi慢病毒载体,为进一步研究TLR4信号通路在急性肺损伤中的作用奠定基础。%Objective To construct the lentiviral vector-mediated small interfering RNA(siRNA) of TLR4, and examine the silent effect on the rat alveolar macrophage NR8383. Methods 4 siRNA sequences targeting TLR4 gene were designed. After synthesis, annealing and restriction enzyme digestion, TLR4-RNAi were ligated with GV115, transformed into competent bacterial cells, and confirmed by PCR and DNA sequencing. 293 T cells were co-transfected. The four
Lenti-Toll样受体4-siRNA的构建及对大鼠肺泡巨噬细胞NR8383的影响
