N-甲基-D-天冬氨酸受体2B亚基/RNAi慢病毒载体的构建及鉴定

目的前扣带皮层(anterior cingulate cortex,ACC)神经元N-甲基-D-天冬氨酸受体2B亚基(N-methyl-D-aspar-tate,NR2B)对晚期癌痛患者的恶性情绪体验形成具有重要作用。构建靶向NR2B基因的RNA干扰(RNA interference,RNAi)慢病毒表达载体,可为下调ACC神经元NR2B靶基因的表达提供有力工具。方法构建靶向目的基因NR2B的RNAi重组慢病毒表达载体并酶切鉴定,再以NR2B/RNAi慢病毒表达载体和NR2B的基因表达质粒共转染293T工具细胞,以Westernblot方法检测其对目的基因NR2B的敲减效率。结果酶切鉴定NR2B/RNAi慢病毒表达载体Ⅰ、Ⅱ及Ⅲ构建成功。West-ern blot结果显示,NR2B/RNAi慢病毒表达载体Ⅰ、Ⅱ及Ⅲ均对NR2B靶基因有敲减效果,但以NR2B/RNAi慢病毒表达载体Ⅲ效率最高。结论成功构建可高效沉默NR2B靶基因的NR2B/RNAi慢病毒表达载体,为下调ACC神经元NR2B的基因表达治疗晚期癌痛患者的恶性情绪体验提供了有力工具。

Construction of lentiviral recombinants expressing shRNA targeting N-methyl-D-aspartate gene

Objective N-methyl-D-aspartate in anterior cingulate cortex is relevant to the dispirital mood of cancer patients with pain.The aim of this study is to construct shRNAs-expressing lentiviral recombinants targeting the rat N-methyl-D-aspartate(NR2B) gene.Methods Firstly the shRNAs-expressing lentiviral recombinants targeting the rat NR2B gene were constructed and then identified by PCR analysis.The shRNAs-expressing lentiviral recombinants targeting the rat NR2B gene were then transfected into 293T cells with the NR2B gene-expressing plasmid.Western blot was employed to assess the gene silencing efficacy of these recombinants.Results PCR analysis and sequencing showed that shRNAs were correctly inserted into the lentiviral vector and three recombinants were constructed successfully.All of these three recombinants could achieve gene knockdown effects,and the recombinants Ⅲ had the most significant gene silencing effect among them.Conclusion shRNAs-expressing lentiviral recombinants targeting the NR2B gene have been successfully constructed and can be used in transgenic analgesic research.