人NHE-1基因miRNA干扰载体构建、鉴定及干扰效应评价  

Construction of human NHE-1 miRNA expression plasmid and it's interference effect evaluation

作　　者：黄美风[1] 方伯言[2] 

机构地区：[1]辽宁医学院研究生院,辽宁锦州121001 [2]辽宁医学院附属第一医院神经内科,辽宁锦州121001

出　　处：《中国现代医学杂志》2010年第18期2721-2724,2728,共5页China Journal of Modern Medicine

基　　金：辽宁省教育厅科研基金资助项目(No:20060524)

摘　　要：目的构建人钠氢交换蛋白-1(NHE-1)基因的miRNA特异性干扰表达载体并转染293细胞,筛选其有效性。方法设计4对miRNA oligo,依次通过退火、连接,构建含NHE-1前体miRNA的重组干扰质粒pcDNATM 6.2-GW/NHE-miR和阴性对照质粒pcDNATM 6.2-GW/neg-miR,并转染293细胞,经Re-al-Time PCR评价干扰效应,筛选出干扰效应最佳的靶序列。结果测序证实目的片段正确插入载体中,成功构建4个人NHE-1基因miRNA干扰载体;Real-Time PCR证实完成干扰效应评价,并根据NHE-1基因转录调控区及其蛋白分子结构功能区评价结果,得到NHE1-4是最佳干扰片段。结论成功构建针对人NHE-1基因的miRNA特异性有效干扰质粒,为后续NHE-1基因腺病毒miRNA表达载体的构建及其在调控APP裂解相关酶类的功能研究奠定了基础。【Objective】 To construct specific miRNA expression vectors targeting human NHE-1 gene and detect the suppression efficiency of the constructed vectors in 293 cell line.【Methods】Four pairs of specific pre-miRNA single stranded DNA oligos for NHE-1 were designed and synthesized,then via annealing and ligating in order,the recombined interfering plasmid pcDNATM 6.2-GW/NHE-miR and negative control plasmid pcDNATM 6.2-GW/neg-miR were constructed.The recombined plasmids were transfected into 293 cell line.The efficiency was detected by Real-Time PCR.【Results】Human NHE-1 gene in 293 cells was found have high expression through PCR detec-tion.The miRNA oligos of NHE-1 were correctly cloned into the pcDNATM6.2-GW/miR plasmids and confirmed by DNA sequencing.Four miRNA RNAi vectors targeting human NHE-1 gene had been constructed successfully.De-tected by Real-Time PCR,NHE1-4 is the best efficiency target sequence.【Conclusion】Specific miRNA RNAi ex-pression vector targeting human NHE-1 gene had constructed successfully.This will lay a foundation for the con-struction of miRNA RNAi adenovirus expression vector targeting human NHE-1 gene and the study in the functions of regulating APP cleavage enzymes.

关 键 词：钠氢交换蛋白-1 干扰载体 干扰效应 

分 类 号：R741.02[医药卫生—神经病学与精神病学]