RNA干扰Neuropilins-2基因真核表达载体的构建及其鉴定  

Construction and effect of Neuropilins-2 eukaryotic expression vector for RNA interference

作　　者：周琪[1] 梁后杰[2] 阎晓初[1] 彭秋平[2] 周进明[2] 吴峰[1] 钟大平[2] 边志衡[2] 

机构地区：[1]第三军医大学西南医院病理科,肿瘤科重庆400038 [2]第三军医大学西南医院肿瘤科,重庆400038

出　　处：《第三军医大学学报》2007年第8期691-694,共4页Journal of Third Military Medical University

基　　金：国家自然科学基金(30572159)~~

摘　　要：目的构建Neuropilins-2(NRP2)基因RNA干扰(RNAi)的真核细胞表达载体。方法以NRP2为靶基因,以pGenSil-l质粒为载体,设计构件重组体,根据GenBank数据库提供的NRP2基因核苷酸序列,按照Tuschl设计原则,选择设计两条带发夹结构的核苷酸序列,克隆到空载体pGenSil-l中,转化DH5α菌株,提取质粒,进行限制性内切酶酶切鉴定和测序分析。重组pGenSil-NRP2载体转染LOVO细胞48 h,收获细胞,采用Western blotting检测其NRP2蛋白表达。结果经酶切鉴定筛选出的重组体测序结果与目的序列完全一致,重组载体构建成功。重组体pGenSil-NRP2转染LOVO细胞48 h后NRP2蛋白表达显著降低。结论利用RNAi技术可成功构建抑制NRP2表达的小干扰RNA重组体。Objective To construct Neuropilins-2 eukaryotic expression vector for RNA interference. Methods Recombinant targeting on gene NRP2 was designed and established with plasmid pGenSil-1 based on NRP2 cDNA equences of Genomes. Two pairs of oligonucleotides were synthesized according to the Tuschl and inserted into plasmid pGenSil-1 to generate siRNA eukaryotic expression vector, DHSα strains were transformed, plasmid were extracted, and recombinant vectors were identified by the restriction map and the sequence analysis. The recombinant plasmid （pGenSil-NRP2） was transfected into the cultured LOVO cells. At 48 h after transfection, the whole cell protein was extracted, and the protein level was detected by Western blotting with mouse-anti-human NRP2 monoclonal antibody. Results Recombinant plasmids were completely coincided with the designs by the restriction map and the sequence analysis, pGenSi1-NRP2 expression vector into LOVO cells down-regulated the protein level of NRP2 at 48 h after transfection. The recombinant eukaryotic expression vector were constructed successfully. Conclusion siRNA recombinant can be constructed successfully by RNAi technique for inhibiting NRP2 expression.

关 键 词：NRP2 RNA干扰 真核表达载体 

分 类 号：Q503[生物学—生物化学] Q782