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

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.