[1]周琪,梁后杰,阎晓初,等.RNA干扰Neuropilins-2基因真核表达载体的构建及其鉴定[J].第三军医大学学报,2007,29(08):691-694.
 ZHOU Qi,LIANG Hou-jie,YAN Xiao-chu,et al.Construction and effect of Neuropilins-2 eukaryotic expression vector for RNA interference[J].J Third Mil Med Univ,2007,29(08):691-694.
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RNA干扰Neuropilins-2基因真核表达载体的构建及其鉴定(/HTML )
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《第三军医大学学报》[ISSN:1000-5404/CN:51-1095/R]

卷:
29卷
期数:
2007年第08期
页码:
691-694
栏目:
论著
出版日期:
2007-04-30

文章信息/Info

Title:
Construction and effect of Neuropilins-2 eukaryotic expression vector for RNA interference
作者:
周琪梁后杰阎晓初彭秋平周进明吴峰钟大平边志衡
第三军医大学西南医院:病理科,肿瘤科
Author(s):
ZHOU Qi LIANG Hou-jie YAN Xiao-chu PENG Qiu-ping ZHOU Jin-ming WU Feng ZHONG Da-ping BIAN Zhi-heng
Institute of Pathology, Department of Phymatology, Southwest Hospital, Third Military Medical University, Chongqing 400038, China
关键词:
NRP2RNA干扰真核表达载体
Keywords:
Neuropilins-2 RNA interference eukaryotic expression vector
分类号:
Q503;Q782
文献标志码:
A
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。
Abstract:
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-l to generate siRNA eukaryotic expression vector, DH5α 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. pGenSil-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.

参考文献/References:

周琪,梁后杰,阎晓初,等. RNA干扰Neuropilins-2基因真核表达载体的构建及其鉴定[J]. 第三军医大学学报, 2007, 29(8):691-694.

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更新日期/Last Update: 2008-10-28