基础医学与临床2011，Vol.31Issue(8)：922-927,6.
BMP4-siRNA重组腺病毒的构建及鉴定
Construction and identification of the recombinant adenovirus expressing BMP4-siRNA
摘要
Abstract
Objective To construct a recombinant adenovirus expressing siRNA target sites for human BMP4.Methods Six pairs of oligonucleotides containing siRNA target sites for human BMP4 using Dharmacon's siDESIGN program were subcloned into the Sfi I site of pSOS, which has been inserted the coding regions of human BMP4, resulting in pSOS-sirBMP4. Then the pSOS-sirBMP4 were transfected into HEK-293 cells, and GFP signal level was used to assess the silencing efficiency of different siRNA target sites. Four pairs of oligonucleotides were chosed and subcloned into shuttle plasmid pSES-HUS and authenticity of PCR amplified sequences and the oligonucleotide cassettes were verified by DNA sequencing. The correctly identified recombinant plasmid was linearized with Pme I and transformed into BJ5183-Ad-easy competent cells containing adenovirus backbone vector to produce recombinant adenovirus DNA by homologous recombination. Then the recombinant adenovirus DNA was linearized with Pac I and transfected into 293 cells to make adenovirus. the silencing efficiency of recombinant adenovirus was assess by RT-PCR and Western blot in MDA-MB-231 cell line. MTT assay was used to detect the proliferation of MDA-MB-231 cells transfected with pAd-sirBMP4. Results Four pairs of oligonucleotides with high silencing efficiency were chosed and subcloned into shuttle plasmid pSES-HUS to make recombinant adenovirus. After infected with the recombinant adenovirus BMP4-siRNA, the expression of BMP4 sharply decrease in the human breast cancer cell line MDA-MB-231. Conclusion The recombinant adenovirus expressing siRNA target sites for human BMP4 has been successfully constructed and may be used to silence the expression of BMP4 in MDA-MB-231 cell line effectively.关键词
BMP4/siRNA/腺病毒/乳腺癌/骨转移Key words
BMP4/ siRNA/ adenovirus/ breast cancer/ osseous metastasis分类
医药卫生引用本文复制引用
郭丹,黄佳祎,孙治君..BMP4-siRNA重组腺病毒的构建及鉴定[J].基础医学与临床,2011,31(8):922-927,6.基金项目
重庆市卫生局(2010-2-157) （2010-2-157）
