医学分子生物学杂志2011，Vol.8Issue(4)：334-339,6.DOI:10.3870/j.issn.1672-8009.2011.04.011
MEKK3基因siRNA重组腺病毒表达载体的构建及其对胃腺癌AGS细胞MEKK3的表达抑制
Construction of MEKK3 siRNA Recombinant Adenovirus Vector and Its Inhibitory Effect on MEKK3 in Gastric Adenocarcinomas AGS Cells
摘要
Abstract
Objective To construct a small interfering RNA ( siRNA ) adenovirus expression vector targeting MEKK3 and to study its effect on the inhibition of MEKK3 in gastric adenocarcinoma AGS cells. Methods Three different siRNA template DNA sequences of MEKK3 gene were designed by Genscript siRNA design software. The corresponding DNA fragments were synthesized in vitro , annealed and then cloned into the pRNAT-H1. 1/Adeno shuttle vector. The recombinant shuttle vector was confirmed by DNA sequencing, then transformed into Escherichia coli BJ5183 carrying backbone plasmid PAdeasy-1 to obtain adenovirus plasmid through homologous recombination. The adenovirus plasmid was transfected into 293A cells to form adenovirus particles. The harvested adenovirus particles were transduced into AGS cells. Western blot was carried out to analyze the suppression of MEKK3 in AGS cells. Results DNA sequencing and western blot confirmed the effective silence effect on MEKK3 by two MEKK3 siRNA adenovirus expression vector pRNATH1. 1/Adeno-MEKK3 siRNA3 , with a suppression ratio up to 89 ％. Conclusion The recombinant pAd-MEKK3-siRNA expression vector effectively targeting MEKK3 provides a tool for further investigation of MEKK3 function in gastric adenocarcinoma cells.关键词
腺病毒载体/小干扰RNA/MEKK3基因Key words
adenovirus vector/ siRNA/ MEKK3 gene分类
生物科学引用本文复制引用
钱凤英,兰风华,董荔红,黄俏佳..MEKK3基因siRNA重组腺病毒表达载体的构建及其对胃腺癌AGS细胞MEKK3的表达抑制[J].医学分子生物学杂志,2011,8(4):334-339,6.基金项目
福建省自然科学基金(No.2009J01181),南京军区医药卫生科研基金(No.08MA100) （No.2009J01181）
