实用临床医药杂志2013，Vol.17Issue(3)：1-4,27,5.DOI:10.7619/jcmp.201303001
人Hiwi miRNA干扰质粒的构建及其鉴定
Construction and identification of miRNA recombinant eukaryotic expression vectors of Hiwi
摘要
Abstract
Objective To construct the miRNA eukaryotic expression vectors of HIWI, and explore its activity of inhibiting the gene and protein expression in human Hepatocellular carcinoma 7721 cell. Methods Four specific micro interference RNA expression vectors specific to Hiwi and one non-homologous negative control vector were designed and constructed. After identification, restriction analysis and DNA sequencing were conducted, the miRNA vectors and the control one were inserted into pcDNA TM6. 2-GW/EmGFPmiR vector. The recombinant miRNA vectors were transfect-ed into 7721 cell. The expression of Hiwi gene miRNA was determined by RT-PCR and Western blot was used to examine the expression of target protein. Results Sequencing suggested that eukaryotic expression vectors targeting Hiwi gene possessed correct read frame and nucleotide sequence. RT-PCR and Western blot results showed that the level of mRNA and target protein of Hiwi in transfected 7721 cells significantly reduced compared to the controls. Conclusion MiRNA eukaryotic expression vectors targeting Hiwi are constructed successfully, which will provide the foundation for study of Hiwi gene function in hepatocellular carcinoma.关键词
Hiwi基因/miRNA干扰/真核表达质粒/肝癌Key words
Hiwi gene/ miRNA/ plasmid construction/ hepatocellular carcinoma分类
医药卫生引用本文复制引用
郝波,蒋建霞,施瑞华,张国新,成道祥..人Hiwi miRNA干扰质粒的构建及其鉴定[J].实用临床医药杂志,2013,17(3):1-4,27,5.基金项目
国家自然基金项目(NSFC81101800) （NSFC81101800）
江苏省南京市科技发展研究项目资助(2010ZD220) （2010ZD220）
