山东医药2016，Vol.56Issue(26)：13-16,4.DOI:10.3969/j.issn.1002-266X.2016.26.004
人SNCG基因shRNA慢病毒载体质粒的构建及鉴定
Construction and identification of shRNA Ientivirus vector pIasmid of human SNCG gene
摘要
Abstract
Objective To construct short hairpin RNA (shRNA)lentiviral vector plasmid of human SNCG gene and to detect the changes of SNCG mRNA and protein expression in human endometrial carcinoma cells HEC-1-A and Ishikawa after transfection.Methods Three SNCG gene specific shRNA sequences were designed,which were SNCG-KD1,SNCG-KD2 and SNCG-KD3,respectively.Using Lipofectamine 2000 to construct a lentiviral vector plasmid in 293T cells.Hu-man endometrial carcinoma HEC-1-A and Ishikawa cells were transfected with SNCG-KD1,SNCG-KD2 and SNCG-KD3 lentiviral vector plasmids.Using real-time PCR to detect the expression of SNCG mRNA,and Western blotting to detect SNCG protein expression.Results Age I and EcoR I were used to connect the three groups of positive transformation.The specific bands were in agreement with the predicted results by 1% agarose gel electrophoresis.The results of the analysis and identification of Lite Chromas (VERSION 2.01)gene sequencing were in agreement with GeneBank database align-ment,which showed that the synthesis of three pairs of shRNA Oligo DNA SNCG sequences SNCG-KD1,SNCG-KD2 and SNCG-KD3 inserted correctly.After the three lentiviral vectors were transfected into HEC-1-A and Ishikawa cells,the ex-pression of SNCG mRNA and protein in HEC-1-A and Ishikawa cells was significantly decreased (P <0.05).Conclusion Three shRNA SNCG lentiviral vector plasmids are successfully constructed and transfected into HEC-1-A and Ishikawa cells,which may effectively knock down the SNCG gene and inhibit the expression of SNCG mRNA and protein in HEC-1-A and Ishikawa cells.关键词
SNCG基因/短发夹干扰RNA/慢病毒载体质粒/子宫内膜癌/基因沉默Key words
SNCG gene/short hairpin RNA/lentivirus vector plasmid/endometrial carcinoma/gene silencing分类
医药卫生引用本文复制引用
李文怡,范余娟,徐红,范江涛,孙丹..人SNCG基因shRNA慢病毒载体质粒的构建及鉴定[J].山东医药,2016,56(26):13-16,4.基金项目
国家自然科学基金资助项目（81460396）。 （）
