人SNCG基因shRNA慢病毒载体质粒的构建及鉴定  

Construction and identification of shRNA lentivirus vector plasmid of human SNCG gene

作　　者：李文怡[1] 范余娟[1] 徐红[1] 范江涛[1] 孙丹[1] 

机构地区：[1]广西医科大学第一附属医院,南宁530022

出　　处：《山东医药》2016年第26期13-16,共4页Shandong Medical Journal

基　　金：国家自然科学基金资助项目(81460396)

摘　　要：目的构建人SNCG基因短发夹干扰RNA(shRNA)慢病毒载体质粒,并观察用其转染后人子宫内膜癌HEC-1-A和Ishikawa细胞SNCG mRNA和蛋白的表达变化。方法设计3种SNCG基因特异性shRNA序列SNCGKD1、SNCG-KD2、SNCG-KD3,用脂质体Lipofectamine 2000在293T细胞构建成慢病毒载体质粒。用SNCG-KD1、SNCG-KD2、SNCG-KD3慢病毒载体质粒转染人子宫内膜癌HEC-1-A和Ishikawa细胞,用实时荧光定量PCR法检测SNCG mRNA,用Western blot法检测SNCG蛋白。结果利用AgeⅠ和EcoRⅠ双酶切连接转化后的3组阳性转化子,1%琼脂糖凝胶电泳,特异条带与预计结果相符。Chromas Lite比对分析基因测序结果,鉴定结果与Gen Bank数据库一致,表明合成的3对SNCG shRNA序列SNCG-KD1、SNCG-KD2、SNCG-KD3插入正确。HEC-1-A和Ishikawa细胞转染SNCG-KD1、SNCG-KD2、SNCG-KD3慢病毒载体质粒后SNCG mRNA和蛋白的表达均显著降低(P均<0.05)。结论成功构建了3种SNCG shRNA慢病毒载体质粒,用其转染HEC-1-A和Ishikawa细胞后可有效沉默SNCG基因表达。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 

分 类 号：R737.33[医药卫生—肿瘤] Q782[医药卫生—临床医学]