人SNCG基因shRNA慢病毒载体质粒的构建及鉴定

摘要

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.%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 SNCG-KD1、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 Age Ⅰ和 EcoR Ⅰ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 GenBank 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。