人生长因子受体结合蛋白2相关的接头蛋白1基因重组慢病毒RNA干扰载体的构建及鉴定

Construction and identification of human growth factor receptor binding protein 2 associated binding 1 gene recombinant lentivirus RNA interference vector

目的 探讨构建人类生长因子受体结合蛋白2相关的接头蛋白1（Gab1）基因重组慢病毒RNA干扰载体的方法,研究其对人脐静脉血管内皮细胞株EA.hy926的沉默效率.方法 针对人类Gab1基因设计5个靶点的小干扰RNA （siRNA）序列,并分别合成靶序列的Oligo DNA,退火形成双链DNA,与经Hpa I和Xho I限制性内切酶双酶切后的GV118载体连接,产生Gab1-RNA干扰（RNAi）转化子,聚合酶链反应（PCR）筛选阳性克隆并进行测序鉴定.将测序正确的Gab1-RNAi、pHelper 1.0和pHelper 2.0质粒共转染239T细胞,包装产生LV-Gab1-RNAi慢病毒并测定其滴度.将获得的慢病毒分别转染EA.hy926细胞,通过荧光显微镜观察绿色荧光蛋白（GFP）的表达,测定其转染效率;通过反转录-聚合酶链反应（RT-PCR）检测转染后的EA.hy926细胞中Gab1 mRNA表达水平来验证慢病毒干扰载体的基因沉默效率.结果 经PCR分析和测序证实,成功构建LV-Gab1-RNAi慢病毒载体;病毒滴度为3×10^9 TU/ml;荧光显微镜下转染组细胞GFP荧光表达强烈,转染效率达70％以上;RT-PCR证实干扰组细胞Gab1 mRNA表达水平（0.088 ±0.003）较对照（0.947±0.087）组和空白组（0.999±0.067）显著降低（P＜0.01）.结论 成功构建人Gab1基因RNAi慢病毒载体并能在293T细胞中扩增获得足够的病毒滴度,能明显抑制Gab1基因在EA.hy926细胞株中的表达.

Objective To construct and identify a lentiviral vector harboring RNA interference （RNAi） sequence targeting human growth factor receptor-binding protein 2 associated binding protein 1 （Gab1） gene and investigate its silenced effect on Gab1 gene in EA.hy926 cells.Methods Five small interfering RNAs （siRNAs） based on the sequence of Gab1 mRNA in the Genbank were designed and synthesized.The designed and synthesized single-stranded primers were annealed to double-stranded oligo sequences and subcloned into linear GV118 lentiviral plasmid digested by enzyme Hpa I and Xho I to produce Gab1-RNAi lentiviral vectors.After the positive clone was chosen and confirmed by polymerase chain reaction （PCR） and DNA sequencing,Gab1-RNAi lentiviral vectors with pHelper 1.0 and pHelper 2.0 were co-transfected into 293T cells to package lentiviral particles.The recombinant lentivirus interfering vectors were transfected into EA.hy926 cells.Transfection efficiency was evaluated with expression of green fluorescent protein （GFP） determined by fluorescent microscope.The expression of Gab1 in EA.hy926 cells was detected using reverse transcription-polymerase chain reaction （RT-PCR）.Results PCR analysis and DNA sequencing demonstrated that the RNAi sequence targeting the human Gab1 gene was successfully inserted into the lentiviral vector,and the titer of vitrus was 3×10^9 TU/mL.The transfection ratio of EA.hy926 cells was up to 70%.The expression level of Gab1 mRNA （0.088 ± 0.003） was significantly decreased in the Gab1-shGab1 transfected groups as compared with the control groups （0.947 ±0.087,P 〈0.01） and the blank groups （0.999 ±0.067,P 〈0.01）.Conclusion The recombinant lentivirus RNAi vectors expressing human Gab1 gene were successfully constructed and could effectively inhibit the expression of Gab1 gene in EA.hy926 cells.