靶向Egr-1基因RNA干扰慢病毒载体的构建及鉴定  

Construction and identification of early growth response-1 RNA interference lentiviral vector

作　　者：张魁[1] 曹剑[1] 董然[1] 

机构地区：[1]北京市心肺血管疾病研究所首都医科大学附属北京安贞医院心外科,北京100029

出　　处：《中华实用诊断与治疗杂志》2013年第12期1185-1188,共4页Journal of Chinese Practical Diagnosis and Therapy

基　　金：国家自然科学基金(81070202)

摘　　要：目的构建靶向人早期生长反应因子(early growth response-1,Egr-1)基因RNA干扰(RNA interference,RNAi)慢病毒载体。方法针对Egr-1基因设计并合成1条Egr-1过表达序列和4条干扰序列,过表达序列与双酶切Ubi-MCS-3FLAG载体连接,干扰序列与双酶切hU6-MCS-CMV-EGFP连接,DNA测序鉴定重组载体。共转染人脐静脉血管内皮细胞后(OE-RNAi-1,OE-RNAi-2,OE-RNAi-3,OE-RNAi-4)Western blot检测Egr-1蛋白表达,筛选有效干扰靶点,实时定量荧光PCR检测Egr-1mRNA,验证干扰效果。结果测序表明Egr-1过表达及RNAi慢病毒载体构建成功;Western blot结果显示,OE-RNAi-4组Egr-1蛋白表达抑制率最高(P<0.05);实时荧光定量PCR验证结果显示,OE-RNAi-4组Egr-1mRNA表达受到明显抑制(P<0.05)。结论本研究成功构建了靶向Egr-1基因RNAi慢病毒载体,为进一步研究Egr-1在静脉桥血管再狭窄中早期内皮功能失调作用机制及基因治疗提供了实验依据。Objective To construct early growth response-1 （Egr-1） RNA interference （RNAi） lentiviral vector. Methods One over-expression （OE） and four RNA interference lentiviral vectors were designed and synthetized for Egr-1. OE lentiviral vector was linked with enzyme cleavage carrier of Ubi-MCS 3FLAG, and RNAi lentiviral vector was linked with enzyme cleavage carrier of hU6-MCS-CMV-EGFP. The recombined carriers were confirmed by DNA sequencing, and virus was collected and virus titer was measured. After human umbilical vein endothelial cell were co- transfected （OE-RNAi-1, OE-RNAi-2, OE-RNAi-3, OE-RNAi-4）, the expression of Egr-1 was detected by Western blot, and the most effective target vector was chosen. Egr-1 mRNA was confirmed by real-time quantitative PCR. Results DNA sequencing revealed that OE and RNAi recombinanted lentiviral vectors were constructed successfully. Western blot demonstrated that the inhibition rate of Egr-1 protein in OE-RNAi-4 group was higher than that in the other groups （P〈0.05）. Real-time quantitative PCR confirmed that Egr-1 mRNA in OE-RNAi-4 group was significantly inhibited （P〈0.05）. Conclusion The Egr-1 RNAi lentiviral vector has been constructed successfully, which provides a foundation for further study on mechanism and gene therapy of endothelial dysfunction in vein graft.

关 键 词：冠状动脉旁路移植术 静脉桥血管再狭窄 早期生长反应因子-1 RNA干扰 

分 类 号：R654.2[医药卫生—外科学]