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诱骗受体3基因RNAi慢病毒载体构建、包装及鉴定

发表时间:2016-09-15  浏览量:1662  下载量:426
全部作者: 赵团结,许英晨,伍冀湘
作者单位: 首都医科大学附属北京同仁医院普通外科
摘 要: 为构建诱骗受体3(decoy receptor 3,DcR3)的RNA干扰(RNA interference,RNAi)慢病毒载体,并对其干涉效果进行鉴定,设计合成了3条针对DcR3基因的shRNA干扰序列,构建于慢病毒载体质粒中,转化大肠杆菌感受态细胞,筛选阳性克隆进行菌落PCR鉴定,对PCR鉴定阳性的克隆进行测序分析,将目的质粒感染293T细胞实时定量PCR(real-time quantitative PCR,RT-qPCR)测定包装的病毒滴度,转染HepG2细胞RT-qPCR筛选干扰效果最佳的载体。PCR结果显示,扩增的目的基因DcR3已成功插入pLKD-CMV-G&PR-U6-shRNA载体,阳性克隆测序结果显示与目的基因序列一致,目的质粒转染48 h后,80%以上293T细胞表达绿色荧光,RT-qPCR法测定包装的病毒滴度为分别为6.58×108, 4.60×108, 4.63×108 IU/mL,其中慢病毒载体3干扰效果最好,达到61.44%.
关 键 词: 外科学;诱骗受体3;RNA干扰;shRNA;慢病毒载体
Title: Construction, packaging and identification of RNAi lentiviral vectors carrying the decoy receptor 3 gene
Author: ZHAO Tuanjie, XU Yingchen, WU Jixiang
Organization: Department of General Surgery, Beijing Tongren Hospital, Capital Medical University
Abstract: To construct, package and identify RNA interference (RNAi) lentiviral vectors carrying the decoy receptor 3 (DcR3) gene, 3 short hairpin RNA interference sequences for DcR3 gene were designed, synthesized and constructed into the lentiviral vector plasmids. The lentiviral vector plasmids were then transformed into competent Escherichia coli cells, and the positive clones were identified by PCR. The identified recombinant vectors were used to infect 293T cells. The titer of packaged virus was determined using real-time quantitative PCR (RT-qPCR) 48 h after infection. PCR analysis showed that the amplified target gene was inserted in the pLKD-CMV-G&PR-U6-shRNA vector. Digestion analysis showed that the reconstructed plasmids were consistent with the theoretical fragment, and the sequencing results showed that the positive fragment was exactly the same as the target gene. Forty-eight hours after infection, more than 80% of cells displayed green fluorescence. RT-qPCR assay showed that the packaged viral titer was 6.58×108, 4.60×108, 4.63×108 IU/mL, respectively. RT-qPCR assay showed that the lentiviral vector 3 knockdowned about 61.44% of the expression of DcR3 mRNA.
Key words: general surgery; decoy receptor 3; RNA interference; shRNA; lentiviral vectors
发表期数: 2016年9月第17期
引用格式: 赵团结,许英晨,伍冀湘. 诱骗受体3基因RNAi慢病毒载体构建、包装及鉴定[J]. 中国科技论文在线精品论文,2016,9(17):1770-1777.
 
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