您的位置:首页  > 论文页面

第三代慢病毒包装体系的建立及大鼠IRF1重组干扰慢病毒的生产

发表时间:2016-08-15  浏览量:1417  下载量:637
全部作者: 耿满满,朱文华,徐 晶,蒋丛姗,吴小颍,王 飔,孟列素,吕社民
作者单位: 西安交通大学基础医学院,环境与基因相关疾病教育部重点实验室
摘 要: 目的:建立安全稳定的第三代慢病毒包装体系并生产出可用于干扰大鼠IRF1基因的干扰病毒颗粒。方法:1)利用带有荧光的高表达质粒对包装的质粒比例、转染试剂进行探索,根据流式细胞仪(fluorescence activated cell sorter,FACS)对病毒滴度的测定结果,建立性价比最高的第三代慢病毒包装体系。根据慢病毒可以整合的特性,建立利用real-time PCR测定没有荧光的慢病毒颗粒滴度的方法。2)设计针对大鼠IRF1的干扰序列并筛选出最佳干扰序列,利用建立好的慢病毒包装体系生产大鼠的IRF1病毒颗粒,并检测滴度。结果:1)成功建立了稳定的第三代慢病毒包装系统和利用real-time PCR 方法检测无荧光病毒的滴度检测方法。2)筛选出针对大鼠IRF1的干扰序列,利用该序列生产出慢病毒颗粒,并测定了滴度以进行下一步的细胞及在体研究。结论:1)第三代慢病毒包装体系已被成功建立。2)获得了针对大鼠IRF1的重组干扰慢病毒。
关 键 词: 分子生物学;第三代慢病毒载体;滴度测定;IRF1
Title: Establishment of the third generation lentiviral vectors and construction of rat’s IRF1 recombinant lentivirus
Author: GENG Manman, ZHU Wenhua, XU Jing, JIANG Congshan, WU Xiaoying, WANG Si, MENG Liesu, LÜ Shemin
Organization: Key Laboratory of Environment and Genes Related to Diseases of Ministry of Education, School of Basic Medical Science, Xi’an Jiaotong University
Abstract: Objective: To establish the safe and stable third generation lentivirus package system and the construction of rat’s IRF1 recombinant lentivirus. Methods: 1) Roundly explored the third generation lentiviral vector (LV) package system about the transfection ratio of the vectors and transfection reagent by the overexpression plasmid with fluorescence which can be assayed by fluorescence activated cell sorter (FACS). Then we have optimized this system in cost saving and highest titers according to the characteristics of LV which can be integrated into host genome. 2) We designed the RNAi sequence targeted to rat’s IRF1 and screened out the interference sequence silenced the gene expression up to 70% and acquired the recombinant interference LV to rat’s IRF1 gene and assayed its titration. Results: 1) The stable third generation LV packaging system is successfully constructed and the titration assay method about the virus without fluorescence by real-time PCR is established. 2) The recombinant lentiviral vector targeted to rat’s IRF1 is constructed and the interference lentivirus is packaged, whose titration is also determined. Conclusions: 1) The third generation LV packaging system is successfully established. 2) The recombinant lentivirus targeted to rat’s IRF1 is acquired.
Key words: molecular biology; the third generation lentiviral vectors; titration assay; IRF1
发表期数: 2016年8月第15期
引用格式: 耿满满,朱文华,徐 晶,等. 第三代慢病毒包装体系的建立及大鼠IRF1重组干扰慢病毒的生产[J]. 中国科技论文在线精品论文,2016,9(15):1577-1586.
 
1 评论数 0
暂无评论
友情链接