基于T7启动子表达系统的呼吸道合胞病毒微型基因组的拯救

中国生物工程杂志

2013, Vol. 33

Issue (9): 1-9

研究报告

基于T7启动子表达系统的呼吸道合胞病毒微型基因组的拯救

袁锐^1,2, 付远辉², 何金生², 焦月盈², 蒋桂云^1,2, 张梅¹, 彭向雷², 阚学通^1,2

1. 安徽医科大学免疫学教研室合肥 230032;
2. 北京交通大学生命科学与生物工程研究院北京 100044

Rescue of the Minigenome of Human Respiratory Syncytial Virus Based on T7 Promoter Expression System

YUAN Rui^1,2, FU Yuan-hui², HE Jin-sheng², JIAO Yue-ying², JIANG Gui-yun^1,2, ZHANG Mei¹, PENG Xiang-lei², KAN Xue-tong ^1,2

1. Department of Immunology, Anhui Medical University, Hefei 230032, China;
2. College of Life Sciences and Bioengineering, Beijing Jiaotong University, Beijing 100044, China

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摘要： 目的:以T7启动子表达系统为基础,通过RNA病毒反向遗传学操作,拯救人呼吸道合胞病毒(human respiratory syncytial virus, RSV)微型基因组。方法:构建可分别表达RSV四种核壳体蛋白的辅助质粒px8δT-PT1-N,px8δT-PT1-P,px8δT-PT1-M2-1和px8δT-PT1-L以及含增强型绿色荧光蛋白(enhanced green fluorecent protein, EGFP)开放读码框(open reading frame, ORF),RSV病毒前导序列(leader region/genomic promotor),转录起始信号(gene start, GS)、转录终止信号(gene end, GE)和尾随序列(trailer region/antigenomic promoter)等顺式作用元件(cis-acting elements)的微型基因组重组质粒pSC11-E,在上述的5个质粒中,均引入T7 RNA多聚酶(T7 RNA polymerase, T7 RNP)启动子,经鉴定正确后,先以pSC11-E转染RSV感染的可组成型表达T7多聚酶的BSR T7/5细胞进行拯救,确定微型基因组编码质粒设计的合理性,而后通过5个质粒共转染BSR T7/5细胞,并通过荧光显微镜观察荧光的表达情况判断拯救成功与否。结果:成功构建了基于T7启动子表达系统的RSV微型基因组5质粒拯救系统,并实现了拯救。结论:该系统的成功构建,有助于今后对RSV病毒开展基因修饰研究。

关键词： 呼吸道合胞病毒; 微型基因组; 反向遗传学

Abstract: Objective:To establish a T7 promoter based reverse genetics system competent for the rescue of human respiratory syncytial virus. Methods:We construct four helper plasmids of px8δT-PT1-N, px8δT-PT1-P, px8δT-PT1-M2-1 and px8δT-PT1-L encoding RSV nucleocapsid proteins, respectively, and one minigenome plasmid of pSC11-E containing open reading frame of the enhanced green flurorecent protein and cis-acting elements including RSV leader region/promoter, gene start, gene end and trailer region/antigenomic promoter. All these plasmids are under the control of T7 promoter and identified by restriction endonucleoase analysis and Western blot. The pSC11-E is rescued by RSV and the above helper plasmids in BSR T7/5 cells, respectively. Then, the fluorescence expression is observed over time with fluorescence microscopy. Results:We successfully constructed a reverse genetics system based 5 plasmids undert the control of T7 promoter and finished the rescue operation to the minigenome of RSV. Conclusion:This system can be further applied to investigate the function of RSV genome by deletion and mutation of its genes.

Key words: Human respiratory syncytial virus Minigenome Reverse genetics

收稿日期: 2013-04-25 出版日期: 2013-09-25

ZTFLH:

Q34

基金资助: 国家重大新药创制科技重大专项资助项目(2013ZX09103-003-011)

通讯作者: 何金生jshhe@bjtu.edu.cn E-mail: jshhe@bjtu.edu.cn

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引用本文:

袁锐, 付远辉, 何金生, 焦月盈, 蒋桂云, 张梅, 彭向雷, 阚学通. 基于T7启动子表达系统的呼吸道合胞病毒微型基因组的拯救[J]. 中国生物工程杂志, 2013, 33(9): 1-9.

YUAN Rui, FU Yuan-hui, HE Jin-sheng, JIAO Yue-ying, JIANG Gui-yun, ZHANG Mei, PENG Xiang-lei, KAN Xue-tong. Rescue of the Minigenome of Human Respiratory Syncytial Virus Based on T7 Promoter Expression System. China Biotechnology, 2013, 33(9): 1-9.

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https://manu60.magtech.com.cn/biotech/CN/ 或 https://manu60.magtech.com.cn/biotech/CN/Y2013/V33/I9/1

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