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  

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

作　　者：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] 

机构地区：[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

出　　处：《中国生物工程杂志》2013年第9期1-9,共9页China Biotechnology

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

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。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 px8T-PT1-N, px88T-PT1-P, px8T-PT1-M2-1 and px8T-lYF1-L encoding RSV nueleocapsid proteins, respectively, and one minigenome plasmid of pSCll-E containing open reading frame of the enhanced green fluroreeent 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 geuome by deletion and mutation of its genes.

关 键 词：呼吸道合胞病毒 微型基因组 反向遗传学 

分 类 号：Q34[生物学—遗传学]