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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 rescue the human respiratory syncytial virus (RSV) minigenome based on the T7 promoter expression system by RNAi reverse genetics. Methods: Construction of helper plasmids px8δT-PT1-N, px8δT-PT1-P, px8δT-PT1-M2-1 and px8δT-PT1-L which can express four kinds of nucleocapsid protein of RSV and enhanced plasmids containing enhanced green fluorescent protein green fluorecent protein (EGFP), open reading frame (ORF), leader region / genomic promoter, gene start (GS), gene end (GE) T7 RNA polymerase (T7 RNP) was introduced into the above five plasmids, respectively, using the mini-genomic recombinant plasmid pSC11-E of cis-acting elements such as a trailer region / antigenomic promoter. ) Promoter. After identified correctly, BSR T7 / 5 cells stably expressing RSV-infected T7 polymerase were transfected with pSC11-E to confirm the rationality of mini-genome coding plasmid design, Transfection of BSR T7 / 5 cells, and fluorescence microscopy observation of the expression of fluorescence to determine the success of the rescue. Results: The RSV minigenome 5 plasmid rescue system based on the T7 promoter expression system was successfully constructed and rescue was achieved. Conclusion: The successful construction of this system will help to carry out gene modification studies on RSV virus in the future.