人呼吸道合胞病毒微型复制子的构建

Construction of minireplicon of human respiratory syncytial virus?

摘要:

摘要：【目的】构建RSV微型复制子（minireplicon）并进行功能学研究。【方法】构建含RSV病毒前导序列（Leader, genomic promoter, Le）、转录起始信号（gene start, GS）、多克隆酶切位点、转录终止信号（gene end, GE）和尾随序列(Trailer, antigenomic promoter, Tr)的基因片段GSGE，通过引入T7 RNA多聚酶（T7 RNA polymerase, T7 RNP）启动子、克隆入载体px8δT和插入增强型绿色荧光蛋白（Enhanced Green Fluorescent Protein, EGFP）等多步操作，获得RSV微型复制子重组质粒px8δT/GSGE1/EGFP和px8δT/GSGE2/EGFP。同时构建可表达大蛋白（large protein，L）的质粒pcDNA3.1/L及表达转录延长/转录终止抑制因子（M2 ORF 1 protein，M2-1）的质粒pcDNA3.1/M2-1。通过脂质体法共转染RSV微型复制子质粒及四种RSV核壳体蛋白质粒至可表达T7 RNP的BSRT7/5细胞系，倒置荧光显微镜及流式细胞仪分析EGFP的表达情况。【结果】成功构建了px8δT/GSGE1/EGFP和px8δT/GSGE2/EGFP及pcDNA3.1/L和pcDNA3.1/M2-1，五质粒共转染BSRT7/5细胞，倒置荧光显微镜及流式细胞仪均观察到绿色荧光的表达。【结论】构建的RSV微型复制子具有转录和复制功能，将有助于进一步开展以RSV反向遗传操作为基础的RSV疫苗研究。

Abstract:

Abstract:［Objective］The reverse genetics technology is an important way to develop genetically engineered attenuated living human respiratory syncytial virus (RSV) vaccine candidates. As the pilot experiment, it is necessary to prepare RSV minireplicon and investigate its biological activity.［Methods］After the gene start and gene end (GSGE) fragment containing the sequence of leader (Le), gene start (GS), multiple cloning sites (MCS), gene end (GE) and trailer (Tr) was synthesized and positioned under the control of T7 RNA promoter, we cloned this fragment further into px8δT vector. Then, the enhanced green fluorescent protein (EGFP) was cloned into the above px8δT vector, and RSV minireplicon plasmids px8δT/GSGE1/EGFP and px8δT/GSGE2/EGFP were finally obtained. Meanwhile, two ORFs of nucleocapsid proteins of large protein (L) and transcription elongation/antitermination factor (M2-1) were cloned and constructed to produce pcDNA3.1/L and pcDNA3.1/M2-1. At the end,we co-transfected a RSV minireplicon plasmid and four nucleocapsid protein plasmids into BSR T7/5 cell lines expressing T7 RNA polymerase by lipofectamine 2000, and analyed the expression of EGFP by inverted fluorescent microscopy and fluorescence activated cell sortor (FACS), respectively.［Results］ The px8δT/GSGE1/EGFP, px8δT/GSGE2/EGFP, pcDNA3.1/L and pcDNA3.1/M2-1 were successfully constructed. After co-transfecting, EGFP can be observed in the transfected BSR T7/5 cells under fluorescent microscopy and by FACS. ［Conclusion］The constructed RSV minireplicon is able to replicate and transcript, which provides a solid foundation for further RSV vaccine study by RSV reverse genetics.