人呼吸道合胞病毒微型复制子功能的初步研究

发布时间：2018-05-24 01:41

本文选题：人呼吸道合胞病毒 + 微型复制子　；参考：《安徽医科大学》2009年硕士论文

【摘要】： 目的:人呼吸道合胞病毒(Human Respiratory Syncytial Virus,RSV)广泛分布于世界各地,是导致婴幼儿严重下呼吸道感染最重要的病毒病原,目前尚无有效的治疗方法。利用RNA病毒反向遗传学操作获得的减毒RSV活病毒,具有稳定的安全性和良好的免疫原性。为开展RSV反向遗传学研究奠定基础,拟先尝试开展RSV微型复制子(minireplicon)构建及功能的初步研究。RSV微型复制子一般包括RSV病毒前导序列(Leader, genomic promoter, Le)、转录起始信号(gene start, GS)、可插入外源基因的多克隆酶切位点(mutiple cloning site, MCS)、转录终止信号(gene end, GE)和尾随序列(Trailer, antigenomic promoter, Tr)。通过引入T7 RNA多聚酶(T7 RNA polymerase, T7 RNP)启动子、克隆入载体px8δT等多步操作,可获得RSV微型复制子质粒,经转染可提供T7 RNP及RSV复制所必须的全部蛋白的细胞,可了解RSV微型复制子能否实现RSV基因组的复制和转录功能。本研究在于构建和表达RSV复制必须的两种重要功能蛋白大蛋白( Large protein,L )和转录延长/转录终止抑制因子(transcription elongation/antitermination factor,M2 ORF 1 protein,M2-1),结合先前构建的RSV微型复制子质粒等,完成RSV微型复制子质粒功能的初步研究。方法:此前我们人工合成了含RSV Le、GS、MCS、GE和Tr的GSGE基因片段,PCR方法分别在GSGE片段的5′和3′端引入T7RNA多聚酶启动子获得GSGE1及GSGE2片段,并分别克隆入载体px8δT,获得RSV微型复制子质粒px8δT/GSGE1和px8δT/GSGE2,进一步将增强型绿色荧光蛋白(Enhanced Green Fluorescent Protein, EGFP)开放读码框(Open Reading Frame, ORF)分别克隆入px8δT/GSGE1和px8δT/GSGE2 ,获得RSV微型复制子重组质粒px8δT/GSGE1/EGFP和px8δT/GSGE2/EGFP。本研究中我们构建了可表达两种核壳体蛋白或聚合酶蛋白的真核表达质粒,包括表达L蛋白的质粒pcDNA3.1/L及表达M2-1蛋白的质粒pcDNA3.1/M2-1。通过脂质体法共转染RSV微型复制子重组质粒及核壳体蛋白质粒(包括先期构建的可表达RSV核蛋白(nucleoprotein, N)及磷蛋白(phosphoprotein, P)的质粒, pcDNA3.1/N和pcDNA3.1/P)至可表达T7 RNA多聚酶(T7 RNA polymerase, T7 RNP)的BSR T7/5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:经限制性内切酶分析及核酸序列分析,证明成功构建了pcDNA3.1/L和pcDNA3.1/M2-1,经RT-PCR及western blot分析证实L及M2-1蛋白有表达,五质粒共转染BSR T7/5细胞,倒置荧光显微镜及流式细胞仪均观察到绿色荧光。结论:成功克隆A亚型RSV Long株L和M2-1基因,并在真核细胞中获得表达。构建的RSV微型复制子具有转录和复制功能,为进一步的RSV反向遗传操作奠定了基础。
[Abstract]:Objective: human Respiratory Syncytial virus (Respiratory Syncytial) is widely distributed all over the world. It is the most important viral pathogen leading to severe lower respiratory tract infection in infants. The live attenuated RSV virus obtained by reverse genetic operation of RNA virus has stable safety and good immunogenicity. To lay the foundation for the research of RSV reverse genetics, A preliminary study on the construction and function of minireplicon.RSV microreplons generally include the leading sequence of RSV virus, genomic promoter, Leer, transcriptional initiation signal, gene startand glutathione, and the polyclonal digesting site of foreign gene, named mutiple cloning. Site, MCSA, transcriptional termination signal gene end, GE) and trailer, antigenomic promoter, Tran. By introducing T7 RNA polymerase T7 RNA polymerase (T7 RNA polymerase) promoter and cloning into vector px8 未 T, the microreplicon plasmid of RSV can be obtained. After transfection, the cells that can provide all the proteins necessary for T7 RNP and RSV replication can be transfected. It is possible to understand whether RSV microreplicators can replicate and transcribe RSV genomes. The purpose of this study was to construct and express two important Large proteins (Large protein L) and transcription elongation/antitermination factor-M 2 ORF 1 protein (M2-1), which are necessary for RSV replication, and to combine with previously constructed RSV microreplicon plasmids. The function of RSV microreplicon plasmid was studied. Methods: previously, we artificially synthesized GSGE gene fragments containing RSV LeGS-GSGS-C GE and Tr to obtain GSGE1 and GSGE2 fragments by introducing T7RNA polymerase promoter into 5 'and 3' end of GSGE fragment, respectively. The RSV microreplicon plasmids px8 未 T/GSGE1 and px8 未 T / GSGE2 were cloned into the vector px8 未 T, respectively. The enhanced green Green Fluorescent Protein, EGFP) open reading frame was further cloned into px8 未 T/GSGE1 and px8 未 T/GSGE2, respectively, and the RSV microreplicon recombination plasmids px8 未 T/GSGE1/EGFP and px8 未 Tr GSGE2EGFPwere obtained. In this study, we constructed eukaryotic expression plasmids expressing two nuclear shell proteins or polymerase proteins, including plasmid pcDNA3.1/L expressing L protein and plasmid pcDNA3.1% M2-1 expressing M2-1 protein. Co-transfection of RSV microreplicon recombinant plasmid and nuclear shell protein particles (including previously constructed plasmids expressing RSV nucleoprotein (N) and phosphoprotein (P) by liposome method, pcDNA3.1/N and pcDNA3.1 / P) to express T7 RNA polymerase T7 RNA polymerase, T7 BSR T7 / 5 cell line, The expression of EGFP was analyzed by inverted fluorescence microscope and flow cytometry. Results: pcDNA3.1/L and pcDNA3.1 / M2-1 were successfully constructed by restriction endonuclease analysis and nucleic acid sequence analysis. The expression of L and M2-1 proteins was confirmed by RT-PCR and western blot analysis. The five plasmids were co-transfected into BSR T7 / 5 cells. Green fluorescence was observed by inverted fluorescence microscope and flow cytometry. Conclusion: the L and M2-1 genes of subtype A RSV Long strain were cloned and expressed in eukaryotic cells. The constructed RSV microreplicon has the functions of transcription and replication, which lays the foundation for further reverse genetic manipulation of RSV.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R373

【共引文献】