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为了研究埃博拉病毒的生物学特性及其与宿主的相互作用,构建无致病基因的复制型埃博拉病毒样颗粒(Transcription and replication competent virus-like particles,trVLPs)成为在BSL-2实验室中进行研究的理想途径。本研究目的是表达和鉴定复制型埃博拉病毒样颗粒(trVLPs)。本研究利用埃博拉病毒7个蛋白的基因组片段(VP40、VP24、GP、VP35、VP30、L、NP)和一个报告基因片段重组构建了表达质粒,瞬时转染293T细胞,通过荧光素酶检测系统测定报告基因的表达活性确定了复制型埃博拉病毒样颗粒(trVLPs)的成功表达。进一步通过超离转染细胞的上清纯化得到病毒样颗粒(trVLPs),在透射电子显微镜下观察到了典型的丝状结构。对病毒样颗粒进行荧光标记并感染Vero细胞,在荧光显微镜下观察到细胞中出现点状和丝状的荧光信号。复制型埃博拉病毒样颗粒的成功表达为进一步研究埃博拉病毒的生物学特性以及与宿主相互作用机制提供了基础。
In order to study the biological characteristics of Ebola virus and its interaction with the host, the construction of pathogenic gene-free replication-competent virus-like particles (trVLPs) Room for research the ideal way. The aim of this study was to express and identify replicative Ebola virus-like particles (trVLPs). In this study, recombinant plasmids were constructed by using the genome fragments of seven Ebola virus proteins (VP40, VP24, GP, VP35, VP30, L, NP) and a reporter gene fragment, transiently transfected into 293T cells and detected by luciferase Systemic determination of the reporter gene expression activity confirmed the successful expression of replicon Ebola virus-like particles (trVLPs). The virus-like particles (trVLPs) were further purified by supernatant transfection of the transfected cells. Typical filamentous structures were observed under a transmission electron microscope. The virus-like particles were fluorescently labeled and infected with Vero cells, and fluorescent and fluorescent spots were observed in the cells under fluorescent microscope. The successful expression of replicative Ebola-like particles provides the basis for further study of the biological characteristics of Ebola virus and its mechanism of interaction with host.