利用人RNA聚合酶ⅰ构建肠道病毒D68的反向遗传系统

摘要：2018年8月，天津大学生命科学学院；环境科学与工程学院(School of Life Sciences, Tianjin University, 92 Weijin Road, Nankai District, Tianjin 300072, China；S

2018年8月，天津大学生命科学学院；环境科学与工程学院(School of Life Sciences, Tianjin University, 92 Weijin Road, Nankai District, Tianjin 300072, China；School of Environmental Science and Engineering, 92 Weijin Road, Nankai District, Tianjin 300072, China)Tao Wang老师研究团队在《Virus Genes》上发表论文：

“A reverse genetics system for enterovirus D68 using human RNA polymerase I”

“利用人RNA聚合酶ⅰ构建肠道病毒D68的反向遗传系统”

Abstract：

Human enterovirus D68 (EV-D68) is a highly contagious virus, which causes respiratory tract infections. However, no effective vaccines are currently available for controlling EV-D68 infection. Here, we developed a reverse genetics system to recover EV-D68 minireplicons and infectious EV-D68 from transfected plasmids using the RNA polymerase I (Pol I) promoter. The EV-D68 minireplicons contained the luciferase reporter gene, which flanked by the non-coding regions of the EV-D68 RNA. The luciferase signals could be detected in cells after transfection and Pol I promoter-mediated luciferase signal was significantly stronger than that mediated by the T7 promoter. Furthermore, recombinant viruses were generated by transfecting plasmids that contained the genomic RNA segments of EV-D68, under the control of Pol I promoter into 293T cells or RD cells. On plaque morphology and growth kinetics, the rescued virus and parental virus were indistinguishable. In addition, we showed that the G394C mutation disrupts the viral 5'-UTR structure and suppresses the viral cap-independent translation. This reverse genetics system for EV-D68 recovery can greatly facilitate research into EV-D68 biology. Moreover, this system could accelerate the development of EV-D68 vaccines and anti-EV-D68 drugs.

摘要：

人类肠道病毒D68 (EV-D68)是一种高传染性病毒，可引起呼吸道感染。然而，目前还没有有效的疫苗可用于控制EV-D68感染。在这里，科研人员开发了一个反向遗传系统，利用RNA聚合酶I (Pol I)启动子从转染的质粒中恢复EV-D68的微型复制子和传染性EV-D68。EV-D68微型复制子包含荧光素酶报告基因，该基因位于EV-D68 RNA的非编码区两侧。转染后细胞中可检测到荧光素酶信号，且Pol I启动子介导的荧光素酶信号明显强于T7启动子介导的荧光素酶信号。在Pol I启动子的控制下，将含有EV-D68基因组RNA片段的质粒转染到293T细胞或RD细胞中，生成重组病毒。在斑块形态和生长动力学上，获救病毒和亲本病毒没有区别。此外，科研人员发现G394C突变破坏了病毒的5'-UTR结构并抑制了病毒的帽独立翻译。该EV-D68的反向遗传系统为EV-D68的生物学研究提供了极大的便利。此外，该系统可加快EV-D68疫苗和抗EV-D68药物的开发。

该论文中，对293T(人胚胎肾293T)细胞和RD(横纹肌肉瘤)的体外培养是使用Ausbian特级胎牛血清完成的。欲了解或购买Ausbian特级胎牛血清可以联系北京缔一生物400-166-8600.