登革病毒2型全长cDNA感染性转录体的构建和鉴定

Construction of Infectious RNA Transcripts from Full-length cDNAs of Dengue 2 Viruse

为构建登革病毒感染性克隆,针对登革病毒2型基因组全长cDNA的体外转录方法及感染性转录体进行研究。采用长链RT-PCR技术,扩增DEN2NGC株全长基因组cDNA,以之为模板,用SP6RNA聚合酶系统制备体外转录RNA转录体,分别经乳鼠脑内接种及电穿孔转染BHK-21细胞,观察其感染效应。并从受染鼠脑和病变细胞中提取总RNA,进行RT-PCR扩增、克隆测序以及电镜观察。结果发现,从感染鼠脑和细胞中经RT-PCR均可扩增出病毒特异的基因片段,大小与预期一致;并从乳鼠脑组织和BHK-21细胞中观察到恢复病毒颗粒。上述结果表明本文成功构建的DEN2 NGC株病毒全长cDNA的体外转录体具有感染性,乳鼠脑内接种途径与电穿孔转染细胞一样可成为体外转录体感染宿主细胞、获得恢复病毒的方法。

To study the method of in vitro transcription and the infectivity of in vitro RNA transcripts of full-length cDNAs of dengue 2 viruse, thus to lay the foundation of constructing infectious dengue virus clone. The full-legnth cDNA of DEN2 NGC was amplified by long RT-PCR. With PCR products as templates, the in vitro RNA transcripts could be prepared by the SP6 RNA polymerase system. The transcripts were transfected into BHK-21 by electroporation and inoculated brain of suckling mice respectively. The effect of infectivity of the transcripts was observed. The specific sequences of DEN2 NGC strain were amplifled through RT-PCR of total RNA extracted from brain of mice and infectious cells and the histopathologic changes of mice brain and cells were observed by electron microscope. The results indicated the specific fragment of the dengue virus could be amplified from infected mice brain and cells, and the virion could be observed. The in vitro RNA transcripts of full-length cDNAs of dengue virus were infectious. The dengue virus could be assembled by infecting brain of newborn mice and transfecting cell.