登革1型病毒广州06株基因组全长感染性克隆的构建与鉴定  

Construction and characterization of an infectious cDNA clone of novel dengue serotype 1 virus Guangzhou 06 strain

作　　者：刘然[1] 韩剑峰[1] 陈水平[1] 于曼[1] 姜涛[1] 邓永强[1] 秦成峰[1] 秦鄂德[1] 

机构地区：[1]军事医学科学院微生物流行病研究所病原微生物生物安全国家重点实验室,北京100071

出　　处：《军事医学科学院院刊》2008年第4期323-327,共5页Bulletin of the Academy of Military Medical Sciences

基　　金：国家自然科学基金资助课题(30770107)

摘　　要：目的:构建本室新分离的登革1型病毒广州06株基因组全长感染性cDNA克隆,为深入研究登革病毒致病机制奠定基础。方法:根据登革1型病毒广州06株基因组全序列中单一酶切位点设计特异引物,分别扩增并克隆5个病毒亚基因组cDNA片段,将其逐一拼接连入pW SK 29低拷贝质粒载体,获得病毒全长cDNA克隆,并将其体外转录为RNA,再转染细胞获得恢复病毒。进一步通过RT-PCR扩增病毒5′和3′非编码区序列和特异性间接免疫荧光对其恢复病毒进行鉴定。结果和结论:构建获得登革1型病毒广州06株基因组全长感染性cDNA克隆,其恢复病毒与野生型病毒具有相似的生物学特性。Objective ： To construct an infectious genomic full-length clone of new isolated dengue serotype 1 virus GZ-06 strain to gain a better understanding of the pathogenesis of dengue virus . Methods ： According to the unique restriction endonuclease cleavage sites in viral genome of denguel virus GZ-06 strain, five cDNA fragments were subcloned respectively, and then its full-length cDNA was ligated into a low-copy plasmid vector pWSK-29. The recovered virus of GZ-06 strain was generated after its transcripts derived from the full-length cDNA clone were electroporated into host cells. For further observation, RT-PCR and IFA were carried out to characterize the properties of recovered virus. Results and Conclusions： Recovered virus derived from the full-length clone exhibited biological characteristics similar to its parental virus in terms of cytopathogenesis in mosquito C6/36 cell, feature of IFA and 5′ and 3′ terminal sequence specificity.

关 键 词：登革热病毒 全长CDNA克隆 恢复病毒 

分 类 号：R373.33[医药卫生—病原生物学]