标题: 探讨登革热二型病毒PL046之质体建构及报导基因的选择
Construction of dengue virus PL046 strain genomic cDNA and reporter chimera
作者: 赖建□
Chien-Hsueh Lai
杨昀良
Yun-Liang Yang
生物科技学系
关键字: 登革热;质体建构;dengue;cDNA clone
公开日期: 2005
摘要: 登革热病毒是全长11kb的正单股形RNA病毒,属黄质病毒科(Flaviviridae)黄质病毒属(Genus Flavivirus)。目前对于登革热病毒在组装过程中,如何正确选择病毒RNA包覆至结构性蛋白的机制并不清楚。
为瞭解登革热病毒组装讯号所在的位置,实验计画先获得登革热病毒全长的cDNA clone,再将登革热病毒的结构性及非结构性基因,分别与报导基因(lacZ)作重组DNA,置于CMV启动子的控制。以含有病毒基因序列(结构性或非结构性基因)及报导基因的报导质体对细胞进行转染,藉细胞转录出含报导基因及病毒基因序列的RNA,再以登革热病毒感染被转染的细胞,产生病毒复制及组装所需之蛋白;任一段带有组装讯号的RNA在细胞中应可被组装至病毒颗粒。将过程中产生的病毒收集后,再次感染细胞,进行报导基因活性测试;若测得酵素活性则表示该重组基因上所带有的病毒基因序列具有组装时所需要的组装讯号。
根据前人之研究,在抗生素浓度减半及低温的培养状况下,筛选具有登革热二型病毒全长基因序列质体的E. coli,进行一连串质体建构、报导基因的选择及表现,建构出含有报导基因及病毒基因序列(结构性或非结构性基因)的质体,期望能为整个实验建立可行的基本架构。
结果显示,所获得的登革热病毒全长cDNA clone置于CMV启动子下游时,并无法表现出具有感染性的登革热病毒。在以X-gal staining分析含有部份病毒基因的报导质体表现时发现,当lacZ与病毒基因分别在不同ORF(bicistronic)的状况下,lacZ位于上游时,产生的蓝色细胞数最多;当lacZ位于下游的ORF并在前方加入IRES时,产生的蓝色细胞数只有在位于上游时的二十分之一。未来在进行组装讯号分析时,建议采用bicistronic的方式,并尝试利用其它的报导基因,改变侦测的状况,期望能提升报导基因所表现之活性被侦测的情形。
Dengue virus (genus Flavivirus, family Flaviviridae) is a single- stranded, positive-sense RNA virus with an 11-kb genome. It is not clear how dengue viruses select viral RNA packaging into structural protein during encapsidation.
The goal of these studies was to develop a dengue virus packaging system for possible future use in defining the dengue virus RNA packaging signal. First, we have to obtain full length cDNA clone of dengue virus type 2. Then, the viral structural or nonstructural cDNA were ligated with lacZ reporter gene separately, and were driven by CMV promoter. BHK-21 cells were transfected by plasmids that contain partial sequence of dengue genome (structural or nonstructural genes) and lacZ reporter gene. The plasmids would produce RNAs containing either the structural or nonstructural sequence of dengue genome alone with the reporter gene. Then, the cells were infected with dengue virus type 2, which would provide viral proteins necessary for viral packaging and replication. Any RNAs contains viral packaging signal would assemble with the viral structural proteins. The media were collected, and used for infecting new BHK-21 cells. If the activity of the reporter gene expresses in BHK-21 cells, this would suggest that the partial sequence of dengue viral genome may contain the signals for viral packaging.
For the propagation of plasmids, the method of Sriburi et al. was followed. They propagated E. coli at room temperature (20-25□C) under low level of antibiotic selection to construct entire cDNA of a dengue serotype 2 virus genome. I have proceeded througth a series of plasmid constructions, and selection and expression of the reporter genes in an attempt to build the foundation of the dengue viral packaging signal research.
The results showed that the full length cDNA clones of dengue virus type 2 driven by CMV promoter did not produce infectious virions. Analyses of expression of the reporter gene with partial sequence of dengue genome revealed that when lacZ located upstream and in a bicistronic unit with the viral sequence, the expression was the best. When lacZ was driven by an IRES and was located downstram, the amounts of the expression was only one-twentieth of that in upstream. In the future, reporter gene expression shall observe the bicistronic manner, and use of other reporter genes may vary the detection method and increase the usefulness of the construct.
URI: http://140.113.39.130/cdrfb3/record/nctu/#GT009228533
http://hdl.handle.net/11536/76951
显示于类别:Thesis


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