探討登革熱二型病毒PL046之質體建構及報導基因的選擇

Abstract

登革熱病毒是全長11kb的正單股形RNA病毒，屬黃質病毒科（Flaviviridae）黃質病毒屬（Genus Flavivirus）。目前對於登革熱病毒在組裝過程中，如何正確選擇病毒RNA包覆至結構性蛋白的機制並不清楚。 為瞭解登革熱病毒組裝訊號所在的位置，實驗計畫先獲得登革熱病毒全長的cDNA clone，再將登革熱病毒的結構性及非結構性基因，分別與報導基因（lacZ）作重組DNA，置於CMV啟動子的控制。以含有病毒基因序列（結構性或非結構性基因）及報導基因的報導質體對細胞進行轉染，藉細胞轉錄出含報導基因及病毒基因序列的RNA，再以登革熱病毒感染被轉染的細胞，產生病毒複製及組裝所需之蛋白；任一段帶有組裝訊號的RNA在細胞中應可被組裝至病毒顆粒。將過程中產生的病毒收集後，再次感染細胞，進行報導基因活性測試；若測得酵素活性則表示該重組基因上所帶有的病毒基因序列具有組裝時所需要的組裝訊號。 根據前人之研究，在抗生素濃度減半及低溫的培養狀況下，篩選具有登革熱二型病毒全長基因序列質體的E. coli，進行一連串質體建構、報導基因的選擇及表現，建構出含有報導基因及病毒基因序列（結構性或非結構性基因）的質體，期望能為整個實驗建立可行的基本架構。 結果顯示，所獲得的登革熱病毒全長cDNA clone置於CMV啟動子下游時，並無法表現出具有感染性的登革熱病毒。在以X-gal staining分析含有部份病毒基因的報導質體表現時發現，當lacZ與病毒基因分別在不同ORF（bicistronic）的狀況下，lacZ位於上游時，產生的藍色細胞數最多；當lacZ位於下游的ORF並在前方加入IRES時，產生的藍色細胞數只有在位於上游時的二十分之一。未來在進行組裝訊號分析時，建議採用bicistronic的方式，並嘗試利用其它的報導基因，改變偵測的狀況，期望能提昇報導基因所表現之活性被偵測的情形。

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.