Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/52675
標題: 假性狂犬病毒感染細胞內vhs蛋白特性之研究
Characterization of Virion Host Shutoff (vhs) Protein in Pseudorabies Virus-Infected Cells
作者: 張天傑
關鍵字: pseudorabies virus (PRV)
基礎研究
畜牧獸醫類
Virion host shutoff (vhs) protein
UL41 gene
recombinant virus
Northern blot
Southern blot
cytokines
monoclonal antibody
TAP purification system.
假性狂犬病毒
vhs
UL41基因
重組病毒
北方墨點法
南方墨點法
細胞素
單源抗體
TAP載體
摘要: 假性狂犬病毒(pseudorabies virus,PRV)是.疹病毒科的阿爾伐.疹病毒亞科內之一員,可以感染許多的哺乳類動物,豬為其天然宿主。前人研究指出,單純.疹病毒(Herpes simplex virus,HSV)之UL41基因產物virion host shutoff(vhs)蛋白,是病毒顆粒tegument的成分之一。在病毒感染細胞早期便會被釋放出來降解宿主的mRNA,進而抑制宿主細胞蛋白質的合成,使得病毒能在有利的情況下進行複製。目前已有研究發現當HSV感染宿主細胞後,vhs蛋白會幫助HSV逃避細胞內非特異性的防禦機制以及減少抗HSV的IFN-α及IFN-β的活化,而使得病毒能在宿主細胞內複製生長。為了研究假性狂犬病毒的致病機轉,本實驗室已經選殖出假性狂犬病毒TNL株的UL41基因,且證實了此一基因產物確為vhs蛋白,同時於in vitro及in vivo的條件下具有RNase的活性,且能夠抑制PRV病毒斑的形成。我們以螢光蛋白基因取代vhs蛋白基因再利用綠色螢光來篩選vhs蛋白基因缺損之病毒株。最後利用PCR及南方墨點法來篩選具螢光的病毒並證實是vhs蛋白基因缺損病毒。本計畫擬分三年完成。第一年計畫重點在探討vhs蛋白基因缺損病毒的特性。我們將利用北方墨點法及蛋白質定量的方式來探討vhs蛋白基因缺損病毒是否具有RNase的活性,之後再利用one-step growth curve來分析vhs蛋白基因的缺損是否影響病毒的生長複製。第二年計畫重點在比較分析vhs蛋白基因缺損病毒株及強毒株感染細胞後對細胞內細胞素產生的影響。我們將利用半定量RT-PCR及北方墨點法來分析vhs蛋白跟炎症反應有關的細胞素之間的關係,並以此方法來探討vhs蛋白是如何幫助PRV來躲避宿主細胞的免疫反應。我們亦擬進行vhs蛋白之單源抗體的製備,並利用免疫沉澱法來尋找在病毒感染細胞內是否具有vhs蛋白基因的調控蛋白。第三年計畫重點在以in vivo的方式尋找可調控vhs活性之宿主細胞蛋白,將利用TAP載體於細胞內表現具有功能性之vhs融合蛋白質,藉由與vhs蛋白融合之tag進行蛋白複合體的純化,以期獲得與vhs產生交互作用之宿主細胞蛋白,得以進一步瞭解vhs蛋白於感染細胞內的功能,進而推測出PRV感染細胞後之可能作用模式。
Pseudorabies virus (PRV), a member of alphaherpesvirinae, infects many mammalian animals. Swine is its natural host. PRV can cause severe syndromes in piglets but only slight clinical signs in finishing pigs. Thus the adult pigs become carriers of the PRV. Virion host shutoff (vhs) protein is a component of the tegument of alphaherpesviruses, including PRV. It mediates the degradation of mRNA and thereafter shuts off host protein synthesis upon infection. In our previous study, we cloned the vhs gene and demonstrated that the recombinant vhs protein exhibited its biological function, ribonuclease activity. From our previous study, we found that a lot of questions concerning the characteristics and functions of vhs protein in the PRV-infected cells are needed to be solved. Therefore, we planned this three years』 project.In the first year, we will focus on the study of the characteristics of vhs-deletion mutant of PRV. The methods of Northern blot and protein quantitation will be used to investigate the RNase activity of the mutant. One-step growth curve study will also be used to test for the effect of vhs gene deletion on PRV replication in the cell. In the second year, the study will be major on the study of the influence of cytokine production when the vhs gene of PRV is deleted. The methods of semi-quantitative RT-PCR and Northern blot analysis will be used to analyze the correlations between the vhs and cytokine production. In the third year, we will focus our study on searching for the protein which regulates the activity of vhs protein in the PRV-infected cell. The TAP purification system will be applied in this study. By purification of the complex of tagged vhs fusion protein, we expect to identify the protein which reacts with the vhs protein in the PRV-infected cell. The results of this three year』s project will lead us to understand the role of vhs protein during replication of PRV in the cell. Furthermore, this project will provide a clue to understand the role of vhs protein in the pathogenesis of PRV infection.
URI: http://hdl.handle.net/11455/52675
其他識別: NSC96-2313-B005-016-MY3
文章連結: http://grbsearch.stpi.narl.org.tw/GRB/result.jsp?id=1451710&plan_no=NSC96-2313-B005-016-MY3&plan_year=96&projkey=PD9609-1018&target=plan&highStr=*&check=0&pnchDesc=%E5%81%87%E6%80%A7%E7%8B%82%E7%8A%AC%E7%97%85%E6%AF%92%E6%84%9F%E6%9F%93%E7%B4%B0%E8%83%9E%E5%85%A7vhs%E8%9B%8B%E7%99%BD%E7%89%B9%E6%80%A7%E4%B9%8B%E7%A0%94%E7%A9%B6
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