Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/66453
標題: 假性狂犬病毒對猪介白素2基因表現之調控
Regulation of Swine IL-2 Gene Expression orProduction by Pseudorabies Virus Infection.
作者: 鄭珮儀
Cheng, Pei-Yi
關鍵字: pseudorabies virus;假性狂犬病毒
出版社: 獸醫微生物學研究所
摘要: 
假性狂犬病毒(Pseudorabies Virus; PRV)亦稱為猪第一型疱疹病毒,屬於阿爾法疱疹病毒亞科之ㄧ員,是一種具有封套的DNA 病毒。研究指出PRV 基因的表現是層階式的,依據其出現的順序可分為三類,分別是立即早期(immediate-early, IE)、早期(early, E)、及晚期基因(late, L),此三期基因依序出現且彼此調控。前人的實驗證實當猪隻受到 PRV 感染時,其輔助型 T 淋巴球會增加並且會促使IL-2 的分泌,但是關於其真正作用的詳細機轉目前仍不清楚,因此本實驗主要目的即是探討 PRV 對於猪IL-2(sIL-2)啟動子的調節機制。
本試驗首先以PCR 的方式得到全長為 2.2-kb 的 sIL-2 啟動子,接著再將之選殖到CAT 報導載體中。為了更進一步了解 sIL-2 啟動子的功能區域及DNA 的作用位置,我們依序由5’端進行刪除,產生五種不同長度的sIL-2 啟動子,並証實它們皆具有活性。接著再以 superinfection 的方式來探討 PRV 對這些啟動子的調控情形,結果發現在PRV 感染後的6 小時,五種不同長度的sIL-2 啟動子活性皆有明顯增加的趨勢,在12 小時的效果最明顯。之後,我們想更近一步了解此種情形是受病毒哪個時期的基因調控,因此使用了不同的抑制劑來觀察,分別是Phosphonoacetic acid (PAA:抑制DNA合成)、Actinomycin D (AD: 抑制transcription)、及cycloheximide (CHX: 抑制蛋白質合成)。由於病毒IE 蛋白產生並不需要任何病毒蛋白的參與,因此當PAA 及AD 存在時並不會抑制IE 蛋白的合成,但若加入蛋白質抑制劑CHX 時,則病毒IE 蛋白不會產生。結果顯示,當PAA 及AD 存在時,PRV 仍然可以造成sIL-2 啟動子活化,而當CHX 存在時,PRV 則無法活化sIL-2 啟動子,由此結果推測PRV 可能會藉由IE蛋白來調控sIL-2 啟動子。為了更近一步證實此假說,我們將含有五種不同長度sIL-2 啟動子之報導載體分別與含PRV IE180 或EP0 的真核表現載體,利用 LMtk-細胞進行 transient cotransfection,以CAT assay 來進行分析IE180 與EP0 對這些啟動子的調控情形。結果顯示低劑量的IE180 與EP0確實會活化sIL-2 啟動子,但當濃度增加後,則反而有抑制的現象。综合以上結果可知 PRV 會藉由IE180 及EP0 蛋白來調節sIL-2 啟動子,而且此種調控並不受IE like binding site、NF AT、及TFIID 等transcription factorbinding site 的影響,故推測PRV 可能會藉由其他途徑來活化sIL-2 啟動子。

Pseudorabies virus (PRV) is a swine alphaherpesvirus that can cause Aujeszky,s disease. Previous reports indicated that the PRV transcription is regulated in a cascade-like fashion. The genes are classified into three kinetic classes in viral lytic infection: immediate-early (IE), early (E), and late (L). In previous study it was demonstrated that the PRV can stimulate T helper lymphocytes to secrete IL-2 in vitro, but the regulation of the IL-2 activation pathway is still not very clear. In this study, we explored the molecular mechanism of swine IL-2 (sIL-2) promoter regulation. The sIL-2 promoter region of 2.2-kb in length was generated by PCR and was subcloned into a CAT reporter vector. To identify the DNA response element, functional promoter assay with progressively 5'deleted sIL-2 promoter fragments was performed. In a superinfection assay, we transfected various sIL-2 promoter reporter plasmids into LMtk- cells, the results showed that PRV infection could enhance the activity of five different sIL-2 promoters at 6 h postinduction. When the sIL-2 promoter was co-transfected with PRV IE180 or EP0 expression vector, it showed that a low concentration of IE 180 or EP0 could enhance activity of sIL-2 promoter whereas were suppressed by a high dose of IE 180 or EP0. In summary, we demonstrated that the sIL-2 promoter could be regulated by the IE180 or EP0 of PRV.
URI: http://hdl.handle.net/11455/66453
Appears in Collections:微生物暨公共衛生學研究所

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