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Regulation of promoter functions of gE, vhs and swine cytokine genes by pseudorabies virus IE180 and EP0
|關鍵字:||pseudorabies virus;假性狂犬病毒;PRV;transactivator;promoter;transient transfection;轉錄活化因子;啟動子;短暫轉染||出版社:||獸醫學系||摘要:||
假性狂犬病毒（pseudorabies virus；PRV），是屬於-病毒亞科，為目前國內豬重大疾病的致病原之一。基因體為線性雙股的DNA分子，約150 kb，其特性為複製週期(reproductive cycle)短，病毒於培養時傳播快，且通常會造成宿主細胞明顯的細胞病變(cytopathic effect；CPE)，並可於神經系統建立潛伏性感染(latent infection)。先前的研究指出，假性狂犬病的基因表現會依循一種層階式(cascade-like fashion)的調控方式進行，由立即早期(immediate early、α)、早期(early、β)及晚期(late、r)基因依序表現，由上游控制下游基因的表現。其中立即早期蛋白IE180有學者證實其為一個轉錄活化因子(transactivator)，能活化病毒早期及晚期基因的表現；而病毒的早期基因產物EP0也被證實能調控早期及晚期基因的表現，亦屬轉錄活化因子。因此本篇論文首先將針對假性狂犬病毒的IE180與EP0來探討其對晚期基因gE與vhs 啟動子(promoter)的調控情形，進而去了解IE180與EP0在假性狂犬病毒複製的過程中對於本身晚期基因的調控機轉。本實驗以PCR的方式建構含gE或vhs 啟動子之真核表現報導載體。在序列的分析結果發現，gE啟動子上具有GCF、EGR-2、GATA-1、C/EBP gamma及3個Sp1轉錄因子的結合位；而vhs 啟動子上則具有CAAT、TATA及4個SP1等轉錄因子的結合位，同時也發現其具有一個PRV IE180的結合位，但此IE180結合位對於vhs 啟動子是非必須的。
在IE180與EP0對gE與vhs啟動子(promoter)的調控情形方面，我們將含gE或vhs 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-細胞進行短暫轉染(transient transfection)，並以CAT assay來進行分析，結果發現gE與vhs啟動子會受到IE180的誘導活化，但卻會受到EP0的抑制；而如果在高劑量IE180的誘導下vhs啟動子的活性反而會受到抑制。另外，本實驗先將含gE或vhs 啟動子之報導載體送入LMtk-細胞中表現24小時後，再以superinfection的方式來觀察；結果發現PRV確實會活化gE與vhs啟動子。
此外，本病毒有由親神經性轉變為親內臟型的傾向，常會造成豬隻淋巴系統受損、免疫抑制，而併發呼吸道、消化道或全身性之感染。因此本研究亦探討當假性狂犬病感染宿主後，IE180與EP0對宿主細胞的免疫反應會引發何種變化，希望能藉由本報告分析結果可作為未來假性狂犬病疫苗之研發有較多的參考依據。本實驗以PCR的方式建構含IL-1β、IL-2、TNF-α或TNF-β啟動子之真核表現報導載體。首先以superinfection的方式來觀察PRV對這四個啟動子的影響，結果發現PRV會活化IL-2與TNF-α啟動子。另外我們將含IL-1β、IL-2、TNF-α或TNF-β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 transfection並以CAT assay來進行分析，結果發現IL-1β、IL-2與TNF-α的啟動子會受到低劑量IE180(1 ug)的誘導活化，但高劑量(3 ug或5 ug)卻反而會抑制；而在EP0方面，低劑量的EP0(1 μg)會活化IL-1β與TNF-α啟動子的表現，但高劑量(3 ug或5 ug)下只會抑制TNF-α啟動子的表現；EP0不管劑量多寡對IL-2與TNF-β啟動子並無明顯的影響。
Pseudorabies virus (PRV), one of the pathogen of pig diseases, is a member of α-herpesvirinae. The genome is a double-stranded DNA of approximately 150 kb. Its replication cycle and the associated cytopathic effects on cultured cells are easily observed. After infection, PRV can establish a latency in nervous system. In previous studies, it is shown 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). The IE180 appears to function as a transactivator and can activate the expression of early and late genes. The early gene product EP0, with a transactivating function, also regulates the early and late genes. In this study, we explored the regulation of IE180 and EP0 on promoters of viral late gene, gE and vhs. We used PCR to clone the gE and vhs promoters and their sequences were determined. These DNA segments, which contain gE or vhs promoters, were subcloned into a chloramphenicol acetyltransferase (CAT) reporter gene, respectively; both reporter gene constructs exhibited promoter activity in transient transfection. After sequence analysis, we found a GCF site, an EGR-2 site, a GATA-1 site, a C/EBP gamma site and three Sp1 binding sites in the gE promoter. For the vhs promoter, we found it contains a CAAT site, a TATA box, four Sp1 binding sites and a PRV IE180 binding site. The roles of these cis-acting elements were dissected by mutational analysis. The regulation of the gE and vhs promoter by IE180 and EP0 was examined by using co-transfection. Results demonstrated that both the gE and vhs promoters were activated by IE180 whereas they were inhibited by EP0 protein. However, the vhs promoter was suppressed by high dose of IE180. Furthermore, by combining transfection and PRV infection, it was found that latter PRV superinfection could enhance the activity of gE and vhs promoter, indicating that viral product(s) could stimulate these promoters. In addition, the relationship between PRV and expression of cytokine genes was investigated in this study. By a simple PCR method, the promoters of cellular IL-1β, IL-2, TNF-α and TNF-β genes were obtained and their identities were confirmed by DNA sequencing; then they were subcloned into a CAT reporter gene, respectively. Using transient assay, results showed that low concentration of IE180 could enhance the activity of IL-1β, IL-2, or TNF-α promoter whereas these promoters were suppressed by high dose of IE180. The promoter of TNF-was not responsible to IE180. By similar assay, we found that the IL-1β and the TNF-α promoters were stimulated by low dose of EP0. However, the TNF-α promoter was suppressed by high dose of EP0. The promoters of IL-2 and TNF-β were not influenced by the EP0.
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