2009-08-012024-05-14https://scholars.lib.ntu.edu.tw/handle/123456789/656842摘要：蝦白點病毒（WSSV）是ㄧ個大的含有外套膜的雙股圓形 DNA病毒，基因組約有300 kb，對蝦的致死率高達90-100%。由於缺乏蝦的細胞株，對於蝦白點病毒基因表現的調控機轉目前大都不甚瞭解。計畫總主持人羅教授的研究室首先利用cycloheximide處理蝦，然後以microarray及RT-PCR的技術找到三個蝦白點病毒的極早期基因ie1, ie2及ie3。其中ie1基因的啟動子在昆蟲Sf9細胞中具有很高的表現，可能是因為STAT結合在ie1基因的啟動子上所造成的。本研究計畫主要的目的是要探討蝦白點病毒的極早期基因之調控機制。首先由序列分析發現在ie1啟動子上游-102到-97及ie2 啟動子上游-352到-347的區域含有Sp1結合序列。另外也發現在蝦（Penaeus monodon）的基因庫中有一個EST序列（HPA-N-S01-EST0038），與斑馬魚的Sp1有同源序列，而斑馬魚的Sp1蛋白質序列和人類的Sp1蛋白質在DNA結合區域有極高的相似度，顯示不同來源的Sp1蛋白質可能都會結合在類似的DNA序列上。目前已知，人類的Sp1是一種會結合在很多GC序列（GC boxes）的廣泛存在且多功能的蛋白質，會影響細胞週期及末端分化等細胞功能。而Sp1的辨識序列在許多細胞與病毒基因的啟動子上都可以找到。 Sp1也會藉由與TAFII130結合而活化沒有TATA序列的啟動子。而ie2與ie3的啟動子並不含TATA序列，因此Sp1很可能對ie2及ie3的轉錄作用是必要的。本研究目標是要（1）選殖與分析P. monodon的Sp1蛋白質；（2）利用昆蟲細胞證明蝦的Sp1對蝦白點病毒極早期基因ie1與ie2的調控作用；（3）證明蝦的Sp1調控ie2的轉錄作用。本研究將會闡述蝦白點病毒的基因表現調控機制，並能發展新的策略以便能抑制蝦白點病毒感染蝦及在蝦的細胞內複製。<br> Abstract: White spot syndrome virus (WSSV) is a large enveloped virus with about 300 kb double-stranded circular DNA that kills shrimps with a mortality rate of up to 90-100%. Because the lack of a shrimp cell line, the molecular mechanism that regulates the transcription of WSSV genes remain largely unknown. Professor Lo’s laboratory first identified three immediate-early genes, ie1, ie2, and ie3, of WSSV by using microarray and RT-PCR analysis from cycloheximide-treated shrimps. Among these immediate-early genes, ie1 displays high transcription activity in Sf9 insect cells, which can be attributed to the binding of STAT to the ie1 promoter. The main goal of this study is to analyze the regulation of immediate-early genes of WSSV. Sequence analysis reveals that two consensus Sp1-binding elements are present in the region from –102 to –97 and –352 to –347 in the ie1 and ie2 promoters, respectively. Moreover, one expressed sequence tag (EST, HPA-N-S01-EST0038) in shrimps, Penaeus monodon, is homologous to the zebra fish Sp1. Comparison of Sp1 amino acid sequence from zebra fish and H. sapiens revealed a high degree of sequence identify in the DNA-binding domain of the proteins, suggesting that Sp1 from different species bind to similar DNA sequences. As is well known, human Sp1 is a ubiquitous and versatile protein that binds to GC-rich recognition elements (GC boxes) to influence different cellular functions such as cell cycle progression and terminal differentiation. Additionally, Sp1 recognition elements are distributed widely in various promoters of cellular and viral genes. Sp1 also interacts with TAFII130 to initiate transcription of TATA-less promoters. The ie2 and ie3 promoters do not contain TATA sequences, therefore, Sp1 may be necessary for the transcription of ie2 and ie3. The aims of this research are to (1) clone and analyze the recombinant PmSp1 from P. monodon; (2) examine the effect of PmSp1 on the transcription of ie1 and ie2 of WSSV in insect cells; (3) demonstrate PmSp1 involves in the regulation of the ie2 gene. This investigation will elucidate the molecular mechanisms that regulate the expression of WSSV genes and develop new strategies to inhibit infection and replication of WSSV in P. monodon.極早期基因蝦白點病毒Sp1Penaeus monodon宿主與病毒交互作用Immediate-early geneswhite spot syndrome virus (WSSV)Sp1Penaeus monodonhost/virus interaction