2013-08-012024-05-18https://scholars.lib.ntu.edu.tw/handle/123456789/705731摘要：白點症病毒（WSSV）是一個大的外套膜病毒，含有約300 kb的雙股DNA，感染蝦會導致其高達90-100％的致死率。由於缺乏蝦的細胞株，因此對於白點症病毒基因的轉錄調控的分子機制仍有很多未知。我們最近的研究在草蝦中選殖到第一個kruppel-like factor (PmKLF)。KLF是含有高度相似性的zinc-finger轉錄因子，對轉錄很重要。免疫螢光分析顯示GFP-KLF融合蛋白質主要表現在昆蟲細胞SF9的細胞核中聚集成點狀。此現象也在被白點症病毒感染或沒有感染的白蝦中被發現。將被白點症病毒感染的白蝦中的KLF抑制其表現，會延遲蝦的死亡率，同時也會降低病毒的複製及ie1極早期基因的表現。因此KLF對白點症病毒的感染扮演重要的角色。此外，本研究室也選殖到白蝦的KLF，命名為LvKLF，其與PmKLF具有高達93%的同源性。在白蝦中將LvKLF抑制後，再感染白點症病毒，多數的病毒極早期基因都明顯地被抑制。依據以上這些初步成果，本計畫將證實(1) KLF蛋白質的功能性區域；(2) KLF如何參與 ie1的轉錄；(3) KLF如何調控WSSV140, 108及164等極早期基因。本研究將對KLF如何調控白點症病毒極早期基因的分子機制有更深入的瞭解，對於開發新的策略以預防白點症病毒的感染及複製將會有很大的助益。<br> Abstract: White spot syndrome virus (WSSV) is a large enveloped virus with about 300 kb-double-stranded circular DNA genome that kills shrimps with a mortality rate of up to 90-100%. Because the lack of a suitable shrimp cell line, the molecular mechanism that regulates the transcription of WSSV genes remains largely unknown. Our recent study cloned the first kruppel-like factor (PmKLF) in Penaeus monodon. Kruppel-like factors (KLFs) are highly related zinc-finger proteins that have crucial roles in transcription. Immunofluorescence analysis revealed that GFP-KLF fusion protein is located in the nucleus as dots in an insect cell line, Sf9. Localization of PmKLF in the nucleus is also observed in the hemolymph from white spot syndrome virus (WSSV)-infected and WSSV-uninfected Litopenaeus vannamei. Knockdown of the expression of PmKLF transcript in WSSV-infected shrimp resultes in delayed cumulative mortalities, suggesting that PmKLF is important to WSSV infection. Moreover, inhibition of PmKLF expression reduces WSSV replication and ie1 expression. Additionally, the homolog of PmKLF in L. vannamei has been cloned and names as LvKLF that displays 93% identity. Injection of LvKLF dsRNA into L. vannamei inhibits the transcription of WSSV IE genes. Based on these findings, the aims of this research are to (1) examine the functional domains in KLF; (2) demonstrate the involvement of KLF in ie1 transcription; (3) elucidate how KLF regulate transcription of WSSV108, 140, and 164. This investigation will elucidate the molecular mechanisms by which KLF regulates the expression of WSSV immediate-early genes and develop new strategies to inhibit infection and replication of WSSV in shrimps.白點症病毒KLF蛋白質極早期基因WSSVKLFimmediate-early genes