白點症病毒晚期基因p150表現之研究

Abstract

白點症病毒(white spot syndrome virus, WSSV)台灣分離株Taiwanisolate,AF440570)所預測的開放譯讀區WSSV067經由研究，命名為p150基因，並將此基因進行分析比對。p150基因全長3,906 bp，可轉錄出1,301個胺基酸，經由電腦分析P150蛋白是屬於酸性蛋白，預測的分子量大小為143.9 kDa，其蛋白胺端(N-terminus)具有兩個glutamic acid-rich domain，羧端(C-terminus)具有三個穿越膜區域(transmembrane region)。p150基因的轉錄本(transcript)大小約為4.3 kb，此基因於白點症病毒感染後12小時的蝦體才能被檢測出，是屬於晚期表現基因，其加聚腺嘌呤位置(Poly A addition site)在加聚腺嘌呤訊號下游第15個核?酸處，與目前已知的白點症病毒基因rr1、DNApol以及預測的開放譯讀區WSSV202、WSSV217比較，在這個區域內可以找到一個保守性序列(T/A)ACATGTA(T/A)A(T/A)，其中包含了一段可折疊相對應的序列ACATGT。經由分析白點症病毒基因p150在轉錄、轉譯方面的序列特性，推測其基因的表現可能是利用病毒本身所特有的機制調控。另外經由試管內轉錄轉譯實驗，表現p150全長基因，可得分子量約150 kDa的蛋白產物。以昆蟲細胞株Sf9進行轉染作用，可觀察到P150蛋白存在於細胞質中，並可能與某種胞器結合，經由共軛焦顯微鏡觀察，確認與溶小體及粒線體無關，但可能具有與細胞內質網結合的特性，推測白點症病毒p150基因可能和病毒顆粒的形成有關。

One of the white spot syndrome virus (WSSV) genes, p150, that encodes an acidic protein was identified and sequenced from the predicted open reading frame, WSSV067, from the Taiwan isolate. The p150 gene that contains 3,906 nucleotides potentially encodes a protein of 1,301 amino acids with two glutamic acid-rich domains in the N-terminus and three transmembrane regions in the C-terminus. The predicted molecular weight of P150 is 143.9 kDa. The transcript of p150, which was detected at 12 hr post-infection and reached a maximal level at 18 hr, belonged to a late gene of WSSV. The mRNA size of p150 gene is about 4.3 kb, and the poly A addition site is 15 bp downstream of the poly A signal. When compare the region between poly A signal and poly A addition site of known WSSV genes of p150, rr1, DNApo1 and the predicted open reading frame WSSV202 and WSSV217, a consensus sequence was found, (T/A)ACATATA(T/A)A(T/A). The product of in vitro transcription and translation of p150 gene was detected as a 150 kDa protein. Immunofluorescent staining of P150 protein in insect cell line showed that the P150 localized in cytoplasm, and might bind to some organelles. P150 did not associate with lysosome and mitochondria according to the observation under Confocal microscope, but might be colocalized with endoplasmic reticulum (ER) under fluorescence microscope. The P150 of WSSV may participate in virus assembly.