抗感染性胰臟壞死病毒核醣核酸酵素之研究

Abstract

感染性胰臟壞死病毒(Infectious Pancreatic Necrosis Virus; IPNV)是一種高度傳染性的病毒，在臺灣感染性胰臟壞死病毒，造成吳郭魚、鰻魚、虱目魚及文蛤等嚴重的損失。因此如何有效的防禦感染性胰臟壞死病毒是當前水產養殖界的重要課題之一。在本篇論文中，設計出具有高度專一性的鎚頭型核醣核酸酵素(Hammerhead Ribozyme)及D型肝炎病毒核醣核酸酵素(Hepatitis D virus Ribozyme)，期能順利切割感染性胰臟壞死病毒的核醣核酸。本實驗選用感染性胰臟壞死病毒的A段1346至1776的基因片段，經由不同的核酸限制酵素的切割，產生出不同的線形的質體，利用Sp6聚合?來進行活體外轉錄，合成出部份IPNV正股RNA，當做受質RNA；另一方面核醣核酸酵素的合成是直接利用含T7?動子序列之去氧核醣核酸模版，進行活體外轉錄反應。結果發現，欲取得多量的RNA時，必先審慎評估T7?動子下游之6個核?酸序列。在設計的14個核醣核酸酵素中，僅有RT-1與RT-3n兩個鎚頭型核醣核酸酵素能夠切割受質核醣核酸，此時的反應溫度為37℃及含12mM的MgCl2。同時為了要符合生物體內生理狀況則降低濃度的鎂離子的濃度由12mM至6mM，亦有不錯的反應效果。另一方面為了要提高切割標的RNA的反應成效，則嚐試著提高溫度，可使得原本於37℃無法進行切割反應的RT-7在50℃時可反應；然而適量地添加變性劑或PEG 6000亦可增加切割反應。

Infectious pancreatic necrosis virus (IPNV) is contagious and widespread fish disease. In Taiwan aquaculture, IPNV has often caused heavy damage to the aquacultures industry for tilapia, eel, milk fish and clam. Development of effective control technology for IPNV infection has obviously become an important issue in fisheries development program and also imperative to continuing growth of culture industry. In this study, I designed highly specific hammerhead ribozyme and hepatitis delta virus ribozyme, synthesized the ribozyme RNAs and assayed the trans-cleavage activity of these ribozymes on the partial sense RNA of IPNV. Ribozyme RNAs were synthesized by T7 RNA polymerase transcription of synthetic DNA template. The downstream (+1 to +6) of T7 promoter was found to be a key factor to RNA production. Both RT-1 and RT-3n. (hammerhead ribozyme) were capable to cleave substrate RNA in 12mM MgCl2 at 37℃. To match in the physiological environment of organism, decreasing the concentration of magnesium ion from 12mM to 6mM obtained good results. On the other hand, in order to elevate the efficiency of cleavage on the target RNA, increasing the reaction temperature and adding denaturants or PEG 6000 can improve the cleavage reaction. RT-7 was able to cleave substrate RNA in 12mM MgCl2 at 50℃, that was uncut at 37℃.