鎚頭型核醣核酸醃於低溫下切割傳染性胰臟壞死病毒之VP5 RNA

Abstract

傳染性胰臟壞死病毒(Infectious Pancreatic Necrosis Virus, IPNV)為兩段雙股核醣核酸科(Birnaviridae) 病毒。其為一傳染性高且分佈廣泛的魚類病毒，在臺灣的水產養殖上造成相當大的損失。因此如何防治此病毒的危害為當前相當重要的課題。在IPNV的基因組中，A段基因體(segment A)於5'端有一個444 bp的small ORF，可轉譯出一17 kDa的病毒蛋白VP5，在病毒感染寄主細胞初期表現，以延後細胞自戕(apoptosis)現象，其功能可能與 anti-apoptosis有關。本實驗嘗試在魚類最適生長溫度下，設計對VP5 RNA進行切割之鎚頭型核醣核酸醃，並針對其切割作用活性加以研究。 以GCG之MFold程式預測VP5 RNA的二級結構，共設計了16個鎚頭型核醣核酸醃。於37℃時共有5個核醣核酸?可對VP5 RNA進行切割反應，其中RZ-1、-5、-7、-8位於套環區，而RZ-16則位於榦狀區。將切割反應溫度降至25℃, RZ-5、-7、-8仍具切割活性。鎂離子在切割作用中為一重要輔助因數，而RZ-5與RZ-8於3 mM低濃度鎂離子時仍具相當切割活性。二價金屬離子除Mg2+外包括Ca2+、Mn2+、Cu2+、Co2+與Cd2+皆可於進行切割作用時當做輔助因數。於反應中添加PEG-6000及Urea或formamide皆可增加切割反應之效率。反應之切割效果與溫度的提升呈正相關，而RZ-5與RZ-8兩組核醣核酸?皆可於17℃的低溫下作用，顯示其未來在ex vivo或in vivo之應用潛力。

Infectious pancreatic necrosis virus (IPNV) is one of the contagious and widespread fish diseases. IPNV belongs to the Bimaviridae family of virus, which has bisegmented ds-RNA genomes and often causes seriously damages to the aquaculture in Taiwan. VP5 is encoded from the small ORF located at 5'end of A segment. VP5 protein had been demonstrated to play a dual role on the regulation of virus replication and up-regulation of the survival factor Mcl-1 during IPNV infection. In this study, we designed highly specific hammerhead ribozymes to test the cleavages of VP5 RNA. Sixteen predicted cleavage sites (14 in loop and 2 in stem) of hammethead ribozyme were se1ected according to the secondary structure of VP5 cDNA sequence by GCG Mfold program . The ribozyme RNAs and VP5 RNA (Partial sense RNA of IPNV) were both synthesized by in vitro transcription. Five (4 in loop and 1 in stem) out of 16 hammethead ribozymes (predicted cleavage site) possess the trans-cleavage activity at 37℃ in vitro. The trans-cleavage activities of RZ-5 and RZ-8 are concentration-dependent ofdivalent-cation (Mg++） fashion at 25℃ in vitro. Divalent-cations including Ca++, Co++， Mn++, Cd++，and Cu++ as Mg++ act as cofactors in trans-cleavage reaction of hammethead ribozymes. The addition of chemical reagents (PEG-6000, urea, and formamide) can improve the cleavage of VP5 RNA by hammethead ribozymes (RZ-5 & RZ-8). The trans-cleavage capabilities of RZ-5 and RZ-8 at low temperature ( 17℃ and 25℃), revealing that these two hammethead ribozymes have strong potential for ex vivo or in vivo trans-cleavage in fish cells to prevent IPNV infection.