| Summary: | 碩士 === 國立成功大學 === 生物學系碩博士班 === 92 === In 1998, an epidemic of hand-foot-and-mouth disease caused by enterovirus 71 (EV71) affected thousands of children in Taiwan. In a few cases, more serious symptoms were developed, such as polio-like paralysis, encephalitis, sometimes even resulting in death. Currently, there is no specific treatment or vaccine for this disease. Recently, RNA interference (RNAi) provide an alternative approach with 21-nucleotide small interfering RNA (siRNA) to specifically down-regulate cellular as well as viral gene expression in mammalian cells. In this study, using EV71 infection as a model system, we demonstrated that long double-strand RNA (dsRNA)-mediated RNAi can be effectively used to achieve specific silencing of EV71 replication. First, in order to maintain a better RNAi effect, pol II promoter was used to drive long dsRNA. Two different vectors, pTCY being drived with a β-actin promoter can express gene stably in the cells, and p2-5A being drived with a 2',5'-oligoadenylate synthetase gene promoter can express gene only after interferon induction, were constructed. In addition, previous researches indicated that EV71 2A protease plays an important role in cleavage of EV71 polyprotein. Thus, EV71 2A gene was selected as a target sequence for RNAi. Two dsRNA constructs, pTCY-ds2A and p2-5A-ds2A, containing 450 bp sense and anti-sense fragments of EV71 2A gene were generated. After transfection of pTCY-ds2A and p2-5A-ds2A to Vero cells, two stable clones, Vero/pTCY-ds2A-2 and Vero/2-5A-ds2A-2, were selected for further investigation. These two transfected cell clones expressing ds2A produced fewer virus particles and the inhibitory effects of virus replication were higher (1000-fold) than those of the non transfected Vero cells. Further experiment of detection of the expression of EV71 3D gene, which encodes viral RNA polymerase for EV71 replication, by RT-PCR showed that both Vero/pTCY-ds2A-2 and Vero/2-5A-ds2A-2 produced low amount of 3D mRNA. The phenomena of the DNA fragmentation after EV71 infection was also reduced. The duration of EV71-specific RNAi activity in both Vero/pTCY-ds2A-2 and Vero/2-5A-ds2A-2 were maintained for 11~14 days. Western blotting analysis for detection of PKR expression showed low levels in both transfected cell clones without virus challenge. These observations indicated that long dsRNA could not induce an interferon-stimulated gene, PKR. Furthermore, to confirm that the anti-viral effect was specific from RNAi, Vero/pTCY-ds2A-2,Vero/2-5A-ds2A-2 and Vero/pTCY-dsE1A were used for EV71 infection. The result revealed that the susceptibilities of Vero/pTCY-dsE1A and non transfected Vero cells to EV71-induced cell death were the same. In summary, this study has shown that the inhibition of viral 2A gene expression by long dsRNA-mediated RNA interference might ultimately be useful in treatment of EV71 replication.
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