| Summary: | 碩士 === 長庚大學 === 生物醫學研究所 === 100 === Enterovirus 71 (EV 71), the newest member of Enteroviridae, is notorious for its etiological role in epidemics of the hand-foot-and-mouth disease, particularly in association with fatal neurological complications in young children. During the EV 71 viral RNA replication, the interaction among host factors and viral 3Dpoly, an RNA-dependent RNA polymerase is crucial for the product of newly viral progeny. It has been reported that the 3D RNA polymerase mutation (I251T) exhibited the temperature sensitive behaviors in the cell culture system. However, little is known with the mechanisms among the 3D polymerase temperature sensitive mutation and host factor. In this study, we construct the yeast expression 3D polymerase with an insertion of Renilla luciferase which allow us to monitor the viral RNA polymerase activity. After yeast transformation, the positive yeast transformants expressed 3D polymerase under the driver by GAL10 promoter. Next, the detection of Renilla luciferase activities was remained increased when the GAL 1 promoter was been turned off, indicating the ability of 3D RNA polymerase expressed in yeast. Altogether, the yeast expressed recombinant EV 71 3D polymerase exhibits a bona fide viral specific function according to the results obtained from Western blot and Renilla luciferase analyses.
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