| Summary: | 碩士 === 中國醫藥大學 === 醫學檢驗生物技術學系碩士班 === 95 === Severe acute respiratory syndrome coronavirus (SARS-CoV) causes a life-threatening atypical pneumonia. SARS CoV is a positive-sense single strand RNA enveloped virus, that encodes two large nonstructural polypeptides, pp1a and pp1ab. Papain-like protease (PLpro) that resides within nsp3 is involved in the proteolytic processing on the LNGG sites at the boundary regions between nsp1, nsp2, and nsp3. Recently, PLpro has been demonstrated to have the de-ubiquitining enzymatic ability on the ISG15-polyubiquitin chains and ubiquitin-AMC substrates. In this study, we intend to generate recombiment SARS PLpro protease and to analyse its cellular response. Then, 2D gel electrophoresis/mass spectrometry was used to differentiate proteomic profiling among mock cells and PLpro-expressing cells treated with type I interferon. In vivo signaling pathway assay indicated that type I interferon-induced signal transduction pathway was inhibited by SARS-CoV PLpro, while the LPS-induced NF-κB signaling was not significantly inhibited in PLpro-expressing cells. The real time RT-PCR assay indicated the expression of PKR gene was blocked by SARS PLpro in the interferon treated cells. In addition, the Enzyme-linked immunoassay revealed that expression of the interferon-α, in the interferon-β treated cell was inhibited by SARS PLpro. The 2D gel and MALDI-TOF/MS proteomic profiling were performed and the different expressed protein spots were identified. Up-regulated proteins in interferon-α treated SARS PLpro-expressing cell were heat shock cognate71 kDa protein、heat shock 27 kDa protein、chloride intracellular channel protein 1、Ran-binding protein 1、peroxiredoxin-6, while down-regulated protein was NADH dehydrogenase iron-sulfur protein 3. In this study, we provide the bio-informatics about the proteomics change and biochemical mechanism in the SARS PLpro expressing cells which will be helpful for developing diagnostic and therapeutic approaches to the SARS infection.
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