臺灣博碩士論文加值系統

登革熱病毒的第三非結構蛋白(NS3)在病毒複製上扮演極重要的角色，若能抑制它的活性就能抑制病毒的複製，因此本實驗便嘗試以NS3的蛋白活性為基礎建立篩藥系統。根據NS3 trans切割NS4b-NS5的特性，我們構築了一受質蛋白(Pin-NS4b5-1D7)，其N端具有biotinylated peptide (Pin)，中間含有NS4b-NS5的切割點，C端為maltose binding protein epitope，可被1D7 monoclonal antibody辨認。當Pin-NS4b5-1D7被由大腸桿菌生產並經過純化的登革熱病毒蛋白(NS2b3pro)切割後，將其產物固定於敷有avidin的96孔平盤，以1D7 monoclonal body進行ELISA，將會因為帶有tag的片段被洗去而沒有訊號﹔若蛋白活性被抑制，則受質蛋白不被切割，則會有訊號。本論文已完成蛋白(NS2b3pro)的純化，受質蛋白(Pin-NS4b5-1D7)的純化，並確定本純化的蛋白對受質蛋白具有活性。
根據NS3須在NS2b的輔助下才能發揮最大活性，以及NS3 cis切割NS2b-NS3的特性，本實驗設計了另一套系統。蛋白D-NS3pro，其N端帶有D epitope，可被6A1 monoclonal antibody辨識，其後連接NS2b-NS3的切割點序列以及NS3 protease domain，並且於C端接有6× His tag。而cofactor方面，構築了N端為GST連接NS2b cofactor domain的融合蛋白(GST-NS2b)。GST-NS2b與D-NS3pro反應後，將促使D-NS3pro進行cis cleavage，然後將產物固定於敷有Ni2+的96孔平盤後，以6A1進行ELISA後，由於帶有tag的片段被洗去，於是沒有訊號產生﹔若蛋白活性被抑制，則蛋白不進行cis cleavage，如此便有訊號。本論文已完成蛋白D-NS3pro的純化，GST-NS2b的純化，並確定D-NS3pro在GST-NS2b的輔助下具有活性，且已初步進行反應條件最佳化。

Non-structural protein 3 (NS3) of dengue virus, which is a helicase and a protease, is a critical enzyme involved in virus replication. Inhibiting the protease or the helicase activity will interrupt virus replication. For developing the therapy of dengue virus infection, we developed an in vitro system for screening protease inhibitors of NS3. The protease complex NS2b3pro containing the hydrophilic domain of NS2b linked to NS3 protease domain with a histidine tag at the C-terminus was expressed in E. coli and purified with Ni2+ column. The substrate Pin-NS4b5-1D7 consists of an NS3 protease trans cleavage site flanked by the last 25 residues of NS4b and the first 77 residues of NS5. Besides the NS3 substrate region, the Pin-NS4b5-1D7 is a protein with a biotinylated peptide at the N-terminus and an epitope recognized by a monoclonal antibody 1D7 at the C-terminus. Theoretically through its biotin in the N-terminal region, Pin-NS4b5-1D7 could be bound to ELISA plates coated with avidin, whereas through its C-terminal tag, the same molecule could be recognized by monoclonal antibody 1D7. When the protease is added to Pin-NS4b5-1D7, the proteolytical product (NS5-1D7 fragment) was released from the biotinylated fusion protein, and as a result, the binding of monoclonal antibody 1D7 decreases. In this study, the recombinant protease NS2b3pro and the substrate Pin-NS4b5-1D7 have been prepared and characterized.
We also took advantage of that NS2b is essential for the cis cleavage activity of NS2b-NS3 and designed an unique system to monitor the cis activity. The NS3 protease portion was tagged by a hexa-histidine stretch at the C-terminus and a D epitope and a few NS2b C-terminal residues at the N-terminus. This protease precursor can be followed by immobilization on Ni2+ coated plates and recognized by monoclonal antibody 6A1. When NS2b was provided from GST-NS2b, the protease was activated and self cleaved. As a result, the D epitope and the following residues of NS2b C-terminus were released out of NS3pro protein. We have characterized and optimized the assay conditions in a Western blotting format in this study.

目錄
ABSTRACT1
中文摘要2
壹、緒論3
貳、實驗材料與方法7
參、實驗結果15
肆、討論24
伍、圖31
陸、表53
柒、附圖57
捌、參考文獻59

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