乙酰膽鹼酯酶主要存在於神經肌肉接頭處和中樞神經系統的膽鹼能突觸處，是神經遞質傳遞過程中極其重要的膽鹼水解酶。研究表明，阿茲海默病人的大腦通常呈現出乙酰膽鹼酯酶的異常表達和分佈，並伴隨著β澱粉樣蛋白的沉澱。目前，乙酰膽鹼酯酶抑製劑是治療阿茲海默症的主要臨床藥物。 / 在本研究中，我們利用Bac-to-Bac 桿狀病毒表達系統分別使人類和雞泡魚的重組乙酰膽鹼酯酶基因在家蠶幼蟲裡得到了高效的表達。我們將乙酰膽鹼酯酶的cDNA序列克隆到pFastBac{U+2122} Dual質粒的多角體蛋白啟動子下游。為了易於監控蛋白表達水平，橙色熒光蛋白的cDNA序列也被克隆到同一個質粒的p10啟動子下游。此外，我們將多聚組氨酸標籤加在了乙酰膽鹼酯酶基因的碳端，從而使蛋白的純化效率得到了顯著提高。我們通過皮下注射含有乙酰膽鹼酯酶的重組bacmid對五齡期的家蠶幼蟲進行了病毒轉染。感染後約4-7天，重組乙酰膽鹼酯酶在蠶蟲內成功得到了表達。酶促反應動力學研究表明，重組乙酰膽鹼酯酶的活性與來自相同物種的天然乙酰膽鹼酯酶基本相似。這種高效率、低成本、高產量的蛋白表達方法可以為我們提供大量的重組乙酰膽鹼酯酶，用於體外篩選治療阿茲海默症的乙酰膽鹼酯酶抑製劑。 / 隨著對阿茲海默症分子學水平上的進一步了解，研究提出乙酰膽鹼酯酶可能通過外周陰離子位點誘導β澱粉樣多肽聚集, 從而形成澱粉樣纖維。因此，理想的乙酰膽鹼酯酶抑製劑應該既有抑制乙酰膽鹼酯酶的活性，又可以對抗β澱粉樣蛋白沉澱的毒性, 從而達到神經保護的作用。因此，我們採用AutoDock Vina軟件對ZINC數據庫內的天然化合物進行了兩輪虛擬篩選，篩選出的化合物理論上是可以同時作用於催化位點和外周陰離子位點。接下來，我們將對候選化合物進行體外驗證。 / Acetylcholinesterase (AChE: EC 3.1.1.7) is the acetylcholine-hydrolyzing enzyme that plays an essential role on cholinergic neurotransmission at the synapses of the brain and at the neuromuscular junctions. Abnormal expression and localization of AChE have been observed together with Aβ deposits in the brain of Alzheimer’s disease (AD) patient. Currently, AChE inhibitors are clinically used as drugs for AD treatment. / In this study, we demonstrated high-level expressions of functional recombinant human AChE and Tetraodon nigroviridis AChE using Bac-to-Bac baculovirus expression system in silkworm Bombyx mori larvae. The cDNA of AChE was cloned into the polyhedrin (PH) promoter of the plasmid pFastBac{U+2122} Dual. To monitor the level of expression, the cDNA coding for an orange fluorescent protein (OFP2) was cloned downstream to the p10 promoter of the same vector. We engineered a polyhistidine-tag (His-tag) tail to the C-terminal of each AChE gene to facilitate the purification. Transfection was carried out by subcutaneous injection of the recombinant bacmid DNA containing the AChE gene into the silkworm larvae of 5th instar. Approximately 4-7 days of post-infection, the recombinant AChE was expressed in the hemolymph of infected larvae. The kinetic studies showed that the biological activities of the recombinant AChEs were comparable to that of natural ones from other sources. This rapid, low-cost, and high yield production method could provide us sufficient amount of recombinant AChE for in vitro screening of AChE inhibitors for AD treatment. / Further advances in understanding the molecular basis of AD have proposed that AChE promote the assembly of Aβ peptide into amyloid fibrils through interaction at the peripheral anionic site of AChE. Consequently, new classes of AChE inhibitors are expected to be able to inhibit the active site of AChE and, at the same time, to protect neurons from Aβ toxicity. Therefore, we applied two rounds of virtual screening of ZINC database using AutoDock Vina to obtain new potential inhibitors which might be able to targeting both of the active and peripheral sites of AChE. The compounds with good performances in both of the two rounds of screening would be validated by the sequential in vitro tests. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Li, Shuo. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 105-124). / Abstracts also in Chinese. / Acknowledgements --- p.I / Abstracts (English) --- p.II / Abstracts (Chinese) --- p.IV / Table of Contents --- p.VI / List of Abbreviations --- p.IX / List of Figures --- p.XII / List of Tables --- p.XIV / Chapter Chapter 1 Introduction --- p.1 / Chapter 1.1 --- Acetylcholine mediated neutotransmission in nervous system --- p.1 / Chapter 1.2 --- Acetylcholineterase --- p.2 / Chapter 1.3 --- Comparison of AChE and BChE --- p.3 / Chapter 1.4 --- Molecular sturcture of AChE --- p.5 / Chapter 1.5 --- Molecular diversity of AChE --- p.7 / Chapter 1.5.1 --- Regulation at transcriptional level --- p.8 / Chapter 1.5.2 --- Regulation at post-transcriptional level --- p.11 / Chapter 1.5.3 --- Regulation at post-translational level --- p.12 / Chapter 1.6 --- Classic functions of AChE --- p.15 / Chapter 1.7 --- Non-classic functions of AChE --- p.18 / Chapter 1.8 --- Diseases associated with AChE --- p.19 / Chapter 1.8.1 --- Myasthenia gravis --- p.19 / Chapter 1.8.2 --- Alzheimer's disease --- p.20 / Chapter 1.8.2.1 --- Pathogenesis of AD --- p.20 / Chapter 1.8.2.2 --- Treatments for AD --- p.22 / Chapter 1.9 --- Silkworm larvae as biofactory for protein expression --- p.24 / Chapter 1.10 --- Traditional baculovirus expression system --- p.26 / Chapter 1.11 --- Bac-to-Bac baculovirus expression system --- p.29 / Chapter 1.12 --- Virtual screening with AutoDock Vina --- p.29 / Chapter 1.13 --- Project overview and the aim of study --- p.31 / Chapter Chapter 2 --- Materials and Methods --- p.33 / Chapter 2.1 --- Materials --- p.33 / Chapter 2.1.1 --- Chemicals and Reagents --- p.33 / Chapter 2.1.2 --- Primers --- p.35 / Chapter 2.1.3 --- Antibodies --- p.35 / Chapter 2.1.4 --- Silkworms --- p.35 / Chapter 2.2 --- Methods --- p.36 / Chapter 2.2.1 --- Construction of the expression cassette --- p.36 / Chapter 2.2.1.1 --- Preparation of E.coli competent cells --- p.36 / Chapter 2.2.1.2 --- Transformation --- p.36 / Chapter 2.2.1.3 --- Agarose gel electrophoresis --- p.37 / Chapter 2.2.1.4 --- Gene clean --- p.37 / Chapter 2.2.1.5 --- Subcloning of target genes --- p.38 / Chapter 2.2.1.6 --- Plasmid DNA extraction --- p.40 / Chapter 2.2.1.7 --- Quantification of plasmid DNA by spectrophotometer --- p.41 / Chapter 2.2.1.8 --- Plasmid DNA sequencing --- p.41 / Chapter 2.2.2 --- Generation of recombinant bacmid DNA --- p.42 / Chapter 2.2.2.1 --- Transposition --- p.42 / Chapter 2.2.2.2 --- White/Blue screening --- p.42 / Chapter 2.2.2.3 --- Extraction of recombinant bacmid DNA --- p.42 / Chapter 2.2.2.4 --- Analysis of recombinant bacmid DNA by PCR --- p.44 / Chapter 2.2.3 --- Transfection of silkworm larvae --- p.45 / Chapter 2.2.3.1 --- Raising silkworm larvae --- p.45 / Chapter 2.2.3.2 --- Preparation of transfecting solution --- p.45 / Chapter 2.2.3.3 --- Transfection of silkworm larvae --- p.45 / Chapter 2.2.3.4 --- Collection of hemolymph after protein expression --- p.46 / Chapter 2.2.3.5 --- Oral infection of sikworm larvae --- p.46 / Chapter 2.2.4 --- Purification of AChE --- p.47 / Chapter 2.2.4.1 --- Nickel-chelating afinity chromatography --- p. 47 / Chapter 2.2.4.2 --- Determination of protein concenttration by BCA assay --- p.47 / Chapter 2.2.4.3 --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.48 / Chapter 2.2.4.4 --- Western blot --- p.49 / Chapter 2.2.5 --- Kinetic analysis of AChE --- p.50 / Chapter 2.2.5.1 --- Ellman assay --- p.50 / Chapter 2.2.5.2 --- Curve fitting --- p.51 / Chapter 2.2.6 --- Virtual screening --- p.51 / Chapter Chapter 3 --- Expression of recombinant AChEs in silkworm larvae --- p.55 / Chapter 3.1 --- Construction of the expression cassette --- p.55 / Chapter 3.1.1 --- Human AChE and Tetraodon nigroviridis AChE --- p.55 / Chapter 3.1.2 --- Amplification of target genes from the parent vectors --- p.56 / Chapter 3.1.3 --- Insertion of target genes into pFastBac Dual --- p.58 / Chapter 3.2 --- Generation of recombinant bacmid DNA --- p.60 / Chapter 3.2.1 --- Phenotype verification --- p.60 / Chapter 3.2.2 --- PCR analysis of the recoombinant bacmid DNA --- p.64 / Chapter 3.3 --- Expression of AChE in silkworm larvae --- p.66 / Chapter 3.3.1 --- Raising silkworms --- p.66 / Chapter 3.3.2 --- High-level expression of AChE in silkworm larvae --- p.68 / Chapter 3.4 --- Oral infeciton --- p.72 / Chapter Chapter 4 --- Analysis of the recombinant AChEs --- p.73 / Chapter 4.1 --- Purification of recombinant AChEs by nickel-chelating affinity chromatography --- p.73 / Chapter 4.2 --- SDS-PAGE and western blot analysis of the recombinant AChEs --- p.76 / Chapter 4.3 --- Kinetic studies of recombinant AChEs --- p.79 / Chapter 4.4 --- Virtual screening --- p.84 / Chapter Chapter 5 --- Discussion and conclusion --- p.95 / Chapter 5.1 --- Demonstration of high-level expression of recombinant AChEs by Bac-to-Bac baculovirus expression system --- p.95 / Chapter 5.2 --- Oral infection of silkworm larvae --- p.98 / Chapter 5.3 --- Characterization of recombinant AChEs --- p.98 / Chapter 5.4 --- Discovery of new AChE inhibitors by virtual screening --- p.100 / Chapter 5.5 --- Future works --- p.101 / Chapter 5.6 --- Other applications --- p.102 / Chapter 5.7 --- Conclusion --- p.102 / References --- p.104 / Appendix I --- p.125 / Appendix II --- p.127 / Appendix III --- p.129
| Identifer | oai:union.ndltd.org:cuhk.edu.hk/oai:cuhk-dr:cuhk_328507 |
| Date | January 2012 |
| Contributors | Li, Shuo., Chinese University of Hong Kong Graduate School. Division of Biomedical Sciences. |
| Source Sets | The Chinese University of Hong Kong |
| Language | English, Chinese |
| Detected Language | English |
| Type | Text, bibliography |
| Format | electronic resource, electronic resource, remote, 1 online resource (xiv, 131 leaves) : ill. (some col.) |
| Rights | Use of this resource is governed by the terms and conditions of the Creative Commons “Attribution-NonCommercial-NoDerivatives 4.0 International” License (http://creativecommons.org/licenses/by-nc-nd/4.0/) |
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