Academic literature on the topic 'Silkworms – Larvae'

碩士<br>中國文化大學<br>生物科技研究所<br>91<br>The digesting, transformation, absorption, transport and utilization, of chlorophylls and their physiological function in animals are still a myth. It is worthy to study if chlorophyll- or porphyrin-related compounds (CRCs or PRCs) play any role in animal. In this study, the red fluorescent protein (RFP) of silkworm (Bombyx mori) was employed to explore the above question. RFP has been known to be antivirus in the digestive juice of silkworm. Absorption spectrophotometer, high performance liquid chromatography, Thornber and MARS electrophoresis system and western blotting were hired to analyze the biochemical characteristics of mulberry leaf、RFPI in the digestive juice, and RFPII on the digestive tract. Data show that mulberry leaf、RFPI and RFPII are very different. By using a reversed-phase HPLC system chlorophyll a and b (Chl a and b) were found in RFPI purified according to Hou and Chiu (1986). The result is different from that of Hayashiya (1978) and Uchida and Hayashiya (1981), who reported that RFPI contained only chlorophyllide a (Chlide a).The Thornber and MARS electrophoresis systems and western blotting show both RFPI and RFPII contain four apoproteins of pigment-protein complexes of mulberry leaf, including RCIa、LHCIIa、LHCIIb and LHCIIc. Combination of the above data suggest that pigments and proteins of RFP I and RFPII might be totally or partially and directly or indirectly tranformed from pigment-protein complexes responsible for photosynthesis in mulberry leaf. It is therefore suggested that there may be a new model of assimilate nutrients in silkworm larvae.

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。<br>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。<br>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。<br>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.<br>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.<br>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.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Detailed summary in vernacular field only.<br>Li, Shuo.<br>Thesis (M.Phil.)--Chinese University of Hong Kong, 2012.<br>Includes bibliographical references (leaves 105-124).<br>Abstracts also in Chinese.<br>Acknowledgements --- p.I<br>Abstracts (English) --- p.II<br>Abstracts (Chinese) --- p.IV<br>Table of Contents --- p.VI<br>List of Abbreviations --- p.IX<br>List of Figures --- p.XII<br>List of Tables --- p.XIV<br>Chapter Chapter 1 Introduction --- p.1<br>Chapter 1.1 --- Acetylcholine mediated neutotransmission in nervous system --- p.1<br>Chapter 1.2 --- Acetylcholineterase --- p.2<br>Chapter 1.3 --- Comparison of AChE and BChE --- p.3<br>Chapter 1.4 --- Molecular sturcture of AChE --- p.5<br>Chapter 1.5 --- Molecular diversity of AChE --- p.7<br>Chapter 1.5.1 --- Regulation at transcriptional level --- p.8<br>Chapter 1.5.2 --- Regulation at post-transcriptional level --- p.11<br>Chapter 1.5.3 --- Regulation at post-translational level --- p.12<br>Chapter 1.6 --- Classic functions of AChE --- p.15<br>Chapter 1.7 --- Non-classic functions of AChE --- p.18<br>Chapter 1.8 --- Diseases associated with AChE --- p.19<br>Chapter 1.8.1 --- Myasthenia gravis --- p.19<br>Chapter 1.8.2 --- Alzheimer's disease --- p.20<br>Chapter 1.8.2.1 --- Pathogenesis of AD --- p.20<br>Chapter 1.8.2.2 --- Treatments for AD --- p.22<br>Chapter 1.9 --- Silkworm larvae as biofactory for protein expression --- p.24<br>Chapter 1.10 --- Traditional baculovirus expression system --- p.26<br>Chapter 1.11 --- Bac-to-Bac baculovirus expression system --- p.29<br>Chapter 1.12 --- Virtual screening with AutoDock Vina --- p.29<br>Chapter 1.13 --- Project overview and the aim of study --- p.31<br>Chapter Chapter 2 --- Materials and Methods --- p.33<br>Chapter 2.1 --- Materials --- p.33<br>Chapter 2.1.1 --- Chemicals and Reagents --- p.33<br>Chapter 2.1.2 --- Primers --- p.35<br>Chapter 2.1.3 --- Antibodies --- p.35<br>Chapter 2.1.4 --- Silkworms --- p.35<br>Chapter 2.2 --- Methods --- p.36<br>Chapter 2.2.1 --- Construction of the expression cassette --- p.36<br>Chapter 2.2.1.1 --- Preparation of E.coli competent cells --- p.36<br>Chapter 2.2.1.2 --- Transformation --- p.36<br>Chapter 2.2.1.3 --- Agarose gel electrophoresis --- p.37<br>Chapter 2.2.1.4 --- Gene clean --- p.37<br>Chapter 2.2.1.5 --- Subcloning of target genes --- p.38<br>Chapter 2.2.1.6 --- Plasmid DNA extraction --- p.40<br>Chapter 2.2.1.7 --- Quantification of plasmid DNA by spectrophotometer --- p.41<br>Chapter 2.2.1.8 --- Plasmid DNA sequencing --- p.41<br>Chapter 2.2.2 --- Generation of recombinant bacmid DNA --- p.42<br>Chapter 2.2.2.1 --- Transposition --- p.42<br>Chapter 2.2.2.2 --- White/Blue screening --- p.42<br>Chapter 2.2.2.3 --- Extraction of recombinant bacmid DNA --- p.42<br>Chapter 2.2.2.4 --- Analysis of recombinant bacmid DNA by PCR --- p.44<br>Chapter 2.2.3 --- Transfection of silkworm larvae --- p.45<br>Chapter 2.2.3.1 --- Raising silkworm larvae --- p.45<br>Chapter 2.2.3.2 --- Preparation of transfecting solution --- p.45<br>Chapter 2.2.3.3 --- Transfection of silkworm larvae --- p.45<br>Chapter 2.2.3.4 --- Collection of hemolymph after protein expression --- p.46<br>Chapter 2.2.3.5 --- Oral infection of sikworm larvae --- p.46<br>Chapter 2.2.4 --- Purification of AChE --- p.47<br>Chapter 2.2.4.1 --- Nickel-chelating afinity chromatography --- p. 47<br>Chapter 2.2.4.2 --- Determination of protein concenttration by BCA assay --- p.47<br>Chapter 2.2.4.3 --- Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) --- p.48<br>Chapter 2.2.4.4 --- Western blot --- p.49<br>Chapter 2.2.5 --- Kinetic analysis of AChE --- p.50<br>Chapter 2.2.5.1 --- Ellman assay --- p.50<br>Chapter 2.2.5.2 --- Curve fitting --- p.51<br>Chapter 2.2.6 --- Virtual screening --- p.51<br>Chapter Chapter 3 --- Expression of recombinant AChEs in silkworm larvae --- p.55<br>Chapter 3.1 --- Construction of the expression cassette --- p.55<br>Chapter 3.1.1 --- Human AChE and Tetraodon nigroviridis AChE --- p.55<br>Chapter 3.1.2 --- Amplification of target genes from the parent vectors --- p.56<br>Chapter 3.1.3 --- Insertion of target genes into pFastBac Dual --- p.58<br>Chapter 3.2 --- Generation of recombinant bacmid DNA --- p.60<br>Chapter 3.2.1 --- Phenotype verification --- p.60<br>Chapter 3.2.2 --- PCR analysis of the recoombinant bacmid DNA --- p.64<br>Chapter 3.3 --- Expression of AChE in silkworm larvae --- p.66<br>Chapter 3.3.1 --- Raising silkworms --- p.66<br>Chapter 3.3.2 --- High-level expression of AChE in silkworm larvae --- p.68<br>Chapter 3.4 --- Oral infeciton --- p.72<br>Chapter Chapter 4 --- Analysis of the recombinant AChEs --- p.73<br>Chapter 4.1 --- Purification of recombinant AChEs by nickel-chelating affinity chromatography --- p.73<br>Chapter 4.2 --- SDS-PAGE and western blot analysis of the recombinant AChEs --- p.76<br>Chapter 4.3 --- Kinetic studies of recombinant AChEs --- p.79<br>Chapter 4.4 --- Virtual screening --- p.84<br>Chapter Chapter 5 --- Discussion and conclusion --- p.95<br>Chapter 5.1 --- Demonstration of high-level expression of recombinant AChEs by Bac-to-Bac baculovirus expression system --- p.95<br>Chapter 5.2 --- Oral infection of silkworm larvae --- p.98<br>Chapter 5.3 --- Characterization of recombinant AChEs --- p.98<br>Chapter 5.4 --- Discovery of new AChE inhibitors by virtual screening --- p.100<br>Chapter 5.5 --- Future works --- p.101<br>Chapter 5.6 --- Other applications --- p.102<br>Chapter 5.7 --- Conclusion --- p.102<br>References --- p.104<br>Appendix I --- p.125<br>Appendix II --- p.127<br>Appendix III --- p.129

碩士<br>國立中興大學<br>昆蟲學系<br>82<br>After infecting with the recombinant Bombyx mori nuclear polyhedrosis virus ( BmNPV ), BmHBs324P, which contains hepatitis B surface antigen (HBsAg) genes drived by polyhedrin promoter , the larvae did not show apparent symptom in the initial stage. Later, the infected larvae ceased feeding at 72 hr post-infection. their intersegments became swollen and climbed around the rearing seat. They died at 96 hr post-infection. The dead larvae vomited digestive fluid. The hemolymph of the infected ones became light brown in color. Production of hepatitis B surface antigen in hemolymph was measured after injection of BmHBs324P into 5th instar larvae. The production curve was going up beginning from 60 hr and reached the maximum at 108 hr post-inoculation. The extracellular HBsAg level in BmN cells was similar to that produced in larvae. But intracellular HBsAg increased from 48 hr, and reached the peak at 96 hr post-inoculation. There were several folds differences in HBsAg level between intracellular and extracellular measurements, revealing that the HBsAg proteins were not released out of the cells completely. The HBsAg production in hemolymph increased with an increase in temperature incubating at 15℃ to 30 ℃, but it was very low at 35℃. Injection of 25,000 plaque forming unit( PFU) BmHBs324P into the 5th instar larvae could produce maximun HBsAg in hemolymph. The 5th instar larvae produced the lowest HBsAg at 1 day, but did not show obvious difference from 2 to 5 days after injecting 50,000 PFU into the 5th instar larvae. The HBsAg production in hemolymph of J207 strain was two folds as that of " hwa 2", while variable productions were found among different silkworm strains. These results could be useful for screening high productive strains for producing HBsAg proteins in silkworm larvae.

碩士<br>國立中興大學<br>昆蟲學系<br>84<br>Expression of Hepatitis B Virus Surface Antigen Protein in a Cell Line and Silkworm Larvae with Recombinant Bombyx mori Nuclear polyhedrosis virusThe silkworm (Bombyx mori) larvae and BmN cell line were infected with the recombinant virus BmYFHBs containing hepatitis B surface antigen (HBsAg) gene which was constructed from B. mori nuclear polyhedrosis virus (BmNPV). the cellular aberration was variable in infected cells. Histological sections of larval tissues stained with immunoenzymic method showed HBsAg protein at 48 hr post-infection and polyhedral inclusion bodies at 72 hr post-infection. The epidermal cells became swollen and lysed, and inclus-ion bodies liberated into body cavity at 84 hr post-infection. At frist, the recombinant virus formed in fat body, epidermis, trachea membrane and hemocytes. Later midgut, Malpighian tubules and silk glands were infected. The virus was not detected in muscles. Various silkworm strains were infected with BmYFHBs virus by feeding to the 5th instar. The more susceptible the more HBsAg protein were produced in hemolymph among these strains. The same was found among strains in the same group. Determination of susceptibility to the recombinant virus and production of HBsAg showed that the susceptibility of artificial diet-fed larvae and extracts from whole larva were higher than those fed on mulberry leaves but were not significantly different in hemolymph. Injection of different recombinant viruses into larvae showed increasing HBsAg production with incubation time until the peak on the 4th day post-infection. Microbial contamination in silkworms was measured under aseptically and non-aseptically rearingconditions.More samples were found contaminated under non-aseptically rearing condition. Decrease in feeding number of larvae and diet changing time under aseptical condition may lower microbial contamination. Injection of 5th instars with BmYFHBs resulted in similar HBsAg production in hemolymph and larval body extracts under aseptically and non-aseptically rearing conditions.

Miu Fei Fei.<br>On t.p. "42" are subscripts following the word "MSP-1" in the title.<br>Thesis (M.Phil.)--Chinese University of Hong Kong, 2003.<br>Includes bibliographical references (leaves 117-125).<br>Abstracts in English and Chinese.<br>ACKNOWLEGEMENTS --- p.i<br>ABSTRACT --- p.ii<br>TABLE OF CONTENTS --- p.v<br>LIST OF FIGURE --- p.vii<br>LIST OF ABBREVIATIONS --- p.ix<br>CHAPTERS:<br>Chapter 1. --- BACKGROUND OF MALARIA<br>Chapter 1.1 --- Epidemilogy --- p.2<br>Chapter 1.2 --- Mode of Infection --- p.4<br>Chapter 1.3 --- Conventional Control & Vaccination --- p.9<br>Chapter 1.4 --- Vaccine Candidate PfMSV-142 --- p.16<br>Chapter 1.5 --- Cloning and Expression of pfMSP-142 --- p.26<br>Chapter 1.6 --- Aims of Study --- p.32<br>Chapter 2. --- Materials and Methods<br>Chapter 2.1 --- Materials --- p.32<br>Chapter 2.2 --- Methods --- p.38<br>Chapter 3. --- Construction of recombinants N-PfMSP-142 and C- PfMSP-142<br>Chapter 3.1 --- Construction of C-PfMSP-l42 --- p.51<br>Chapter 3.2 --- Construction ofN-PfMSP-l42 --- p.56<br>Chapter 4. --- Purification with IMAC<br>Chapter 4.1 --- Immobilized Metal Affinity Chromatography (IMAC) --- p.58<br>Chapter 4.2 --- Purification ofN-PfMSP-l42 --- p.61<br>Chapter 4.3 --- Purification profile of N-PfMSP-142 --- p.68<br>Chapter 4.4 --- Purification of C-PfMSP-l42 --- p.70<br>Chapter 4.5 --- Purification profile of C-PfMSP-142 --- p.73<br>Chapter 4.6 --- Expression pattern of recombinants PfMSP-142 --- p.76<br>Chapter 5. --- Purification combined with other chromatography method<br>Chapter 5.1 --- Affinity chromatography --- p.78<br>Chapter 5.2 --- Gel filtration chromatography --- p.80<br>Chapter 5.3 --- Ion exchange chromatography --- p.83<br>Chapter 5.4 --- Conclusion --- p.93<br>Chapter 6. --- Characteristic of IMAC products --- p.94<br>Chapter 7. --- Characteristic of N-hisPfMSP-l42 & C-hisPfMSP-l --- p.42<br>Chapter 7.1 --- Immunogenitcity of N-PfMSP-l42 and C-PfMSP-142 --- p.100<br>Chapter 7.2 --- Competitive ELISA --- p.105<br>Chapter 8. --- Discussion --- p.107<br>REFERENCE --- p.117

Pang, Lap-yin.<br>"42" in title is subscript.<br>"July 2000."<br>Thesis (Ph.D.)--Chinese University of Hong Kong, 2000.<br>Includes bibliographical references (p. 163-173).<br>Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.<br>Mode of access: World Wide Web.<br>Abstracts in English and Chinese.

Chan Ping Kei.<br>Thesis submitted in: December 2001.<br>Thesis (M.Phil.)--Chinese University of Hong Kong, 2002.<br>Includes bibliographical references (leaves 135-143).<br>Abstracts in English and Chinese.<br>ACKNOWLEGEMENTS --- p.i<br>ABSTRACT --- p.ii<br>TABLE OF CONTECTS --- p.v<br>LIST OF FIGURE --- p.viii<br>LIST OF ABBREVIATIONS --- p.xii<br>CHAPTER<br>Chapter 1 --- INTRODUCTION<br>Chapter 1.1 --- Epidemilogy --- p.1<br>Chapter 1.2 --- Malaria disease --- p.1<br>Chapter 1.3 --- Life cycle of Malaria --- p.1<br>Chapter 1.4 --- Current measure to control Malaria --- p.6<br>Chapter 1.5 --- Anti-malaria vaccine candidate --- p.7<br>Chapter 1.6 --- Anti-erythrocytic malaria vaccine MSP-1 --- p.10<br>Chapter 1.7 --- Baculovirus Expression System --- p.20<br>Chapter 1.8 --- hexa-histidine tagged fusion protein --- p.25<br>Chapter 1.9 --- IMAC --- p.26<br>Chapter 1.10 --- Aim of study --- p.26<br>Chapter 2 --- MATERIALS AND METHODS<br>Chapter 2.1 --- Materials --- p.29<br>Chapter 2.2 --- Methods --- p.40<br>Chapter 3 --- CONSTRUCTION AND CHARACTERIZATION OF RECOMBINANT BmNPV CARRYING PfMSP-l42<br>Chapter 3.1 --- Cloning of C-HisPfMSP-l42 into pBM030 --- p.71<br>Chapter 3.2 --- Construction of Recombinant BmNPV Carrying PfMSP-l42 --- p.72<br>Chapter 3.3 --- Purification of Recombinant BmNPVs --- p.78<br>Chapter 3.4 --- In vitro expression of Recombinant --- p.80<br>Chapter 3.5 --- In Vivo Expression of Recombinant PfMSP-l42 Protein --- p.80<br>Chapter 4 --- PURIFICATION OF BmNPV-EXPRESSED RECOMBINANT C- TERMIAL HEXA-HIS-TAGGED PfMSP-l42 PROTEIN<br>Chapter 4.1 --- Nickel ion charged Chelating Sepharose Fast Flow (immobilized metal affinity chromatography) --- p.88<br>Chapter 4.2 --- POROS HS/M (Strong Cation Exchanger) --- p.105<br>Chapter 4.3 --- Combination of chromatographic separations --- p.107<br>Chapter 5 --- CHARACTERIZATION OF RECOMBINANT C-HISPfMSP-l42 PROTEIN<br>Chapter 5.1 --- Proper formation of disulphide bridges in epidermal growth factor (EGF) like domains --- p.115<br>Chapter 5.2 --- Characterization of the integrity of hexa-histidines residue on recombinant PfMSP-142 protein --- p.117<br>Chapter 5.3 --- Immunogenicity of Recombinant C-HisPfMSP-l42 Protein --- p.117<br>Chapter 6 --- DISCUSSION<br>Chapter 6.1 --- Construction of recombinant BmNPV carrying HisPfMSP-l42 --- p.122<br>Chapter 6.2 --- Expression of recombinant HisPfMSP-l42 proteins --- p.123<br>Chapter 6.3 --- Purification of recombinant C-HisPfMSP-l42 protein --- p.125<br>Chapter 6.4 --- Characterization of recombinant C-HisPfMSP-l42 protein --- p.128<br>Chapter 6.5 --- Future prospects --- p.130<br>REFERENCE --- p.135<br>APPENDICES<br>Chapter 1. --- Appearance of Mulberry leaves<br>Chapter 2. --- Biomark 2000 (Beckman) program for sandwich ELISA protocol<br>Chapter 3. --- Nucleotide Sequence of PfMSP-l42 3D7 Isolate<br>Chapter 4. --- Nucleotide sequence of PfMSP-l42 FVO isolate<br>Chapter 5. --- Efficiency of the mAb5.2 immunoaffinity column in purifying the recombinant PfMSP-l42 protein