跳到主要內容

臺灣博碩士論文加值系統

(18.204.48.64) 您好!臺灣時間:2021/08/04 18:54
字體大小: 字級放大   字級縮小   預設字形  
回查詢結果 :::

詳目顯示

我願授權國圖
: 
twitterline
研究生:陳怡妌
研究生(外文):Yi-Ching Chen
論文名稱:epsilon二型榖胱甘肽硫轉移酶與南台灣埃及斑蚊之殺蟲劑抗藥性的相關性
論文名稱(外文):Correlation of glutathione S-transferase epsilon2 and insecticide resistance in Aedes aegypti from South Taiwan
指導教授:何兆美何兆美引用關係
指導教授(外文):Chau-Mei Ho
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:臨床醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:75
中文關鍵詞:埃及斑蚊榖胱甘肽硫轉移酶殺蟲劑抗藥性百滅寧
外文關鍵詞:Aedes aegyptiGlutathione S-transferaseInsecticide ResistancePermethin
相關次數:
  • 被引用被引用:0
  • 點閱點閱:108
  • 評分評分:
  • 下載下載:0
  • 收藏至我的研究室書目清單書目收藏:0
埃及斑蚊是傳播登革熱的主要病媒蚊之一。為了防止疾病的傳播,以合成除蟲菊酯類殺蟲劑控制病媒蚊數量,但許多地區埃及斑蚊對此類殺蟲劑產生抗藥性,使得防治效果大受影響。解毒系統增強為昆蟲產生抗藥性的原因之一,麩胺基硫轉移酶參與解毒過程被證實與抗藥性有關,本研究的目的在於研究台灣台南市和高雄市之埃及斑蚊的抗藥情形及與epsilon二型麩胺基硫轉移酶(GSTε2 )的關係,並以感性品系(NS)與抗性品系(LYR)當做對照組。首先利用世界衛生組織藥膜測試套組篩選出對百滅寧(permethrin)具抗藥性埃及斑蚊品系,接著測量對百滅寧之半致死濃度(LC50),並以酵素活性試驗、半定量PCR、Western blotting等實驗證明台灣埃及斑蚊對百滅寧具抗藥性與total GST、GSTε2的相關性。野外品系中以高雄市苓雅區(LY-O)品系抗性最強;而所有抗性品系的total GST酵素活性皆高於NS,其中活性最強的品系有台南市西區(TNW-O)、高雄市苓雅區(LY-O)、和高雄縣鳳山市(FS-O);抗性品系的GSTε2之mRNA、蛋白表現量皆高於NS。經基因選殖得到GSTε2核甘酸序列全長為669bp,含222個胺基酸,以SDS-PAGE電泳確認分子量約24KD。比對各族群GSTε2之胺基酸序列發現LYR、LY-M、TNE-J、YC-J具胺基酸的點突變,後三者主要變異區為S124Y、I150V、E178A和A198E,經由NCBI Conserved Domains search的結果發現其突變位置為低保守段(low conserved domain),且這三品系的抗性也不高,因此推斷其抗性與序列變異無關。但LYR之rGSTε突變位置為G105S,屬於高保守段(high conserved domain),也是受質之結合位,其酵素活性為NS的1.7倍,因此推測此品系之GST活性升高可能與105點突變有關,進而涉及到較高之解毒作用。
Aedes(Ae.) aegypti is the primary vector of dengue fever. In South Taiwan, indigenious dengue fever usually occur during August to November. Insecticide resistant strains of Ae. aegypti have been detected in many areas, the insecticide resistance has also been linked to the failure of dengue control programs. Increased detoxification of mosquitoes by detoxic enzyme cytochrome P450, glutathione S-transferase, or hydrolase is one of the mechanisms of insecticide resistance. Insect glutathion S-transferases (GSTs) have been reports for implicated in resistance to insecticides. The purpose of the research was to find out the correlation between GSTε2 of Ae. aegypti and permethrin resistance. Laboratory strains NS and LYR were used as susceptible and resistant strains. Mosquitoes from different administrative districts in Tainan and Kaohsiung were screened by WHO testing kit for determing the resistance. Larvae with permethrin resistant strians were used to measure median lethal concentration (LC50) to permethrin. Total GST activity of resistant strains and quantitative PCR, Western blot of GSTε2 among the strains were also implemented. LY-O strain had the highest resistance to permethrin.Total GST activities of all the resistant strains were higher than that of NS, and TNW-O, LY-O and FS-O were the most highest. We also found overexpression of mRNA and protein of GSTε2 in resistant strains. The cloned GSTε2 gene was 669 bp in length and 222 deduced amino acids. Comparisons of GSTε2 amino acid sequences for all strains, site mutations in LY-M, TNE-J and YC-J were found at sites of S124Y、I150V、E178A and A198E, respevtically theses sites located in low conserved domain. The site mutations may not related to the resistance. The G105S mutation of LYR was located in highly conserved domain which considerd as a substrate binding pocket. The in vitro study showed the specific activity of LYR rGSTε2 protein was 1.7 fold higher than NS.
中文摘要 1
英文摘要(Abstract) 3
圖目錄(List of Figures) 8
表目錄(List of Tables) 9
縮寫表(Abbreviations) 10
一、 緒論(Introduction)
1-1病媒與埃及斑蚊 13
1-2 殺蟲劑和抗藥性 13
1-3埃及斑蚊之麩胺基硫轉移酶 16
1-4研究動機 17
1-5實驗設計 18
二、 實驗材料與方法(Material and Method)
2-1 埃及斑蚊
2-1-1 蚊子品系與飼養 20
2-1-2 埃及斑蚊粗蛋白萃取液 20
2-1-3 蛋白濃度測定 21
2-2 殺蟲劑對埃及斑蚊藥效之生物檢定
2-2-1 各種殺蟲劑對埃及斑蚊成蚊之死亡率測定 22
2-2-2 百滅寧(permethrin)對埃及斑蚊幼蟲藥效之測定 23
2-2-3 埃及斑蚊抗性品系之抗藥性維持 23
2-3 埃及斑蚊之GST epsilon2(GSTε2)序列分析
2-3-1 埃及斑蚊RNA製備 24
2-3-2 互補去氧核醣核酸cDNA製備 24
2-3-3 聚合酶連鎖反應(polymerase chain reaction﹔
PCR) 25
2-3-4 DNA電泳分析(electrophoresis) 26
2-3-5 DNA純化(elution) 26
2-3-6 載體之選殖-DNA 接合作用(ligation)及轉形作用 27
2-3-7 DNA與蛋白質序列比對及搜尋 28
2-4純化埃及斑蚊之epsilon二型麩胺基硫轉移酶重組蛋白 28
2-5 埃及斑蚊之GST epsilon2基因表現量分析-半定量
(semiquantitative) RT-PCR 30
2-6 埃及斑蚊之GST epsilon2蛋白表現量分析
2-6-1 一維電泳(electrophoresis) 30
2-6-2蛋白質染色與褪色(protein staining and destaining)33
2-6-3 免疫轉漬分析法(Western blotting analysis) 33
2-7 抗體(rabbit polyclonal GST epsilon 2 antibody)製備
2-7-1 蛋白純化(protein elution) 34
2-7-2 抗體生成(antibody inducement) 34
2-8埃及斑蚊之麩胺基硫轉移酶活性分析
2-8-1 實驗原理 35
2-8-2 以光譜儀偵測各抗性品系之GST活性 37
2-8-3 以酵素連酶偵測品系間GST活性 37
2-8-4埃及斑蚊LYR及NS兩品系之rGSTε2酵素活性分析 38
三、 結果(Result)
3-1 以WHO套組測試各品系埃及斑蚊成蚊之死亡率 39
3-2 各埃及斑蚊品系幼蟲對百滅寧(permethrin)的感受性 39
3-3 各品系埃及斑蚊GST活性分析結果 40
3-4 各抗性品系埃及斑蚊之GSTε2核甘酸與胺基酸序列分析 41
3-5 各抗性品系埃及斑蚊之GSTε2 mRNA表現量比較 41
3-6 各抗性品系埃及斑蚊之GSTε2蛋白表現量比較 42
3-7 rGSTε2蛋白之比活性 42
四、 討論(Discussion) 43
五、 參考文獻(Reference) 48
六、 圖表與說明(詳見圖表目錄)
附錄 67
1. Roberts, D. R., L. L. Laughlin, P. Hsheih, and L. J. Legters. 1997. DDT, global strategies, and a malaria control crisis in South America. Emerg. Infect. Dis. 3:295-302.
2. Gubler, D. J., and G. G. Clark. 1995. Dengue/dengue hemorrhagic fever: the emergence of a global health problem. Emerg. Infect. Dis. 1:55-57.
3. Sanders, E. J., P. Borus, G. Ademba, G. Kuria, P. M. Tukei, and J. W. LeDuc. 1996. Sentinel surveillance for yellow fever in Kenya, 1993 to 1995. Emerg. Infect. Dis. 2:236-238.
4. Meslin, F. X. 1997. Global aspects of emerging and potential zoonoses: a WHO perspective. Emerg. Infect. Dis. 3:223-228.
5. Thompson, D. F., J. B. Malone, M. Harb, R. Faris, O. K. Huh, A. A. Buck, and B. L. Cline. 1996. Bancroftian filariasis distribution and diurnal temperature differences in the southern Nile delta. Emerg. Infect. Dis. 2:234-235.
6. Daniels, T. J., R. C. Falco, I. Schwartz, S. Varde, and R. G. Robbins. 1997. Deer ticks (Ixodes scapularis) and the agents of Lyme disease and human granulocytic ehrlichiosis in a New York City park. Emerg. Infect. Dis. 3:353-355.
7. Jackson, L. A., and D. H. Spach. 1996. Emergence of Bartonella quintana infection among homeless persons. Emerg. Infect. Dis. 2:141-144.
8. 徐爾烈. 登革熱病媒蚊生態分布與調查防治.
9. (WHO)., W. H. O. 1992. Vector resistance to pesticides. 15th Report of the WHO Expert Committee on Vector Biology and Control. . World Health Organ. Tech. Rep. Ser. 818:1-62.
10. 徐爾烈. 1996. 行政院衛生署科技研究發展計畫。登革熱病媒蚊對合成除蟲菊酯抗藥性之研究(Ⅱ).
11. Silverman, J. a. B., D.N. 1993. Glucose aversion in the German cockroach, Blattella germanica. J. Insect Physiol 39:925-933.
12. Young, J. R. 1979. Fall armyworm: control with insecticides. Fla. Entomol. 62:
:130–133.
13. Vinson, G. P., and B. J. Whitehouse. 1971. Effect of corticotrophin (Synacthen, Ciba) on compartmental arrangement of steroids in the rat adrenal cortex. J. Endocrinol. 51:2-3.
14. Ffrench-Constant, R. H., T. A. Rocheleau, J. C. Steichen, and A. E. Chalmers. 1993. A point mutation in a Drosophila GABA receptor confers insecticide resistance. Nature 363:449-451.
15. Lin, T. J., K. T. Huang, and C. Y. Liu. 2006. Determination of organophosphorous pesticides by a novel biosensor based on localized surface plasmon resonance. Biosens Bioelectron 22:513-518.
16. Griffitts, J. S., S. M. Haslam, T. Yang, S. F. Garczynski, B. Mulloy, H. Morris, P. S. Cremer, A. Dell, M. J. Adang, and R. V. Aroian. 2005. Glycolipids as receptors for Bacillus thuringiensis crystal toxin. Science 307:922-925.
17. Strode, C., C. S. Wondji, J. P. David, N. J. Hawkes, N. Lumjuan, D. R. Nelson, D. R. Drane, S. H. Karunaratne, J. Hemingway, W. C. t. Black, and H. Ranson. 2008. Genomic analysis of detoxification genes in the mosquito Aedes aegypti. Insect Biochem. Mol. Biol. 38:113-123.
18. Hayes, J. D., and D. J. Pulford. 1995. The glutathione S-transferase supergene family: regulation of GST and the contribution of the isoenzymes to cancer chemoprotection and drug resistance. Crit. Rev. Biochem. Mol. Biol. 30:445-600.
19. John D. Hayes, J. U. F., and Ian R. Jowsey. 2005. Glutathion transferase. Annu. Rev. Pharmacol. Toxicol .45:51-88.
20. Ranson, H., C. Claudianos, F. Ortelli, C. Abgrall, J. Hemingway, M. V. Sharakhova, M. F. Unger, F. H. Collins, and R. Feyereisen. 2002. Evolution of supergene families associated with insecticide resistance. Science 298:179-181.
21. Hemingway, J., N. J. Hawkes, L. McCarroll, and H. Ranson. 2004. The molecular basis of insecticide resistance in mosquitoes. Insect Biochem. Mol. Biol. 34:653-665.
22. Kasai, S., I. S. Weerashinghe, T. Shono, and M. Yamakawa. 2000. Molecular cloning, nucleotide sequence and gene expression of a cytochrome P450 (CYP6F1) from the pyrethroid-resistant mosquito, Culex quinquefasciatus Say. Insect Biochem. Mol. Biol. 30:163-171.
23. Vaughan, A., and J. Hemingway. 1995. Mosquito carboxylesterase Est alpha 2(1) (A2). Cloning and sequence of the full-length cDNA for a major insecticide resistance gene worldwide in the mosquito Culex quinquefasciatus. J. Biol. Chem. 270:17044-17049.
24. Ranson, H., L. Rossiter, F. Ortelli, B. Jensen, X. Wang, C. W. Roth, F. H. Collins, and J. Hemingway. 2001. Identification of a novel class of insect glutathione S-transferases involved in resistance to DDT in the malaria vector Anopheles gambiae. Biochem. J. 359:295-304.
25. Ding, Y., F. Ortelli, L. C. Rossiter, J. Hemingway, and H. Ranson. 2003. The Anopheles gambiae glutathione transferase supergene family: annotation, phylogeny and expression profiles. BMC Genomics 4:35.
26. Lumjuan, N., L. McCarroll, L. A. Prapanthadara, J. Hemingway, and H. Ranson. 2005. Elevated activity of an Epsilon class glutathione transferase confers DDT resistance in the dengue vector, Aedes aegypti. Insect Biochem. Mol. Biol. 35:861-871.
27. Ortelli, F., L. C. Rossiter, J. Vontas, H. Ranson, and J. Hemingway. 2003. Heterologous expression of four glutathione transferase genes genetically linked to a major insecticide-resistance locus from the malaria vector Anopheles gambiae. Biochem. J. 373:957-963.
28. 2002. DengueNet WHO's Internet-based System for the global surveillance of dengue fever and dengue haemorrhagic fever (dengue/DHF) http://www.who.int/denguenet. Dengue/DHF global public health burden. Wkly Epidemiol Rec 77:300-304.
29. da-Cunha, M. P., J. B. Lima, W. G. Brogdon, G. E. Moya, and D. Valle. 2005. Monitoring of resistance to the pyrethroid cypermethrin in Brazilian Aedes aegypti (Diptera: Culicidae) populations collected between 2001 and 2003. Mem. Inst. Oswaldo. Cruz. 100:441-444.
30. Somboon, P., L. A. Prapanthadara, and W. Suwonkerd. 2003. Insecticide susceptibility tests of Anopheles minimus s.l., Aedes aegypti, Aedes albopictus, and Culex quinquefasciatus in northern Thailand. Southeast Asian J. Trop. Med. Public Health 34:87-93.
31. Rodriguez, M. M., J. Bisset, D. M. de Fernandez, L. Lauzan, and A. Soca. 2001. Detection of insecticide resistance in Aedes aegypti (Diptera: Culicidae) from Cuba and Venezuela. J. Med. Entomol. 38:623-628.
32. Coto, M. M., J. A. Lazcano, D. M. de Fernandez, and A. Soca. 2000. Malathion resistance in Aedes aegypti and Culex quinquefasciatus after its use in Aedes aegypti control programs. J. Am. Mosq. Control Assoc. 16:324-330.
33. Focks, D. A., R. J. Brenner, J. Hayes, and E. Daniels. 2000. Transmission thresholds for dengue in terms of Aedes aegypti pupae per person with discussion of their utility in source reduction efforts. Am. J. Trop. Med. Hyg. 62:11-18.
34. Centers of Disease Control, R.O.C.(Taiwan). Notifiable Infectious Disease Statistics System.
35. Hsieh, Y. H., and C. W. Chen. 2009. Turning points, reproduction number, and impact of climatological events for multi-wave dengue outbreaks. Trop. Med. Int. Health 14:628-638.
36. Perera, M. D., J. Hemingway, and S. P. Karunaratne. 2008. Multiple insecticide resistance mechanisms involving metabolic changes and insensitive target sites selected in anopheline vectors of malaria in Sri Lanka. Malar. J. 7:168.
37. Macoris Mde, L., M. T. Andrighetti, L. Takaku, C. M. Glasser, V. C. Garbeloto, and J. E. Bracco. 2003. Resistance of Aedes aegypti from the state of Sao Paulo, Brazil, to organophosphates insecticides. Mem. Inst. Oswaldo. Cruz. 98:703-708.
38. IuM, G. 1963. Insecticide resistance and vector control. Thirteenth Report of the WHO Expert Committee on insecticides. World Health Organ Tech. Rep. Ser. 265:1-227.
39. Soderlund, D. M., and D. C. Knipple. 2003. The molecular biology of knockdown resistance to pyrethroid insecticides. Insect Biochem. Mol. Biol. 33:563-577.
40. Ranson, H., B. Jensen, X. Wang, L. Prapanthadara, J. Hemingway, and F. H. Collins. 2000. Genetic mapping of two loci affecting DDT resistance in the malaria vector Anopheles gambiae. Insect Mol. Biol. 9:499-507.
41. Prapanthadara, L., Promtet, N., Koottathep, S., Somboon, P., Suwonkerd, W., McCarroll, L., Hemingway, J.,. 2002. Mechanisms of DDT and permethrin resistance in Aedes aegypti from Chiang Mai, Thailand. Dengue Bull 26:185–189.
42. Organization, W. H. 1998. Test procedures for insecticide resistance monitoring in malaria vectors, bio-efficacy and persistence of insecticides on treated surfaces WHO. WHO/CDS/CPC/MAL/98.12
43. Chang, C., W. K. Shen, T. T. Wang, Y. H. Lin, E. L. Hsu, and S. M. Dai. 2009. A novel amino acid substitution in a voltage-gated sodium channel is associated with knockdown resistance to permethrin in Aedes aegypti. Insect Biochem. Mol. Biol. 39:272-278.
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top