跳到主要內容

臺灣博碩士論文加值系統

(44.222.104.206) 您好!臺灣時間:2024/05/29 22:15
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

: 
twitterline
研究生:賴以祥
研究生(外文):I-Hsiang Lai
論文名稱:鳥禽類披衣菌主要外膜蛋白質基因之選殖及其抗原性分析
論文名稱(外文):Cloning and Antigenic Analysis of the Major Outer Membrane Protein Gene from Chlamydophila psittaci
指導教授:廖明輝廖明輝引用關係
指導教授(外文):Ming-Huei Liao
學位類別:碩士
校院名稱:國立屏東科技大學
系所名稱:生物科技研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:75
中文關鍵詞:披衣菌感染症主要外膜蛋白質聚合酶連鎖反應鸚鵡披衣菌流產披衣菌
外文關鍵詞:ChlamydiosisMajor outer membrane proteinpolymerase chain reactionChlamydophila psittaciChlamydophila abortus
相關次數:
  • 被引用被引用:4
  • 點閱點閱:355
  • 評分評分:
  • 下載下載:23
  • 收藏至我的研究室書目清單書目收藏:1
披衣菌感染症(chlamydiosis)是一重要人畜共通傳染病,國內已有鳥禽類、牛、羊等重要家畜禽之感染調查報告。雖然國內對此疾病已建立多項診斷技術,但診斷試劑仍需仰賴進口,診斷本病成本極為昂貴。本研究目的即是利用基因工程方法來大量生產披衣菌之主要外膜蛋白質(major outer membrane protein;MOMP),期能大幅降低成本且安全生產具特異抗原性、免疫原性之蛋白質,作為未來診斷試劑開發用。實驗中利用聚合酶連鎖反應(polymerase chain reaction;PCR)分別增幅Chlamydophila psittaci及Chlamydophila abortus之MOMP蛋白質之VD1、VD2、VD3及VD4等四段基因,經定序確認再分別選殖至pET32a表現載體中,並轉形送入BL21 Star(DE3)菌株內作蛋白質表現。結果所誘導表現出之重組MOMP缺損蛋白質皆存在細菌沉澱物之包涵體蛋白質中,且隨著誘導時間之增長,其表現量有增加之趨勢。將此蛋白質加以純化後進行蛋白質定量測定,並用各種野外及市售披衣菌抗體以西方轉漬法作蛋白質定性分析,以證明重組蛋白質之特異性及抗原性。結果顯示,披衣菌重組MOMP蛋白質之特異性抗原性以VD2及VD3區域較佳,而C. psittaci MOMP VD4可能為鴨主要誘導抗體產生之區域。未來可提供作為開發披衣菌抗原、抗體診斷試劑時之特異免疫蛋白質來源。
Chlamydiosis is an important zoonosis worldwide. Many reports are available on the occurrence of Chlamydiosis of ruminants and avians in Taiwan. Several diagnostic techniques have been developed locally but almost all diagnostic reagents in the market are imported from abroad. The current diagnostic techniques are not only expensive but also hazardous to the diagnostician. The objective of this study was therefore using molecular biology techniques to produce the major outer membrane protein (MOMP) from Chlamydophila psittaci and Chlamydophila abortus. This technique will be able to produce a more specific, safe and cheap diagnostic antigen. In this study the MOMP VD1, VD2, VD3 and VD4 genes were amplified by polymerase chain reaction (PCR). These genes were sequenced and then cloned into pET32a vector. The clones were then transformed into BL21 Star (DE3) for protein expression. The recombinant MOMPs were found in the inclusion bodies. It was also observed that the amount of protein expressed was directly proportional to the incubation time. After purification of expressed protein, the protein concentration was checked by a spectrophotometer followed by western blot for specific and antigenic analysis. The results of this study have shown that the recombinant MOMPs VD2 and VD3 are more specific and antigenic. C. psittaci MOMP VD4 may have the ability to induce antibody production in the duck. The recombinant MOMPs may be used for production of diagnostic reagents.
中文摘要----------------------------------------------- I
英文摘要----------------------------------------------- Ⅱ
誌謝--------------------------------------------------- Ⅲ
目錄--------------------------------------------------- Ⅳ
圖索引------------------------------------------------- Ⅷ
第一章 緒言------------------------------------------ 1
第二章 文獻探討-------------------------------------- 3
2.1 披衣菌之結構與特性--------------------------------- 3
2.2 披衣菌之生活週期----------------------------------- 4
2.3 披衣菌之分類--------------------------------------- 4
2.4 Chlamydophila psittaci----------------------------- 5
2.4.1 流行病學----------------------------------------- 5
2.4.2 鳥類感染之臨床症狀------------------------------- 5
2.4.3 人類感染之臨床症狀------------------------------- 6
2.5 Chlamydophila abortus------------------------------ 7
2.5.1 流行病學----------------------------------------- 7
2.5.2 動物感染之臨床症狀------------------------------- 8
2.6 動物披衣菌感染症之診斷方法------------------------- 8
2.7 披衣菌之抗原特性----------------------------------- 9
2.8 主要外膜蛋白質(MOMP)----------------------------- 10
第三章 材料與方法------------------------------------ 14
3.1 披衣菌來源----------------------------------------- 14
3.2 細胞培養與披衣菌增殖------------------------------- 14
3.3 披衣菌核酸抽取及純化------------------------------- 14
3.4 披衣菌MOMP全長基因選殖定序------------------------- 15
3.4.1 引子設計----------------------------------------- 15
3.4.2 聚合酶鏈鎖反應----------------------------------- 16
3.4.3 PCR產物之純化------------------------------------ 16
3.4.5 MOMP基因之選殖----------------------------------- 17
3.4.6 重組DNA之抽取及鑑定------------------------------ 18
3.4.7 核酸定序及分析----------------------------------- 19
3.5 披衣菌MOMP蛋白質表現------------------------------- 20
3.5.1 引子設計----------------------------------------- 20
3.5.2 聚合酶鏈鎖反應----------------------------------- 21
3.5.3 MOMP基因片段之處理------------------------------- 22
3.5.4 表現載體之製備----------------------------------- 22
3.5.5 MOMP基因之選殖----------------------------------- 23
3.5.6 重組DNA之抽取及鑑定------------------------------ 24
3.5.7 MOMP蛋白質表現----------------------------------- 24
3.6 SDS-PAGE蛋白質電泳--------------------------------- 25
3.7 蛋白質純化----------------------------------------- 26
3.7.1 包涵體蛋白之分離--------------------------------- 26
3.7.2 蛋白質樣品的製備--------------------------------- 27
3.7.3 親和性管柱的製備--------------------------------- 27
3.7.4 蛋白質的純化與復性------------------------------- 27
3.7.5 蛋白質定量--------------------------------------- 28
3.8 披衣菌MOMP蛋白質之抗原性分析----------------------- 29
3.8.1 西方轉漬法--------------------------------------- 29
3.8.2 免疫染色法--------------------------------------- 29
第四章 結果------------------------------------------ 31
4.1 披衣菌MOMP全長基因之分析比對----------------------- 31
4.1.1 基因選殖與定序----------------------------------- 31
4.1.2 核酸序列比對------------------------------------- 31
4.1.3 胺基酸序列比對----------------------------------- 32
4.2 披衣菌MOMP基因蛋白質表現--------------------------- 32
4.2.1 原核表現載體之建構------------------------------- 32
4.2.2 重組蛋白質的表現--------------------------------- 33
4.2.3 重組蛋白質的純化--------------------------------- 34
4.3 蛋白質定量----------------------------------------- 34
4.4 披衣菌MOMP蛋白質之抗原性分析----------------------- 35
第五章 討論------------------------------------------ 55
第六章 參考文獻-------------------------------------- 60
第七章 附錄------------------------------------------ 68
7.1 溶液的配製----------------------------------------- 68
7.2 勝任細胞的製備------------------------------------- 71
7.3 pGEM®-T Easy Vector圖譜---------------------------- 73
7.4 pET32a Vector圖譜---------------------------------- 74
作者簡介----------------------------------------------- 75
田蔚城。2000。生物技術的發展與應用。九洲。第三版。223-241。
葉明穎。1997。台灣地區鴿子披衣菌血清學調查及分離。國立台灣大學獸醫學研究所碩士論文。
張澔任。2000。應用聚合酶連鎖反應偵測Chlamydophila abortus。國立中興大學獸醫微生物學研究所碩士論文。
廖永剛,張秋燕,呂榮修,黎南榮,蔡向榮,劉培柏。1997。本省山羊披衣菌感染之疫情調查。台灣省畜衛所研報。33:143-150。
廖永剛,洪彩蓮,黎南榮,許天來。1998。台灣母山羊流產症之病因探討與疫學研究。台灣省畜衛所研報。34:15-19。
廖永剛,洪彩蓮,李水蓮,魯懿萍,許天來。1999。畜牧獸醫人員鸚鵡披衣菌血清抗體調查。中華民國獸醫學會雜誌。25:74。
魯懿萍,賴以祥,廖明輝,黃旭田,洪信雄。2002。鳥禽類披衣菌症快速診斷技術之開發與應用。中華民國獸醫學會暨台灣畜牧獸醫學會九十一年度聯合學術論文發表會。110。
賴以祥,廖明輝,魯懿萍,黃旭田,洪信雄。2001。台灣南部地區鳥禽類之披衣菌抗原檢測。中華民國獸醫學會暨台灣畜牧獸醫學會九十年度聯合學術論文發表會。90。
謝蕙蓮。2000。台灣地區反芻獸披衣菌血清學診斷及分離之主要外膜蛋白基因分析。國立台灣大學獸醫學研究所碩士論文。
Andersen, A. A. 1996. Comparison of pharyngeal, fecal, cloacal samples for the isolation of Chlamydia psittaci from experimentally infected cockatiels and turkeys. J Vet Diagn Invest 8: 448-450.
Bas, S., Muzzin, P., Ninet, B., Bornand, J. E., Scieux, C. and Vischer, T. L. 2001. Chlamydia serology: comparative diagnostic value of immunoblotting, microimmunofluorescence test, and immunoassays using different recombinant proteins as antigens. J Clin Microbiol 39: 1368-1377.
Batteiger, B. E., Lin P. M., Jones, R. B. and Van Der Pol, B. J. 1996. Species-, serogroup-, and serovar-specific epitopes are juxtaposed in variable sequence region 4 of the major outer membrane proteins of some chlamydia trachomatis serovars. Immun Infect 64: 2839-2841.
Blewett, D. A., Gisemba, F., Miller, J. K., Johnson, F. W. and Clarkson, M. J. 1982. Ovine enzootic abortion: the acquisition of infection and consequent abortion within a single lambing season. Vet Rec 22: 499-501.
Bradford, M. M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of dye binding. Anal Biochem 72: 248-254.
Bush, R. M. and Everett, K. D. E. 2001. Molecular Evolution of the Chlamydiaceae. Int J Syst Evol Microbiol 51: 203-220.
Chae, C., Cheon, D. S., Kwon, D., Kim, O., Kim, B., Suh, J., Rogers, D. G., Everett, K. D. E. and Andersen, A. A. 1997. In situ hybridization for the detection and localization of swine Chlamydia trachomatis. Vet Pathol 36: 133-137.
De Sa, K., Souriau, A., Bernard, F., Salinas, J. and Rodolakis, A. 1995. An oligomer of the major outer membrane protein of chlamydia psittaci is recognized by monoclonal antibodies which protect mice from abortion. Infect Immun 63: 4912-4916.
Everett, K. D. E. and Hatch, T. P. 1995. Architecture of the cell envelope of chlamydia psittaci 6BC. J Bacteriol 177: 877-882.
Everett, K. D. E. and Andersen, A. A. 1997. The ribosomal intergenic spacer and Domain I of the 23S gene are phylogenetic markers for Chlamydia spp. Int J Syst Bacteriol 47: 461-473.
Everett, K. D. E. and Andersen, A. A. 1999. Identification of nine species of the Chlamydiaceae using PCR-RFLP. Int J Syst Bacteriol 49: 803-813.
Everett, K. D. E., Bush, R. M. and Andersen, A. A. 1999a. Emended description of the order Chlamydiales, proposal of Parachlamydiaceae fam. nov. and Simkaniaceae fam. nov., each containing one monotypic genus, revised taxonomy of the family Chlamydiaceae, including a new genus and five new species, and standards for the identification of organisms. Int J Syst Bacteriol 49: 415-440.
Everett, K. D. E., Hornung, L. J. and Andersen, A. A. 1999b. Rapid detection of the Chlamydiaceae and other families in the order Chlamydiales: three PCR tests. J Clin Microbiol 37: 575-580.
Everett, K. D. E. 2000. Chlamydia and Chlamydiales: more than meets the eye. Vet Microbiol 75: 100-126.
Grimes, J. E. and Clark, D. 1986. Chlamydia psittaci infection of pet and other birds in the United States in 1984. J Infect Dis 153: 374-375.
Harrison, G. J. 1989. A practitioner’s view of the problem of avian chlamydiosis. J Am Vet Med Assoc 195: 1929-1928.
Hiroshi, Y., Yuichiro, K., Sadashi, S. and Toshikatsu, H. 1998. Differentiation of Chlamydia species by combined use of polymerase chain reaction and restriction endonuclease analysis. Microbiol Immunol 42: 411-414.
Jones, H. M., Kubo, A. and Stephens, R. S. 2000. Design, expression and functional characterization of a synthetic gene encoding the chlamydia trachomatis major outer membrane protein. Gene 258: 173-181.
Kaltenboeck, B., Kousoulas, K. G. and Storz, J. 1993. Structures of and allelic diversity and relationships among the major outer membrane protein (ompA) genes of the four chlamydial species. J Bacteriol 175: 487-502.
Kaltenboeck, B., Heard, D., DeGraves, F. J. and Schmeer, N. 1997. Use of synthetic antigens improves detection by enzyme-linked immunosorbent assay of antibodies against abortigenic chlamydia psittaci in ruminants. J Clin Microbiol 35: 2293-2298.
Kido, Y., Shirai, M., Ouchi, K. and Nakazawa, T. 2001. Analysis of the serological response to chlamydia pneumoniae in patients with ischemic heart disease by recombinant MOMP-ELISA. J Infect Chemother 7: 180-185.
Kim, S. K. and De Mars, R. 2001. Epitope clusters in the major outer membrane protein of chlamydia trachomatis. Immun Infect 13: 429-436.
Klein, M., Kotz, A., Bernardo, K. and Kronke, M. 2003. Detection of chlamydia pneumoniae-specific antibodies binding to the VD2 and VD3 regions of the major outer membrane protein. J Clin Microbiol 41: 1957-1962.
Lampe, M. F., Wong, K. G., Kuehl, L. M. and Stamm, W. E. 1997. Chlamydia trachomatis major outer membrane protein variants escape neutralization by both monoclonal antibodies and human immune sera. Immun Infect 65: 317-319.
Laroucau, K., Souriau, A. and Rodolakis, A. 2002. Improved sensitivity of PCR for Chlamydophila using pmp genes. Vet Microbiol 82: 155-164.
Loock, M. V., Vanrompay, D., Herrmann, B., Stappen, J. V., Volckaert, G., Goddeeris, B. M. and Everett, K. D. E. 2003. Missing links in the divergence of Chlamydophila abortus from Chlamydophila psittaci. Int J Syst Evol Microbiol 53: 761-770.
LaVallie, E. R., DiBlasio, E. A., Kovacic, S., Grant, K. L., Schendel, P. F. and McCoy, J. M. 1993. A thioredoxin gene fusion expression system that circumvents inclusion body formation in the E. coli cytoplasm. Biotechnol 11: 187-193.
May, S. W., Kelling, C. L., Sabara, M. and Sandbulte, J. 1996. Virulence of feline chlamydia psittaci in mice is not a function of the major outer membrane protein. Vet Microbiol 53: 355-368.
McCafferty, M., Herring, A. J., Andersen, A. A. and Jones, G. E. 1995. Electrophoretic analysis of the major outer membrane protein of chlamydia psittaci reveals multimers which are recognized by protective monoclonal antibodies. Infect Immun 63: 2387-2389.
Moore, F. M., McMilan, M. C., Margeret, L. and Petrak, M. L. 1991. Comparison of culture peroxidase - anti peroxidase reaction and serum latex agglutination methods for diagnosis of chlamydiosis in pet birds. J Vet Med Assoc 199: 71-73.
Mygind, P., Christiansen, G., Persson, K. and Birkelund, S. 2000. Detection of chlamydia trachomatis-specific antibodies in human sera by recombinant major outer membrane protein polyantigens. J Med Microbiol 49: 457-465.
Papouchado, M. L., Valdez, S. N., Ermacora, M. R., Ganan, S. and Poskus, S. E. 1997. Highly sensitive and specific enzyme-linked immunosorbent assays for GAD65 autoantibodies using a thioredoxin-GAD65 fusion antigen. J Immunol Methods 207: 169-178.
Papp, J. R., Shewen, P. E. and Gartley, C. J. 1994. Abortion and subsequent excretion of Chlamydiae from the reproductive tract of sheep during estrus. Infect Immun 62: 3786-3792.
Pospischil, A., Thoma, R., Hilbe, M., Grest, P., Zimmermann, D. and Gebbers, J. O. 2002. Abortion in humans caused by Chlamydophila abortus (Chlamydia psittaci serovar 1). Schweiz Arch Tierheilkd 144: 463-466.
Poppert, S., Essig, A., Marre, R., Wagner, M. and Horn, M. 2002. Detection and differentiation of Chlamydiae by fluorescence in situ hybridization. Appl Environ Microbiol 68: 4081-4089.
Pudjiatmoko, F. H., Ochiai, Y., Yamaguchi, T. and Hirai, K. 1997. Phylogenetic analysis of the genus Chlamydia based on 16S rRNA gene sequences. Int J Syst Bacteriol 47: 425-431.
Sandbulte, J., TerWee, J., Wigington, K. and Sabara, M. 1996. Evaluation of chlamydia psittaci subfraction and subunit preparation for their protectivecapacities. Vet Microbiol 48: 269-282.
Sambrook, J., Frritsch, E. F. and Maniatis, T. M. 1989. Molecular coloning: a laboratory manual. Cold Spring Harbor, New York, 1.40-1.52.
Takahashi, T., Masuda, M., Tsuruno, T., Mori, Y., Takashima, I., Hiramune, T. and Kikuchi, N. 1997. Phylogenetic analyses of Chlamydia psittaci from birds based on 16S rDNA gene sequence. J Clin Microbiol 35: 2908-2914.
Takashima, I., Imai, Y., Itoh, N., Kariwa, H. and Hashimoto, N. 1996. Polymerase chain reaction for the detection of Chlamydia psittaci in the faeces of budgerigars. Microbiol Immunol 40: 21-26.
Tan, T. W., Herring, A. J., Anderson, I. E. and Jones, G. E. 1990. Protection of sheep against Chlamydia psittaci infection with a subcellular vaccine containing the major outer membrane protein. Infect Immun 58: 3101-3108.
Vanrompay, D., Van Nerom, A., Ducatelle, R. and Haesebrouck, F. 1994. Evaluation of five immunoassays for detection of Chlamydia psittaci in cloacal and conjunctival specimens from turkeys. J Clin Microbiol 32: 1470-1474.
Vanrompany, D., Cox, E., Mast, J., Goddeeris, B. and Volckaert, G. 1998. High-level expression of chlamydia psittaci major outer membrane protein in COS cells and in skeletal muscles of turkey. Infect Immun 66: 5494-5500.
Vretou, E., Psarrou, E., Kaisar, M., Vlisidou, I., Montesanto, V. S. and Longbottom, D. 2001. Identification of protective epitopes by sequencing of the major outer membrane protein gene of a variant strain of chlamydia psittaci serotype 1 (chlamydophila abortus). Infect Immun 69: 607-612.
Villemonteix, P., Agius, G., Ducroz, B., Rouffineau, J., Plocoste, V., Castets, M. and Magnin, G. 1990. Pregnancy complicated by severe Chlamydia psittaci infection acquired from a goat flock: case report. Eur J Obstet Gynecol Reprod Biol 37: 91-94.
Wang, F. I., Shieh, H. and Liao, Y. K. 2001. Prevalence of chlamydophila abortus infection in domesticated ruminants in Taiwan. J Vet Med Sci 63: 1215-1220.
Woolhouse, M. E. J., Taylor, L. H. and Hayden, D. T. 2001. Population biology of multihost pathogens. Science 292: 1109-1112.
Wolf, K., Fischer, E., Mead, D., Zhong, G., Peeling, R., Whitmire, B. and Caldwell, H. D. 2001. chlamydia pneumoniae major outer membrane protein is a surface-exposed antigen that elicits antibodies primarily directed against conformation-dependent determinants. Infect Immun 69: 3082-3091.
QRCODE
 
 
 
 
 
                                                                                                                                                                                                                                                                                                                                                                                                               
第一頁 上一頁 下一頁 最後一頁 top
1. 林千惠(民89)。國中小啟智教育教師離職傾向及其相關因素之調查研究。特殊教育研究學刊,19,15-36。
2. 古金英(民90)。建立一個輪調環境下受輪調員工的工作壓力源模式-以銀行業為例。人力資源管理學報,1(3),1-25。
3. 王春源、錢淑芬(民84)。對角色性格差異、角色壓力、工作投入與滿意度之關連性研究。中山人文社會科學期刊,4(1),121-185。
4. 王秀春、林月玲、李碧霞(民86)。台北市某市立醫院護理人員離職原因之探討。護理新象,7(2),658-672。
5. 尹裕君、安妮(民80)。護理人員離職原因與工作滿意度相關性探討。榮總護理,8(4),428-436。
6. 胡庭禎、林春華(民89)。病歷部人員角色壓力與組織承諾關聯性之探討。弘光學報,35,73-95。
7. 邱馨儀(民85)。論學校組織文化與教師組織承諾之互動。人事月刊,32(5),20-27。
8. 陳月枝(民81)。護理人力供給研究。護理雜誌,39(1),35-45。
9. 陳海鳴、余靜文(民89)。企業文化發展與組織承諾的關聯性研究-被購併企業續留員工之觀點。管理與系統七月份,249-270。
10. 陳梅麗、林小玲、連金延、余先敏、蔡欣玲(民89)。醫學中心護理人員工作壓力、離職意願與離職行為之相關性研究。榮總護理,17(3),260-268。
11. 黃英忠、張肇松、杜佩蘭(民91)。組織承諾與工作滿足關係之研究-以公辦民營與公立醫院為例。醫院,35(1),10-24。
12. 楊克平(民78)。台灣地區護理人員流動因素之探討。護理雜誌,36(3),51-64。
13. 劉淑如、鄭靜瑜(民91)。高屏地區護理人員組織承諾之探討。慈濟護理雜誌,1(1),56-65。
14. 潘依琳、張媚(民87)。醫院護理人員之工作特性、成就動機對工作投入、工作滿足與留職意願之影響。中華公共衛生雜誌,17(1),48-58。
15. 蕭伃伶、盧美秀(民85)。臨床護理人員之組織承諾、組織留任與專業留任的相關性研究。護理研究,4(2),137-149。