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系統識別號 U0026-0812200911254028
論文名稱(中文) 減毒株之豬霍亂沙門氏桿菌攜帶豬前胸腺激素基因作為疫苗研發的可能性
論文名稱(英文) Attenuated Salmonella choleraesuis carrying eukaryotic expression vectors encoding porcine prothymosin α for vaccine development
校院名稱 成功大學
系所名稱(中) 微生物及免疫學研究所
系所名稱(英) Department of Microbiology & Immunology
學年度 93
學期 1
出版年 94
研究生(中文) 陳俊成
研究生(英文) Chung-Cheng Chen
學號 s4691112
學位類別 碩士
語文別 英文
論文頁數 44頁
口試委員 指導教授-蕭璦莉
口試委員-朱純燕
口試委員-吳昭良
口試委員-余俊強
中文關鍵字 疫苗  前胸腺激素  沙門氏桿菌病 
英文關鍵字 prothymosin α  live attenuated Salmonella vaccine 
學科別分類
中文摘要   沙門氏桿菌病(salmonellosis)是台灣養豬業常見的細菌性傳染病,其中豬霍亂沙門氏桿菌(Salmonella choleraesuis)是主要的分離株,佔90%以上.豬隻遭到感染後會引起嚴重的胃腸炎,有時產生敗血症,有時產生慢性的腸道損壞而造成延長飼養期間且不經濟,此種疾病也會併發肺炎,若不加以治療處理,常會造成豬隻的高死亡率,對於養豬業的傷害很大.一般的針劑疫苗無法達到有效防疫的效果,目前只能利用豬場管理以及藥物控制來達到防治目的,不過長久依賴抗生素只會造成病原菌的抗藥性,所以新疫苗的發展對養豬業很重要.減毒沙門氏桿菌口服疫苗在最近幾年漸漸引起注意,因為減毒株會侵入宿主組織,刺激分泌性、體液性及細胞性免疫反應.誘發出的保護性免疫反應效果遠比死菌疫苗或次單位疫苗好.過去的研究顯示前胸腺激素(prothymosin α; ProT)具有增強免疫反應的效果,因此我們從豬的腎臟細胞選殖出豬的前胸腺激素(porcine ProT),並將它構築到可以在真核細胞中表現基因產物的pTCY質體,再利用電破法將之轉形到減毒株的豬霍亂沙門氏桿菌(Salmonella choleraesuis subsp. choleraesuis; S.C.S.C.),形成攜帶有由真核細胞啟動子調控表現pProT質體的重組疫苗株,S.C./pProT.利用小鼠為動物模式,採口服方式給予疫苗,口服方式進行攻毒實驗,進行免疫反應及保護力的研究.研究結果顯示S.C./pProT可以引起強烈的抗體反應、脾臟淋巴細胞對S.C.有高度增生反應,脾臟淋巴細胞中T淋巴細胞的比例增加、其中CD4+及CD8+都有增加.經由S.C./pProT免疫過的小鼠存活率提高.期望未來此重組疫苗能應用在豬隻對豬霍亂沙門氏桿菌感染的防治上,對本地的養豬業能有所貢獻.
英文摘要  Salmonella choleraesuis infections in swine cause a septicemic disease resulting in pneumonia and other systemic involvement, with some involvement of the intestinal tract. The causative agent is a Gram-negative, host adapted, facultative intracellular bacterium belonging to the family of Enterobacteriaceae. They spread by direct contact and usually result in high mortality of pigs, leading to economic loss in porcine industry. The use of live-attenuated Salmonella vaccine seems to be a better strategy against Salmonella infection. In this study, prothymosin α (ProT), a highly acidic protein that can upregulate immune responses, was transformed into attenuated S. choleraesuis by electroporation. The attenuated S. choleraesuis harboring eukaryotic expression plasmids encording porcine ProT gene, S.C./pProT, were administered orally to BALB/c mice. Mice immunized with S.C./pProT for three times were protected against subsequent lethal oral challenge of virulent S. choleraesuis, Dadin strain. When mice were immunized with S.C./pProT, strong antibody response was induced, splenocyte proliferation was upregulated, and expanded T cell population in the spleen. Through the strong upregulation of immune responses against virulent Salmonella challenge, survival rate of the immunized mice reaches 50~60%. Therefore, S.C./pProT may be a good candidate vaccine against S. choleraesuis infection in pigs in the future.
論文目次 Abstract…………………………………………………………………………... I
Chinese Abstract…………………………………………………………………. II
Acknowledgement……………………………………………………………….. Ⅳ
Contents………………………………………………………………………….. V
Figure contents…………………………………………………………………... Ⅶ
Appendix………………………………………………………………………… Ⅷ
Abbreviation……………………………………………………………………... Ⅸ
Introduction
Salmonella choleraesuis infection………………………………………….. 1
Bacterium as gene delivering vehicle………………………………………. 2
Prothymosin α gene………………………………………………………… 3
Strategy for developing oral Salmonella vaccine and immunization………. 4
Materials and methods
Bacteria and mice…………………………………………………………... 6
Cloning of the cDNA of porcine prothymosin α.…………………………… 6
Consruction of attenuated S. choleraesuis carrying porcine prothymosine α.…………………………………………………………………………….. 8
Immunization and challenge………………………………………………... 8
Antigen preparation………………………………………………………… 9
Detection of anti-Salmonella antibodies by ELISA………………………... 10
Lymphocyte proliferative response to Salmonella choleraesuis…………… 11
Detection of splenocyte sub-populations…………………………………… 12
Depletion of T cells in vivo…………………………………………………12
Adoptive transfer studies……………………………………………………13
Statistic analysis…………………………………………………………. 14
Results:
Construction of the porcine prothymosin α genes and attenuated Salmonella choleraesuis carrying porcine prothymosine α.……………15
Aliment of DNA and amino acid sequences of porcine prothymosin α
between swine and mouse…………………………………………………15
Detection of antibody responses against Salmonella by ELISA……………16
Induction of lymphocyte proliferative responses in mice by immunization with attenuated S. choleraesuis carrying porcine prothymosine α.…………17
Detection of splenocyte sub-populations……………………………………17
Protection of mice against a lethal Salmonella challenge by oral immunization with S. choleraesuis harboring porcine prothymosine α expression vector……………………………………………………………18
The effects of CD4+ T cell depletion or CD8+ T cell depletion on the efficacy of recombinant attenuated S.C./pProT vaccine……………………19
The effects of adoptive transfer of immunized sera, splenocytes or both sera and splenocytes to naïve mice…………………………………………20
Discussion............................................................................................................... 21
References……………………………………………………………………….. 28
Figures…………………………………………………………………………… 31
Appendix…………………………………………………………………………. 43
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