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研究生:蕭振文
研究生(外文):Jen-Wen Shiau
論文名稱:利用去毒性綠膿桿菌外毒素研發免疫去勢疫苗及基因疫苗
論文名稱(外文):Detoxicated Pseudomonas exotoxin A used for the development of immunocastration vaccine and DNA vaccine
指導教授:宋永義楊惠郎
指導教授(外文):Yung-Yi SungHuey-Lang Yang
學位類別:博士
校院名稱:國立臺灣大學
系所名稱:畜產學研究所
學門:農業科學學門
學類:畜牧學類
論文種類:學術論文
論文出版年:2000
畢業學年度:88
語文別:中文
論文頁數:100
中文關鍵詞:激性腺素釋放素免疫去勢基因疫苗綠膿桿菌外毒素
外文關鍵詞:GnRHimmunocastrationDNA vaccinePseudomonas exotoxin A
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本研究之目的,乃利用去毒性之綠膿桿菌外毒素(Pseudomonas exotoxin, PE),研發豬之免疫去勢疫苗及對抗綠膿桿菌外毒素之基因疫苗。試驗分為三部分,試驗一,使用已去毒性之綠膿桿菌外毒素做為攜帶蛋白(Carrier),與激性腺素釋放素(Gonadotropin-Releasing Hormone, GnRH)接合後,作為豬之免疫去勢疫苗,接種公豬後,探討其對公豬生殖性腺之影響。試驗結果顯示,接種免疫去勢疫苗之公豬能誘發抗-GnRH與抗-攜帶蛋白之抗體,導致公豬性腺與輔性腺發育萎縮、血清中睪固酮下降、睪丸組織切片中無成熟精子存在,有效抑制公豬的性腺發育與生精作用,達到免疫去勢之目的。
試驗二,是利用基因疫苗的方法,將 GnRH基因構築在含有去毒性綠膿桿菌外毒素基因之表現質體(pSecTagC-PE-GnRH),該質體在巨細胞病毒(Cytomegalo Virus, CMV)啟動子驅動下,能夠在真核細胞中進行基因的表現。構築完成的表現質體先行在小鼠之成纖維細胞株(3T3)進行基因之活體外(in vitro)短暫表現,由西氏雜交(Western Blot)證明綠膿桿菌外毒素的表現。該表現質體隨即進行 BALB/c小鼠之肌肉接種(intramuscular, i.m.),結果小鼠產生抗-綠膿桿菌外毒素抗體,但測不到抗-GnRH的抗體產生。由於接種基因疫苗的小鼠誘發高的抗-綠膿桿菌外毒素抗體,為測定疫苗接種小鼠能否產生免疫力而抵抗綠膿桿菌外毒素之攻毒(Challenge),乃使用致死劑量的綠膿桿菌外毒素進行小鼠攻毒試驗。試驗結果顯示,注射基因疫苗的全部小鼠完全耐過毒素之攻毒而存活。而僅接種磷酸鹽緩衝液(Phosphate Buffer Saline, PBS)之對照小鼠則全數於24小時內死亡。由此結果,得知該表現質體在小鼠雖測不到抗-GnRH抗體,但卻能表現抗-綠膿桿菌外毒素之特異性抗體,保護接種小鼠免於毒素之攻毒致死。
試驗三,首先將表現人類IgG的質體改造成為帶有小鼠IgG重鏈與輕鏈變異區(Variable Region)的嵌合IgG,此IgG含有小鼠IgG變異區及人類IgG的穩定區。而後,進一步將 GnRH之核酸序列,構築入此一嵌合IgG重鏈或輕鏈變異區域內第三個互補決定區(Complementarity Determining Region, CDR3),成為具有表現GnRH之嵌合IgG表現質體,該質體 DNA轉殖入BHK(Baby Hamster Kidney)細胞株進行活體外短暫表現,並進行 BALB/c小鼠之免疫接種。試驗結果顯示,利用lipofectamine將該等構築之表現質體DNA轉殖入BHK細胞株,可以表現出IgG,但並未測得GnRH之表現。而質體 DNA亦可在接種的BALB/c小鼠體內表現嵌合IgG抗體,但GnRH之抗體力價則極微弱,但似有因GnRH套數的增加而稍為增加的現象。
由上述之研究結果顯示,利用去毒性綠膿桿菌外毒素研發免疫去勢疫苗及對抗綠膿桿菌外毒素之基因疫苗已獲初步成果,但利用基因疫苗方式生產抗-GnRH的生殖調控疫苗則尚待進一步研究發展。
The aim of this study was conducted to investigate the use of detoxicated Pseudomonas aeruginosa exotoxin A for the development of immunocastrative vaccine and DNA vaccine. The experiment was divided into three parts for trail.
The trail one: GnRH is a self-antigen, in order to obtain an antibody response to GnRH; it has to be linked a non-self immuno-carriers. A truncated Pseudomonas aeruginosa exotoxin A has been modified by gene deletion (rPE) was conjugated chemically with GnRH. The PE- GnRH conjugate was used as a vaccine to immunize boars. The results showed that animals injected with PE-GnRH conjugate successfully induced the production of anti-GnRH and anti-PE antibodies that resulted in the atrophy of the sex organs. A significant decline in sera testosterone level was also observed in all immunized boars and it is similar to castration level until the end of the study. These results demonstrate that PE represented a very attractive carrier system for active immunization against GnRH.
The trail two: a recombinant plasmid, which contains the detoxicated Pseudomonas aeruginosa exotoxin A (PE) and GnRH at C-terminal, was inserted on expression vector pSecTagC. The expression of this bacterial exotoxin in animal cells was demonstrated in 3T3 cell by transient transfection and western blot assay. Recombinant plasmid DNA was then injected intramuscularly to BALB/c mice. Anti-PE specific antibody was found in animals vaccinated with plasmid containing PE-GnRH gene and "detoxicated" recombinant PE protein. Mice vaccinated with DNA were protected from the challenge of lethal dosage of Pseudomonas aeruginosa exotoxin A. Our results indicated that mice vaccinated with DNA encoding PE and GnRH gene could express PE protein in vivo, induce specific immune response, and provide sufficient protective immunity that safeguarded mice from the challenge of lethal dosage of PE toxin. But the anti-GnRH antibody could not be detected by ELISA.
The trail three: the GnRH oligo-nucleotide sequences was constructed into the complementarity determining region (CDR) 3 of the variable domain of the light chain (VL) and heavy chain (VH) of chimeric immunoglobulin G. The constructed expression plasmid GnRH-IgG were used a DNA vaccine to transfect the BHK cells and to immunize BALB/c mice. The results showed that the BHK cells and these mice immunized pGnRH-IgG vaccine produced anti-IgG antibodies but no anti-GnRH antibody titer could be detected by ELISA.
封面
目錄
Ⅰ、中文摘要
Ⅱ、豬之免疫去勢研究
壹、文獻檢討
一、前言
二、生殖內泌素調控與免疫去勢機制
三、免疫去勢用抗原之生產
四、綠膿桿菌外毒素之蛋白質結構與功能
五、免疫去勢對動物繁殖與生長性能之影響
貳、材料與方法
參、結果
肆、討論
Ⅲ、含激性腺素釋放素-去毒性綠膿桿菌外毒素基因疫苗之研究
壹、文獻檢討
一、前言
二、基因疫苗之接種方式與影響因素
三、基因疫苗之研究概況
四、改善基因疫苗效率之策略
貳、材料與方法
參、結果
肆、討論
Ⅳ、含激性腺素釋放素-嵌合免疫球蛋白G基因疫苗之研究
壹、文獻檢討
一、前言
二、抗原性抗體之基因疫苗研究概況
三、以基因疫苗方式表現抗原性抗體
貳、材料與方法
參、結果
肆、討論
Ⅴ、結論
Ⅵ、參考文獻
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