臺灣博碩士論文加值系統

中 文 摘 要
血吸蟲為世界衛生組織列為第二重大之熱帶寄生蟲疾病，三種血吸蟲以日本血吸蟲之致病力最嚴重。日本血吸蟲26KDa 麩氨基硫S-轉移（glutathione S-transferase；GST）已經被認定為一良好的疫苗候選者，本實驗室曾運用RT-PCR技術將台灣株日本血吸蟲26KDa GST的cDNA核酸選殖出來，於大腸桿菌中表現出重組蛋白，實驗證明其具疫苗保護效果。本研究論文進一步探討該疫苗抗原對於不同地域株日本血吸蟲感染預防之適用性。實驗結果顯示，數個不同地域株之日本血吸蟲26KDa GST的cDNA核酸序列極為相似，且所編碼之氨基酸序列完全相同。台灣及中國湖北株日本血吸蟲天然及重組蛋白免疫小鼠後所獲得之減蟲率及交叉保護力相似，顯示此26KDa GST疫苗抗原將可廣泛適用於各個不同地域株。以台灣株日本血吸蟲26KDa GST多源抗體及單株抗體進行被動免疫，分別得到50.2﹪及40﹪之減蟲率，證明本疫苗所誘發之保護效果與體液性免疫具有絕對的關聯。實驗結果亦顯示，弗氏佐劑或鋁佐劑混合26KDa GST免疫誘發之抗體種類不同，應用鋁佐劑未能獲得良好減蟲效果，小鼠血清中所存在之保護性抗體可能與IgG2b有關。此結果顯示疫苗免疫使用之佐劑種類是影響保護效果及誘發不同抗體類別之重要因素。

Abstract
Schistosomiasis is listed as the second most important tropical parasitic disease by World Health Organization. Among the three major human schistosomes, Schistosoma japonicum is the most pathogenic. The cDNA of 26KDa GST from the total RNA of adult worm of a Taiwanese strain of S. japonicum was synthesized and amplified by using RT-PCR technique, followed by the subcloning in our laboratory. The recombinant proteins were then expressed in Escherichia coli and demonstrated to be a good candidate vaccine. In this studies , the suitability of using 26KDa GST as a vaccine candidate against different strains of S. japonicum was evaluated. The results showed that the cDNA sequences of 26KDa GST among different strain of S. japonicum are similar and encode the same amino acid sequences. The worm reduction rate and cross protective immunity in mice immunized with native and recombinant GST26 from Taiwanese and Hupei strain of the parasite had similar results, indicating that the 26KDa GST can be a vaccine condidate against the different strains of S. japonicum. Passive transfer experiment using sera from the mice immunized with recombinant GST26, as well as monoclonal antibodies, showed a worm reduction rate of 50.2% and 40% respectively, indicating the close relationship between the protective immunity induced by GST26 and antibodies. The level of anti 26KDa GST IgG2b antibody in immunized mice with Freund's adjuvant was significantly higher than in the mice using Alum adjuvant. Also, poor reductive rate in mice group was obtained from the Alum adjuvant. This results showed that different adjuvant combined with subunit vaccine might produce different subclasses of antibodies which will lead to different protective effect.

中文摘要………………………………………………………….1
英文摘要………………………………………………………….2
第一章 緒論…………………………………………………… 3
第二章 材料與方法…………………………………………...11
實驗動物………………………………………………………11
生活史維持……………………………………………………11
SjCH及SjCA 26 KDa GST cDNA基因定序分析…………...12
SjT、SjCH 26 KDa rGST之製備…….………………………13
SjCH nGSTs（26~28 KDa）蛋白之製備....................................15
SjT、SjCH 26 KDa rGST與SjCH nGSTs免疫小鼠對SjT及SjCH感染抵抗能力之比較......................................................16
連脢免疫吸附分析（ELISA）...................................................16
西方墨點法（Western blotting）….............................................17
SjT 26 KDa rGST免疫抗血清的取得......................................18
SjT 26 KDa rGST免疫抗血清被動轉移及交叉被動保護實驗………………………………………………………………18
日本血吸蟲26 KDa GST蛋白單株抗體之取得……………..19
單株抗體的被動免疫保護力實驗……………………………20
SjT 26 KDa rGST混合弗氏佐劑或鋁佐劑之免疫效果與抗體誘發種類及效價評估…………………………………………20
統計分析..…………………………………………………….21
第三章 結果………………………………………………..…22
日本血吸蟲26 KDa GST cDNA基因定序分析………………………………………………………………22
SjT、SjCH 26 KDa rGST及SjCH nGSTs（26~28 KDa）之製備………………………………………………………………22
SjT、SjCH 26 KDa rGST與SjCH nGSTs免疫小鼠對SjT及SjCH感染抵抗能力之比較................................................…. 24
SjT 26 KDa rGST免疫抗血清之取得與免疫抗血清被動轉移及交叉被動保護實驗.........................................................…..24
日本血吸蟲26 KDa GST蛋白單株抗體的被動免疫保護力實驗......................................................................................….....25
SjT 26 KDa rGST混合弗氏佐劑或鋁佐劑之免疫效果與抗體誘發種類及效價評估...............................................................26
第四章 討論…………………………………………………..28
致 謝…………………………………………………………..35
參考文獻......................................................................................36
圖表及說明..................................................................................46

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