構築 Burkholderia pseudomallei 熱休克蛋白基因groEL (pcDNA3/groEL) 之 DNA 疫苗，以肌肉注射免疫 BALB/c 小鼠，評估小鼠感染B. pseudomallei與B. cenocepacia 之免疫活性及疫苗保護效力。測量免疫後小鼠血清中anti-GroEL total IgG 與 IgG2a 皆明顯增高；株落增殖試驗中以 GroEL 誘導小鼠脾臟細胞，細胞數量明顯增加，並促使脾臟細胞分泌 IFN-γ 。抗 GroEL 抗體媒介之調理毒殺作用，雖不能消減 B. pseudomallei 增長，卻能抵制 B. cenocepacia。而接種疫苗小鼠尾巴靜脈，注射 B. pseudomallei 刺激免疫後，其肝臟和脾臟之菌落數並沒有減少，免疫7天內死亡之小鼠數量超過 50％；相較之下，接種疫苗小鼠刺激免疫 B. cenocepacia 後器官內菌量明顯減少，且所有小鼠於免疫7天後仍存活。組織學觀察評測接種疫苗小鼠，部份小鼠肝臟呈現急性肝炎症狀；測量肝臟指標酵素，數值高於無接種免疫小鼠組。因此，提議 B. pseudomallei groEL 質體 DNA 免疫 BALB / c 小鼠能誘導 Th1 型免疫反應，對抗感染 B. cenocepacia 具交叉保護作用，但無法對抗 B. pseudomallei 感染。

The immunogenicity and protective efficacy of a DNA vaccine encoding a truncated groEL heat shock gene (pcDNA3/groEL) from Burkholderia pseudomallei was evaluated in vaccinated BALB/c mice infected with B. pseudomallei or B. cenocepacia. After vaccination, the levels of anti-GroEL total IgG and IgG2a were increased in mouse sera. The clonal expansion of the spleen cells increased, and the GroEL protein induced IFN-γ production by spleen cells. The anti-GroEL antibody-mediated opsonic killing effect was not able to eliminate the growth of B. pseudomallei but was able to eliminate the growth of B. cenocepacia. After intravenous challenge of the vaccinated Balb/c mice with B. pseudomallei, the number of bacteria colonizing the in liver and/or spleen was not reduced. Over 50% of vaccinated mice infected with B. pseudomallei died within 7 days post-infection. By contrast, the bacterial loads in organs were significantly reduced if the vaccinated mice were infected with B. cenocepacia. All of vaccinated mice were alive 7 days post-infection. Liver damage, as assessed by histological observation, and abnormalities in the levels of liver enzymes rapidly resolved in vaccinated mice. We suggest that B. pseudomallei groEL plasmid DNA immunization of Balb/c mice induces a Th1-type immune response and provides cross-protection against B. cenocepacia but not against B. pseudomallei infection.

論文審定書 ii
誌謝 iii
中文摘要 iv
英文摘要 v
第 一 章 敘論 1
第一節 Burkholderia致病菌簡介 1
第二節 B. pseudomallei致病機轉與免疫機制 3
第三節 Melioidosis疫苗研發策略 4
第 二 章 研究目的 7
第 三 章 材料與方法 8
第一節 菌株 8
第二節 質體構築 8
第三節 pcDNA3/groEL基因表現 9
第四節 重組GroEL表現與純化 9
第五節 生物資訊分析 11
第六節 小鼠免疫 11
第七節 抗體分析 11
第八節 脾臟細胞增生反應 12
第九節 細胞激素分析 12
第十節 毒殺調理作用試驗 13
第十一節 致死劑量、細菌負荷量與存活試驗 14
第十二節 生化反應與組織學觀察 14
第十三節 統計分析 15
第 四 章 結果 16
第一節 groEL基因分析、選殖與表現 16
第二節 特異性免疫反應之生成與分析 17
第三節 體外巨噬細胞毒殺試驗 17
第四節 存活率試驗 18
第五節 細胞組織學觀察與生化反應試驗 19
第 五 章 討論 20
第 六 章 圖表 21
圖次
圖3-1 小鼠免疫週期表 22
圖4-1 重組GroEL親水性分析 23
圖4-2 GroEL CTLPred軟體分析 24
圖4-3 誘導免疫反應分析 25
圖4-4 脾臟細胞增生試驗 26
圖4-5 誘導細胞激素分析 27
圖4-6 調理毒殺試驗 28
圖4-7 致死劑量試驗 29
圖4-8 存活率試驗 30
圖4-9 細菌負荷量試驗 31
圖4-10 肝臟細胞組織學觀察 32
圖4-11 生化反應試驗 33
表次
表4-1 胺基酸特性分析 34
表4-2 特異性抗體檢測 35
參考文獻 36

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