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研究生:李珮寧
研究生(外文):Pei-Ning Li
論文名稱:鮑氏不動桿菌抗原 NcsP、TonB-R、Pase1 與 Pase2 單株抗體之製備與特性分析
論文名稱(外文):Production and characterization of monoclonal antibodies against the Acinetobacter baumannii antigens NcsP, TonB-R, Pase1 and Pase2
指導教授:楊秋英
指導教授(外文):Chiou-Ying Yang
口試委員:楊明德林念璁
口試委員(外文):Ming-Te YangNien-Tsung Lin
口試日期:2016-07-26
學位類別:碩士
校院名稱:國立中興大學
系所名稱:分子生物學研究所
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:2016
畢業學年度:104
語文別:中文
論文頁數:38
中文關鍵詞:鮑氏不動桿菌單株抗體被動免疫
外文關鍵詞:Acinetobacter baumanniimonoclonal antibodypassive immunization
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鮑氏不動桿菌 ( Acinetobacter baumannii, AB ) 為環境中常在菌,當人體免疫力低下時 AB 菌容易伺機感染,感染部位為呼吸道、泌尿系統、外傷傷口等,嚴重會導致菌血症而死亡,目前治療方式以抗生素為主,但由於 AB 獲取抗藥基因能力強,導致許多廣泛抗藥性菌株生成 (pan-drug resistant AB),使抗生素治療受到限制,而透過疫苗和治療性抗體的研究,期望可達到預防與替代抗生素治療的目的。本研究目的為製備與分析四個本實驗室研究具疫苗潛力的 AB 菌蛋白質 NcsP 、 TonB-R 、 Pase1 、 Pase2 的單株抗體來輔助 AB 菌研究並測試抗體是否具保護力。透過融合瘤細胞技術產生單株抗體,利用 ELISA 及西方墨點法篩選專一性抗體,共取得六株單株抗體,分別為辨認 NcsP 的抗體 N1,辨認 Pase1 的抗體 P1a、P1b、P1c ,辨認 Pase2 的抗體 P2 及同時辨認 TonB-R 、 Pase1 、 Pase2 的抗體 TPP,此六種抗體同型 (isotype) 皆為 IgG1。首先透過競爭型 ELISA 方式,比較辨認 Pase1 的 P1a、P1b、P1c 彼此間結合位置是否有差異,結果顯示 P1b 與 P1c 會彼此競爭與抗原的結合,而 P1a 與 P1b 及 P1a 與 P1c 兩組皆無競爭現象;抗體變異區序列分析顯示 P1b 與 P1c 核苷酸與胺基酸序列有高度一致性,並與 P1a 不同。接下來進行抗體效力測試,首先測試體外殺菌卅抑菌能力,將 N1、TPP 與菌體混合培養 4 小時後計數菌量,與陰性控制組的菌數相比,N1 與 TPP 菌量皆無顯著性下降,接著將 N1 與未免疫小鼠抗體 (pre-immune) 混合,測試抗體是否會利用血清中補體對 AB 菌殺菌,結果顯示不論有無將補體去活性,對於 N1 殺菌能力皆無顯著性影響。最後測試六株單株抗體的被動保護力,由小鼠尾靜脈注射 1 mg 的抗體,兩小時後再由腹腔感染 AB 菌,結果顯示除陽性控制組 (免疫 α-AB) 外,實驗組與陰性控制組存活率無顯著性差異,分別比較其血液、肺臟與脾臟中菌數,實驗組與陰性控制組相比無顯著性差異,其後將 P1a、P1c、P2、N1、TPP 每種各 200 µg 混合並進行被動免疫,同樣由腹腔感染 AB 菌,組織菌量結果顯示實驗組與陰性控制組的血液、脾臟與肺臟中菌數無顯著差異,綜合以上結果,本研究針對四個目標抗原篩選到六株抗體,其中兩株 P1b、P1c 特性相似,雖六株抗體無法直接於體外或體內提供良好的殺菌及保護能力,但可利用抗體特性,將抗體應用於 AB 菌目標抗原的研究與檢測上。

Acinetobacter baumannii, is an opportunistic pathogen that can easily survive in different environments. It may cause respiratory infection, urinary tract infection, wound infection, even can cause bacteremia and lead to death. The leading way to treat A. baumannii infection now is using antibiotics. However A. baumannii can easily acquire antibiotic resistant genes, and pan-drug resistant A. baumannii has emerged. In this study I tried to produce and characterize monoclonal antibodies (mAbs) against four A. baumannii proteins, NcsP, TonB-R, Pase1, Pase2, the protective antigens identified in our laboratory. I got 6 clones applying hybridoma technique, selection by ELISA and Western blot and I named the mAbs N1, P1a, P1b, P1c, P2, TPP. N1 recognizes NcsP; P1a, P1b, P1c recognize Pase1; P2 recognizes Pase2; and TPP recognizes TonB-R, Pase1, Pase2 at the same time. All 6 antibodies are IgG1 isotype. To differentiate the binding site of the three Pase1 antibodies, competition ELISA was carried out. The results showed that P1b and P1c can compete with each other, implicate they may recognize the same epitope. P1a and P1b or P1a and P1c showed no competing on each group. Sequencing of VH and VL regions of mAbs, P1b and P1c exhibited high identity. Next, in vivo and in vitro assay were carried out to test the efficacy of 6 mAbs. To test in vitro efficacy, I mixed TPP or N1 with A. baumannii, incubated for 4 hours and counted the number of colonies. Compared with negative control, N1 and TPP groups displayed no significant decrease of the colony count. Then I mixed N1 with pre-immune serum to test if complement could coordinate with N1 to kill A. baumannii. The results showed whether the complement was inactivated or not there was no significant difference. Passive protective assay was carried out to test in vivo efficacy of 6 mAbs. Mice were injected with mAbs from tail vein 2 hours prior to inoculate intraperitoneally (i.p.) with A. baumannii. The survival rate showed no significant difference between experimental and control groups. Compared bacterial burdens in blood, lung and spleen, there were no significant difference between experimental and control groups. Finally, I injected mice with a mixture of P1a, P1c, P2, N1, TPP, and inoculate i.p. with A. baumannii. There were also no significant difference between experimental and control groups in blood, spleen and lung. In conclusion, 6 mAbs did not display good efficacy either in vivo or in vitro, but they can apply to study and detect target proteins of A. baumannii.

摘要 i
Abstract ii
目次 iii
縮寫字對照表 (Abbreviation) v
壹、 前言 1
一、 緒論 1
二、 抗體介紹 1
三、 AB菌具疫苗潛力之抗原 2
四、 實驗目的 3
貳、 材料與方法 4
一、 單株抗體製備 4
二、 SDS-PAGE 膠體電泳 5
三、 西方墨點電法 (Western blot) 6
四、 酵素免疫分析法 (ELISA, Enzyme-Linked immunosorbent assay) 6
五、 抗體基因定序 6
六、 被動免疫 (passive immunization) 分析 8
七、 抗體殺菌卅抑菌力分析 9
參、 結果 10
一、 單株抗體製備及純化 10
二、 單株抗體特性分析 10
1. 單株抗體基本特性分析 10
2. P1a、P1b、P1c競爭試驗 10
三、 抗體效力 (efficacy) 測試 11
1. 體外 (in vitro) 殺菌卅抑菌力分析 11
2. 體內 (in vivo) 被動保護力分析 12
肆、 討論 14
伍、 參考文獻 16
壹、 圖表 19
表一、研究中使用之核酸引子 19
圖一 以西方墨點法確認免疫反應情形。 20
圖二 ELISA分析各單株抗體之專一性。 21
圖三 單株抗體對目標抗原之辨識情形。 22
圖四 以 ELISA 偵測同型結果。 23
圖五 抗體純化結果。 23
圖六 P1a、P1b、P1c、N1、TPP、P2 ELISA 力價分析結果。 24
圖七 P1a、P1b、P1c 競爭試驗。 25
圖八 P1a、P1b、P1c 輕鏈可變區核苷酸序列比對分析。 26
圖九 P1a、P1b、P1c 輕鏈可變區胺基酸序列比對分析。 27
圖十 P1a、P1b、P1c 重鏈可變區核酸序列比對分析。 28
圖十一 P1a、P1b、P1c 重鏈可變區胺基酸序列比對分析。 29
表二 各抗體輕鏈與重鏈可變區與 germline 基因比較 30
圖十二 TPP、N1體外殺菌卅抑菌力試驗。 31
圖十三 各別免疫P1a、P1b、P1c、P2、N1、TPP後生存曲線。 32
圖十四 以colony PCR 確認小鼠血液中分離之菌株為AB菌。 33
圖十五 分別免疫 P1a、P1b、P1c、P2、N1、TPP 後組織內菌數分析。 34
圖十六 注射單株抗體對AB菌感染小鼠在血液、肺臟以及脾臟生菌數之影響。 35
貳、 附錄 36

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