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    標題: 家禽霍亂巴斯德桿菌單株抗體之製備及其特性研究
    Generation and characterization of monoclonal antibodies against avian Pasteurella multocida
    作者: 陳志瑋
    Chih-Wei Chen
    貢獻者: 邱淑媛
    嘉南藥理科技大學:生物科技研究所
    關鍵字: 單株抗體
    家禽霍亂
    多殺性巴斯德桿菌
    細胞融合
    噬菌體呈現技術
    擬抗原決定位
    Pasteurella Multocida
    Fowl cholera
    Phage libraries
    Monoclonal antibody
    日期: 2007
    上傳時間: 2008-12-03 11:18:39 (UTC+8)
    摘要: 家禽霍亂 ( fowl cholera ) 是一種全球性的禽鳥類疾病,家禽罹患易導致死亡,造成經濟損失。除了良好的衛生管理之外,家禽霍亂可藉由接種疫苗進行預防,而現階段所使用的死菌疫苗之保護力尚未完全。
    有鑒於引起家禽霍亂的巴斯德桿菌 ( Pasteurella multocida ),其關鍵性抗原至今仍未確定,僅知道一些如菌體蛋白及醣類作為抗原經免疫後可提供部分保護力。因此,本研究擬以巴斯德桿菌免疫小鼠製備單株抗體,並利用所產生之12株單株抗體的研究,嘗試辨認禽鳥類巴斯德桿菌之重要抗原。
    本研究之第一部分為家禽巴斯德桿菌單株抗體之製備。BALB/cByJNarl小鼠以家禽霍亂巴斯德桿菌ATCC 12945菌株免疫,將小鼠脾臟B細胞與骨髓瘤細胞F0進行細胞融合,得到12株對禽巴斯德桿菌有專一性的單株抗體,其中10株為IgG,2株為IgM。
    本研究之第二部分為單株抗體性質之探討。12株單株抗體均能與部份巴斯德桿菌之禽分離株有專一性結合及形成免疫沉澱線。小鼠的被動免疫保護試驗,以效價160的抗體中和10倍 LD50劑量之家禽霍亂強毒株FCN後,再經由皮下注射小鼠,部分單株抗體可完全中和家禽霍亂菌株之病原性。在西方墨點染色 ( Western blot ) 中所呈現之抗體結合區域,在蛋白質電泳膠片上並無對應的蛋白質色帶,但在醣染色中則有對應的色帶。以玻醣醛酸酶 ( hyaluronidase ) 處理蛋白質電泳之樣品,會降低西方墨點染色上之抗體結合墨點之分子量,顯示與保護性抗體結合之巴斯德桿菌抗原為非蛋白質物質,且可推斷為莢膜上玻醣醛酸類之多醣成分。
    本研究之第三部分為菌體抗原表位之模擬。將高保護力單株抗體3-4、5及無保護力之2-4與噬菌體胜肽庫進行三次親和性吸附反應後,隨機選取所得的噬菌體放大培養,抽取DNA進行定序,單株抗體3-4得到四條重複性較高的胜肽序列,YRLPAYV ( 8/15 )、DYLTWQWDYWGF ( 5/12 )、FKLPPSPAFFSPT ( 6/11 )、YRLPTYPVESRQ ( 4/11 ) ,在單株抗體5得到二條重複次數較高之胜太序列APAWETV ( 4/15 )、CPQSYPCPTMKP ( 11/16 ),為可能的擬抗原決定位 ( mimotope ) ,其ELISA之親和性結果有待進一步研究。
    總結實驗,以ATCC 12945免疫小鼠後所生產之單株抗體,在抗體特性分析試驗中,發現單株抗體能專一的辨認部分巴斯德桿菌之禽分離株。且由被動保護試驗結果證實,單株抗體有被動保護小鼠之能力。在蛋白質染色、醣染色及西方墨點試驗結果比較下,推斷單株抗體所變認之抗原可能是一類似玻醣醛酸之多醣類物質。
    Fowl cholera (FC) is a worldwide avian disease that resulted in significant financial loss. It can be prevented by vaccination using inactivated bacterins. However, the key immunogens of avian Pasteurella multocida (Pm) have not been illustrated well enough to provide completely protection, although certain cell components such as proteins and carbohydrates were known being able to provide partially protections. The aim of this study is trying to better illustrate on immunogens of avian pasteurellosis by generating antibody-secreting hybridoma cells and studying on the characters of antibodies produced from these cells.
    Two IgM and 10 IgG secreting hybridoma cell lines were obtained by fusing B cells collecting from ATCC 12945 immunized BALB/cByJNarl mice with a myeloma cell line F0. The antibodies generated by these cell lines were specifically reacted with avian strains of Pasteurella multocida, determined by ELISA. They also formed precipitation with most of the ELISA-positive-reaction cells by double immuno-diffusion reaction. Seven of the monoclonal antibodies revealed 100% protection rate while neutralizing with 10 LD50 of a high-virulent fowl cholera Taiwan isolate FCN in a titer of 160.
    The antigenic components of ATCC 12945 identified by 12 monoclonal antibodies by Western blotting were found unable to be stained with coomassie blue protein stain. However, they were recognized by a staining procedure that was used to identify glycoproteins, indicating that the antigenic components contained carbohydrate. The antigenic bands on Western blotting decreased to the position corresponding to 14.4 kDa after hyaluronidase treatment, indicating that the antigens were composed of hyaluronic acid. The antibodies reacted with crude capsule extract (CCE) of ATCC 12945, but not with the capsule-free cell. In comparison with CCE treated with pepsin or tryptin, the hyaluronidase-treated CCE loss its reactivity with most of the monoclonal antibodies indicating that antibodies were reacted with hyaluronic acid rather than protein components of the capsule.
    Four and two repeating peptide sequences that were supposed to mimic the conformation of antigens (mimotopes) were obtained from antibodies 3-4 and 5, respectively by a panning procedure with commercial phage libraries. No repeating sequence was obtained from monoclonal antibody 2-4, the one with low protection rate.
    In conclusion, 12 avian specific monoclonal antibodies against Pasteurella multocida were obtained in this study. Certain of them provided good passive protection in mice model. These antibodies reacted with carbohydrate-containing components that were sensitive to hyaluronidas. No peptide sequence of Pasteurella multocida was obtained by performing epitope mapping with the antibodies. The results indicated that hyaluronic acid plays an important role as the immunogen of avian pasteurellosis.
    關聯: 校內校外均不公開
    顯示於類別:[生物科技系(所)] 博碩士論文

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