Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/21513
標題: Analysis of variable region of anti- Neisseria meningitidis monoclonal antibody
抗奈瑟氏腦膜炎球菌 (Neisseria meningitidis)抗體變異區之分析
作者: 劉中玉
Liu, Chung-Yu
關鍵字: Neisseria meningitidis
奈瑟氏腦膜炎雙球菌
出版社: 分子生物學研究所
摘要: A panel of hybridomas secreting antibodies against Neisseria meningitidis (NM) serogroup B (GBM) or serogroup Y (GYM) was generated previously in this laboratory. The main theme of this thesis was to characterize some of these hybridoma antibodies. The first part of this study was focused on three anti-GBM clones. To ensure the monoclonality, hybridoma cells from three original wells were subcultured separately in soft agarose. The binding of the antibody from the subcloned cells to GBM was evaluated by ELISA and the clone with highest productivity was chosen from each well for further characterized. Among these three antibodies, 172-33, 43-38, and 89-16, 172-33 showed the strongest binding activity to GBM with no cross-reactivity to the human neuroblastoma cell IMR-32, while both 43-38 and 89-16 bound to both GBM and IMR-32. To revealing the possible antigen of these antibodies, Western blot analysis was performed with the total protein extracts of GBM. A protein band corresponding to the opacity protein was recognized strongly by 172-33. While no band was visualized by 43-38, almost all protein bands were reactive with 89-16. These results indicated that 43-38 may recognize the capsular polysaccharide of GBM and 89-16 is a polyreactive antibody. Furthermore, the epitope of the 172-33 was mapped by phage display technology to contain the conserved sequence DIWKQA. In the second part of this study, the variable region sequences of five hybridoma antibodies, three against GBM and two against GYM, were determined. The DNA encoding the variable regions were obtained by RT-PCR using a set of primers corresponding to the first framework-region sequences and four primers with sequence complementary to the J gene segments. The VH sequences of four antibodies except for the polyreactive antibody 89-16, each with a distinct specificity, are almost identical. However, the VL sequences of the five antibodies are different significantly. These results implied that the VL region may play a more significant role than that of VH in the formation of antibody combining site in the anti-NM antibodies.
本研究論文主要針對本實驗室前人已製備的抗奈瑟氏腦膜炎雙球菌抗體進行分析,主要工作分為兩部分:第一部份是以B群菌免疫小鼠製備得到的三個孔盤的融合瘤細胞為主。首先將細胞養在soft agarose進行單株化,以ELISA分析單株化細胞與B群菌的結合情形,從中選取分泌能力最好的一株,分別命名為172-33、43-38與89-16。ELISA分析這三株抗體發現172-33對B群菌之結合能力最好,且對人類神經元母細胞IMR-32沒有交叉反應;而43-38與89-16不但能與B群菌之結合,同時對IMR-32亦有明顯的結合情形。Western blot分析此三株單株抗體對B群總蛋白之結合情形,結果顯示抗體172-33能專一的辨識免疫原上之Opa蛋白;89-16與許多蛋白皆有反應;而43-38則未有任何蛋白條帶出現,此結果顯示抗體43-38的抗原可能是夾膜多醣,而89-16則是屬於一種多反應(polyreactive)抗體。利用噬菌體展示技術探討抗體172-33之抗原表位,結過顯示抗體172-33辨識的基本構造可能含有DIWKQA序列。 本論文的第二部份是分析五株抗奈瑟氏腦膜炎雙球菌抗體的變異區基因,其中三株為抗B群菌,兩株抗Y群菌。以抗體的第一骨架區序列及抗體J基因片段互補序列的引子進行RT-PCR得到主導抗體變異區的DNA片段。序列分析結果顯示,儘管各抗體之專一性都不一樣,有四株抗體其重鏈變異區幾乎相同;而各抗體之輕鏈變異區序列差異極大。此結果顯示在決定這些抗體的專一性而言,亦即在形成抗體接合位構造上,輕鏈變異區較重鏈變異區重要。
URI: http://hdl.handle.net/11455/21513
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