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題名 | Immunochemical Studies on β[feb5]-Bungarotoxin=臺灣雨傘節腱前神經毒(β-Bungarotoxin)之免疫化學研究 |
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作者 | 楊振忠; 詹鴻霖; Yang, Chen-chung; Chan, Hong-lin; |
期刊 | 動物研究學刊 |
出版日期 | 20000400 |
卷期 | 39:2 2000.04[民89.04] |
頁次 | 頁79-90 |
分類號 | 388.796 |
語文 | eng |
關鍵詞 | 臺灣雨傘節蛇毒; 腱前神經毒; 單株抗體; 融合融合瘤; 雙專一性單株抗體; 胜冴疫苗; Bungarotoxin; Presynaptic neurotoxin; Monoclonal antibody; Hybrid hybridoma; Bispecific monoclonal antibody; Peptide vaccine; |
中文摘要 | β1-Bungarotoxin(β1-Bgt)是雨傘節蛇毒中最主要且最具毒性的腱前神經毒。這個帶有磷脂[]活性的毒蛋白由兩個不相同的多胜[]經由隻硫鍵的連結所組成。其一稱為A鏈,它由120個胺基酸所組成;另一稱為B鏈,由60個胺基酸所構成。A鏈的氨基酸序列類似於蛇毒或哺乳類胰臟所產生的磷脂[]序列;而B鏈可與特定目標細胞膜做專一性之結合並且有阻絕電位敏感性鉀通道的能力。利用定量沉澱反應以及抗體Fab片段與毒蛋白所形成之可溶性複合物分析得知每分子β1-Bgt含有七個主要的抗原決定基,其中五個位於A鏈上,二個個位於B鏈上。此外,23個β1-Bgt的單株抗體被製備出來,其中有7個可抑制毒蛋白酵素活性達百分之七十以上,並能中和毒蛋白的毒性。藉著表位分析,得知這些中和抗體的表位是位於A鏈蛋白序列31-37,46-51,91-98以及100-106。將能認知毒蛋白表位46-51及100-106的融合瘤二度融合,所產生的融合融合瘤可分泌雙專一性單株抗體,此種抗體可引起免疫複合物之形成以及增進親合力效價,並可高度中和β1-Bgt的生物活性。更進一步,將上述的這四段抗原決定基合成胜[]來免疫老鼠,再施以高劑量的β1-Bgt,發現免疫後的老鼠對毒蛋白有相當的耐受性。 |
英文摘要 | β1-Bungarotoxin (β1-Bgt) is the main and the most toxic isoform in the β-Bgt family. The toxin consists of 2 dissimilar polypetide chains: the A chain with 120 amino acid residues and the B chain with 60 amino acid residues, cross-linked by an interchain disulfide bond and possessing weak phospholipase A2 (PLA2) activity. The amino acid sequence of the A chain is homologous with those of PLA2 enzymes from snake venom and the mammalian pancreas, while the B chain serves as a recognition subunit of the toxin towards a specific target presynaptic membrane and blocks the voltage-gated potassium channel. The numbers of antigenic determinants on β1-Bgt, A chain, and B chain were determined to be 7,5, and 2, respectively, by the quantitative precipitin precipitin reactions and analysis of the molecular weight of the soluble complex formed from β1-Bgt and Fab fragments of the purified antibody. Twenty-three stable monoclonal antibodies (mAbs) were prepared against β1-Bgt, seven of which could inhibit more than 70% of PLA2 activity of β1-Bgt and could neutralize the toxicity of the toxin. Continuous epitopes of β1-Bgt were mapped, and the result indicates that the A chain sequences 31-37, 46-51, 91-98, and 100-106 are the neutralizing epitopes of β1-Bgt. A hybrid hybridoma that produces bispecific mAb, which recognizes 2 different epitopes on the A chain of β1-Bgt at peptide sequences 46-51 and 100-106, has been obtained by fusing 2 hybridoma cell lines. The combination of hispecific mAb, with its 2 corresponding epitopes of β1-Bgt, not only facilitates immuno-complex formation and enhances avidity, but also highly neutralizes the biological activity of β1-Bgt. Furthermore, mice immunized with BSA-conjugated A-chain-peptide sequences A(31-37), A(46-51), A(91-98), or A (100-106) were protected from a high-dose β1-Bgt challenge. |
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