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博碩士論文 etd-1113112-160654 詳細資訊
Title page for etd-1113112-160654
論文名稱
Title
點帶石斑魚介白素-1β蛋白之多株抗體製備及其特性分析
Production and characterization of polyclonal antibody against Epinephelus coioides interleukin-Production and characterization of polyclonal antibody against Epinephelus coioides interleukin-1β
系所名稱
Department
畢業學年期
Year, semester
語文別
Language
學位類別
Degree
頁數
Number of pages
56
研究生
Author
指導教授
Advisor
召集委員
Convenor
口試委員
Advisory Committee
口試日期
Date of Exam
2012-09-12
繳交日期
Date of Submission
2012-11-13
關鍵字
Keywords
融合蛋白、抗血清、脂多醣、介白素-1β、點帶石斑魚
antiserum, lipopolysarcharide, fusion protein, IL -1β, Epinephelus coioides
統計
Statistics
本論文已被瀏覽 5692 次,被下載 587
The thesis/dissertation has been browsed 5692 times, has been downloaded 587 times.
中文摘要
點帶石斑魚(Epinephelus coioides) 為臺灣南部的重要養殖魚之一,目前台灣石斑魚苗養殖受病原感染問題嚴重,感染死亡率高,造成嚴重經濟損失。因此,如何早期發現疫情進而防止疫情發生,便顯得十分地重要。介白素-1 (IL-1) 屬於促發炎性細胞激素(proinflammatory cytokine)可與抗發炎性細胞激素,形成體內回饋機制,維持宿主免疫衡定,因此其基因表現之提升可做為個體炎症反應之指標。本研究以點帶石斑魚受精卵萃取總RNA,經以反轉錄酵素-聚合酶鏈鎖反應增幅出介白素-1β(IL- 1β)之cDNA片段,將之選殖至表現載體pGEX4T-3,並以原核系統表現其融合蛋白,經大量純化後,施打於紐西蘭白兔製備多株抗體。研究中並利用脂多醣誘發石斑魚頭腎組織表現IL- 1β,再以所製備抗體進行抗體特異性確認。西方墨點法結果顯示,此抗體可檢測在點帶石斑魚頭腎組織蛋白萃取物中,由脂多醣刺激誘導產生的IL- 1β蛋白質。免疫組織化學染色分析結果也顯示,此抗體可在脂多醣誘導的點帶石斑魚頭腎組織冷凍切片中,偵測到大量IL- 1β之表現,而在沒有脂多醣誘導的組織切片中,只偵測到極少量的IL- 1β。總結而言,本研究所製備之抗體可做為點帶石斑魚免疫反應之檢測試劑,有助於應用在魚體病原感染免疫反應機制之探討。
Abstract
Grouper (Epinephelus coioides) is one of the important farmed fish in the southern Taiwan. However, grouper aquaculture in Taiwan has a serious problem of infection, especially in grouper larvae breeding stage. The infection resulted in very high mortality, which causes massive economic loss. Therefore, early detecting the presence of pathogen is critical for preventing epidemic outbreak. Interleukin-1 (IL-1) is one of proinflammatory cytokines that form a feedback control loop with anti-inflammatory cytokines to maintain the homeostasis of host immune response. The increase of IL-1 expression could be an indicator of pathogenic insult. In this study, total RNA of Epinephelus coioides fertilized egg was extracted for reverse transcription-polymerase chain reaction (RT-PCR) to amplify cDNA of IL -1β. The cDNA amplified was then cloned into pGEX4T-3 for the expression and purification of GST-IL-1β fusion protein. GST-IL-1β fusion protein purified was then used to immunize New Zealand white rabbit for generation of antiserum against IL-1β. Western blot result confirmed the specificity of antiserum as the immune serum, but not the preimmune serum, detected the immunogen GST-IL-1s. Further experiments using live Epinephelus coioides injected with or without lipopolysarcharides (LPS) further confirmed that this antiserum could detect a massive increase of IL-1β protein after the injection of LPS in either protein lysate by western blotting or in frozen tissue section of head kidney by immunohistochemistry. In summary, we successfully generated a rabbit specific antiserum against IL-1β of Epinephelus coioides , which could be a useful reagent for future analysis of fish immune response upon pathogen infection.
目次 Table of Contents
致謝 3
中文摘要 4
Abstract 6
前言 8
實驗目的 19
材料與方法 20
結果與討論 35
參考文獻 39
圖表 45
未來與展望 55
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