Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/92622
標題: 製備以及應用重組蛋白質於多株抗體之生產
Production and application of recombinant proteins in production of polyclonal antibodies
作者: 蘇尹芃
Yin-Peng Su
關鍵字: 重組蛋白質;多株抗體;包涵體;recombinant protein;polyclonal antibody;inclusion body
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摘要: 
大腸桿菌系統是目前運用最為廣泛的重組蛋白質生產技術,以其所生產之蛋白質,或以重組蛋白質為抗原所生產的抗體已成功的應用於各式基礎研究或是臨床之診斷與治療。但由於大腸桿菌表現系統缺乏真核細胞的轉譯後修飾作用,此先天差異影響其生產的重組蛋白質之折疊;因此對於重組蛋白質是否具有原始蛋白之構型以及其他特質仍有許多探究的空間。本研究的目的是利用大腸桿菌系統表現GFP,EGFP兩種報導性蛋白質以及來源於orf病毒具有dsRNA結合能力的E3L蛋白質,分別純化其可溶性與不可溶性的蛋白、用以免疫小鼠以產生多株抗體。並藉由西方墨點法、酵素連結免疫吸附分析與免疫螢光染色等技術分析其免疫辨識之效價。結果顯示,生產之抗體除了能辨識原核系統產生之抗原外,亦能清楚辨識表現在真核中的抗原;此外,利用不可溶性GFP重組蛋白質生產之抗體仍能有效辨識可溶性GFP抗原,而其效果表現與可溶性抗原生產之抗體所差無幾;另一方面,我們也發現了不論是可溶性或是不可溶性之GFP抗體皆能辨識真核系統所產生之EGFP抗原。

Currently, E. coli is the most applicable system to produce foreign recombinant proteins. Proteins or sera acquired from animals immunized with antigenic recombinant proteins made from this prokaryotic expressing system have been broadly implemented to basic researches or clinical diagnosis and therapy. However, the defect of post-translational modification might affect protein folding; hence, whether the recombinant protein produced from E. coli represents the original conformation or maintains biological function require further investigation. In this study, E.coli expression system was exploited to produce recombinant GFP, EGFP, and orf virus E3 protein with the ability to bind dsRNA. The soluble and insoluble fractions of target proteins were purified and used as antigenic proteins for immunization of mice to peoduce polyclonal antibodies that were then characterized by western blot, ELISA and immunofluorescent analysis. The results demonstrated that the polyclonal antibodies contained the capacity of identifying antigens produced in both prokaryotic and eukaryotic systems. Furthermore, the serum produced from mice immunized with insoluble fraction of GFP recombinant protein could evidently detect GFP antigen to a similar extent to that made from soluble counterpart. In addition, both the polyclonal antibody arose from the soluble and insoluble fraction of the GFP can cross reactive with EGFP protein expressed by eukaryotic expression system.
URI: http://hdl.handle.net/11455/92622
其他識別: U0005-2201201411030300
Rights: 同意授權瀏覽/列印電子全文服務,2016-01-23起公開。
Appears in Collections:生命科學院碩士在職專班

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