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標題: CBD融合蛋白質應用於檢驗試劑的開發
Application of CBD fusion proteins in the development of immuno-diagnostic reagents
作者: 陳靜美
Chen, Ching- Mei
關鍵字: Cellulose-Binding Domain(CBD);纖維素結合功能區;Fusion protein;Foot and mouth disease virus (FMDV);diagnostic reagents;融合蛋白質;口蹄疫病毒;檢驗試劑
出版社: 獸醫學系
將纖維素水解酵素中纖維素結合區獨立出來與其他蛋白或短的 (peptide) 形成融合蛋白,能使融合蛋白具備與纖維素材質結合的能力。本研究為探討利用Cellulose Binding Domain (CBD) 融合蛋白作為檢驗試劑的可行性,選擇口蹄疫病毒O台灣血清型外鞘蛋白VP1的G-H loop,將其基因密碼殖入CBD基因密碼之下游,並利用T7啟動子于宿主大腸菌中大量表現融合蛋白,經由西方墨點偵測,檢查其是否可被豬的口蹄疫病毒O台灣血清型免疫抗血清偵測。藉不同質體的構築,比較G-H loop的長度及環化與否對抗體反應的影響,結果發現G-H loop的環化與長度均會影響抗體反應訊號的強度。其次,藉殖入Asia 1 血清型的G-H loop,檢查O台灣血清型的抗血清與G-H loop之間的反應專一性,結果發現O台灣血清型的抗血清只與O台灣血清型 G-H loop反應,不與Asia 1 血清型反應。最後,進行表現宿主大腸菌的高密度大量培養,降低誘導表現融合蛋白質的isopropyl-β-D-thiogalactoside (IPTG) 最終濃度到20 μM,並藉其他生長條件的控制包括:供氧量、溫度、振盪轉速及pH值等,以提高細胞濃度,增加融合蛋白的產量。SDS-polyacrylamide gel electrophoresis (SDS-PAGE) 結果顯示融合蛋白質的表現量最高可達總蛋白約30 %。經DE52陰離子交換色層分析、nickel-nitrilotriacetic acid (Ni-NTA) 親和性色層分析及Mono Q陰離子交換色層分析等步驟純化自pCM6合成的融合蛋白,得到約99 % 純度的蛋白質,將可被利用為免疫檢驗法之抗原基質。

The cellulose-binding domain (CBD) of cellulase remains functional when it is isolated from cellulase and forms fusion protein with other proteins or peptides. The fusion protein gains the property of cellulose-binding activity. In order to develop CBD fusion protein into diagnostic reagent for viral infection, this study chose to express the G-H loop of the O Taiwan serotype FMDV capsid protein VP1 as fusion protein with CBD. The fusion protein expressed in Escherichia coli from the T7 promoter was examined on Western blot for its reactivity with the antiserum against the O Taiwan serotype of FMDV. By comparing reactivity of different recombinant fusion proteins with the antiserum, I observed that the length of the G-H loop and its cyclization played a major role. I also constructed a recombinant plasmid to express a CBD-fusion protein with the G-H loop of Asia 1 serotype and found that it did not react with antiserum against the O Taiwan serotype. Furthermore, I conducted overexpression of one of the fusion proteins by growing E. coli to high cell density. By minizing the final concentration of the inducer IPTG to 20mM and manipulating other growth conditions, such as oxygen supply, temperature and pH of the medium, I was able to reach a protein expression level of 30 %. I was also able to purify the fusion protein produced from the plasmid pCM6 with a purity of 99 % through three column chromatography steps.
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