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標題: 以螺旋胜&;#32957;連接子接合免疫調節蛋白
Conjugation of Immunoregulatory Proteins byHelix-Forming Linker
作者: 曾慈瀅
Tseng, Tzu-Ying
關鍵字: recombinant polymerase chain reaction Escherichia coli
出版社: 獸醫學系暨研究所
摘要: 摘 要 在本研究中我們將A 蛋白以螺旋連接子分別接合B 蛋白或C 蛋白,本研 究所使用的螺旋連接子由胺基酸序列 EAAAK 重複三次組成 (EAAAK)3,共包 含15 個胺基酸。我們利用重組聚合&;#37238;鏈反應(recombinant polymerase chain reaction, recombinant PCR)將螺旋連接子分別接合A 蛋白與B 蛋白或C 蛋白, 得到A-B 與A-C 兩個基因融合片段,將此二片段分別構築到原核表現質體 pET28 並將質體送入大腸桿菌誘導重組蛋白表現。將rA-B 與rA-C 純化後,經 MALDI-TOF 質譜儀分析鑑定並根據A、B 以及C 的功能進行蛋白的活性測試。 結果顯示,rA、rA-B 或rA-C 皆能趨化周邊血液單核球(peripheral blood mononuclear cells, PBMCs),此外rA-B 能與表現B 蛋白受器之巨噬細胞或纖維 母細胞細胞株結合,另一方面,rA-C 能刺激周邊血液單核球的增生。整體而言, (EAAAK)3 做為連接子能保有rA-B 中的A 與B 之活性或rA-C 中的A 與C 之 活性。
Abstract In this study, we linked A (A) with B (B) or with C (C) by a 15-amino acid helixforming linker, (EAAAK)3. By performing recombinant polymerase chain reaction (PCR), DNA sequence of (EAAAK)3 linker was introduced between that of A and B or C to generate DNA fragments encoding A-B and A-C. Both DNA fragments were cloned respectively into pET28 and transformed into Escherichia coli to induce recombinant proteins expression. Purified recombinant A-B (rA-B) and A-C (rA-C) proteins were further analyzed and by matrix-assisted laser desorption inoization-time of flight mass spectrometry (MALDI-TOF MS) to verify their identities. Furthermore, based on the functions of A, B, or C, we performed protein activity assays. Recombinant A (rA), rA-B and rA-C all have chemotactic effect on peripheral blood mononuclear cells (PBMCs). In addition, rA-B could bind to B receptor-expressing cells, such as macrophages and fibroblast cell line. On the other hand, rA-C could stimulate the proliferation of PBMCs. Taken altogether, our results indicated that (EAAAK)3, as a peptide linker could retain the activities of A and B in rA-B or A and C in rA-C.
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