Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/98195
標題: 雞介白素- 2 融合細胞素在原核與家禽系統之表現與運送
Expression and Transport of Chicken Interleukin-2 Fusion Cytokines in Prokaryotic and Avian Systems
作者: 黃胤銓
Yin-Chuan Huang
關鍵字: 介白素-2;融合蛋白質;細胞穿透胜肽;interleukin-2;fusion protein;cell penetrating peptide
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摘要: 
細胞素為一群天然的蛋白質,可做為免疫系統細胞間溝通之重要調節者。。本研究的目標便是將雞的介白素-2融合細胞素於原核以及家禽表現系統中表達以及運送。介白素-2 主要由輔助型 T 細胞分泌,具有調控免疫反應之功能,特別是刺激許多不同種類的白血球增生。我們利用重組 PCR 技術將具有白血球趨化能力的雞基質細胞衍生因子-1 與 IL-2 藉由胜肽連接子序列連接,將 SDF1-IL2 序列接入原核表現載體 pET32a 中,並利用大腸桿菌表現菌株 BL21 (DE3) 表現雞的重組融合細胞素。經誘導後,我們收取SDF1-IL2 蛋白並使用鎳離子螯合親合層析法純化。之後分別以趨化試驗與增生試驗評估 SDF1-IL2的生物活性。另一方面, 我們致力運用細胞穿透胜肽 (CPP) 建立融合細胞素真核表現與運送系統。CPP 過去已被用於將生物分子攜入許多種人類細胞株中,然而,雞的系統仍未被測試。我們首先以人類細胞株 CNE2 做為進行可行性測試,確認 CPP 可以攜帶 pEGFP-N1 進入細胞中。我們繼而構築了真核表現之雞的融合細胞素質體 pVAX- 以及 pEGFP-SDF1-IL2,並且證明 CPP 能夠運送這些質體進入雞細胞株 DF-1 中並且表現。未來這些雞的重組融合細胞素可以應用於獸醫領域中。

Cytokines are natural proteins which can serve as principal mediators for communication between cells of the immune system. The specific aims of this study were to express and transport chicken interleukin-2 (IL-2) fusion cytokines in prokaryotic and avian systems. IL-2, is an important cytokine mainly produced by T helper cells to modulate immune responses, in particular, to stimulate the proliferation of a variety of leukocytes. In this study, chicken stromal-derived factor-1 (SDF-1) that can attract leukocytes to target locations was fused to IL-2 with a peptide linker by employing recombinant polymerase chain reaction (PCR) technique. Subsequently, the resultant SDF1-5PA-IL2 DNA fragment was cloned to a prokaryotic expression vector, pET32a, to express recombinant chicken fusion cytokine in Escherichia coli BL21 (DE3). After induction, we harvested SDF1-IL2 fusion cytokine and purified the protein by nickel (Ni2+) chelation affinity chromatography. We evaluated the bioactivities of SDF1-IL2 based on chemotactic and proliferating activities of SDF-1 and IL-2, respectively. On the other hand, we endeavored to establish an eukaryotic fusion cytokine expression and transport system by using cell penetrating peptides (CPPs). CPPs have been used to introduce biomolecules into a number of human cell lines. However, chicken system has not been tested yet. As a feasibility test, we confirmed that CPP could carry pEGFP-N1 into a human cell line, CNE2. Subsequently, we constructed eukaryotic chicken IL-2 fusion protein expression plasmids (pVAX- and pEGFP-SDF1-IL2) and verified that CPP could transport these plasmids into chicken cell line, DF-1, and express fusion cytokines in avian system. In the future, these chicken fusion cytokines can be applied in veterinary medicine.
URI: http://hdl.handle.net/11455/98195
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