Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/24029
標題: 抗Indoleamine-2,3-Dioxygenase多株、單株及單鏈抗體之製備、分析及應用
Preparation, characterization and application of anti-human indoleamine-2,3-dioxygenase antibodies
作者: 黃雅芳
Huang, Ya-Fang
關鍵字: IIndoleamine-2,3-dioxygenase;Indoleamine-2,3-dioxygenase;Single-chain variable fragment;SKOV-3;Tryptophan;單鏈抗體;卵巢癌細胞;Tryptophan
出版社: 生物化學研究所
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摘要: 
Indoleamine-2,3-dioxygenase(IDO)是一種存在於細胞質內蛋白質酵素,能將Tryptophan催化形成Kynurenine。腫瘤細胞若受到發炎反應因子如IFN-γ刺激時,就會誘導大量表現IDO,促使腫瘤細胞周圍Tryptophan濃度降低,導致免疫細胞走向凋亡。此外,Tryptophan被代謝後所產生的一系列有毒代謝產物,對淋巴細胞也會造成毒害,使得腫瘤細胞有免疫逃脫機會。
由於IDO的大量表現會使得腫瘤細胞免於遭受免疫系統的攻擊,因此如能利用自身免疫系統來中和IDO的活性,則達到抗癌效果。本研究主要在製備抗Indoleamine 2,3-diogenase之多株抗體、單株抗體及單鏈抗體,期望能運用抗體與抗原結合的原理,中和IDO在體內的生物活性。本實驗使用卵巢癌細胞 (SKOV-3)以RT-PCR實驗選殖出ido基因,將選殖到原核表現質體pET-28a,以大腸桿菌表達目標蛋白his-IDO,利用親和性管柱純化IDO重組蛋白後,來免疫小鼠,製備抗his-IDO多株抗體,後利用融合瘤逐步稀釋技術來製造單株抗體。實驗結果發現,his-IDO重組蛋白可和抗his-IDO多株抗體及單株抗體做專一性的結合,此外也發現其效價高,可偵測到卵巢癌細胞(SKOV-3)中高度表現IDO。而單株融合瘤細胞之VH及VL以聚合酶連鎖反應技術篩選出來接上連接子(Linker),製成單鏈抗體cDNA(Single-chain variable fragment, scFv),期望未來可以運用在癌症醫療上作為輔助性治療之用。

Indoleamine-2,3-dioxygenase(IDO), an intracellular enzyme, catalyzes the rate-limiting step in tryptophan catabolism to the kynurenine pathway. The tumor cells, after an expressed high level of IDO by the inducing of interferon-γ (IFN-γ), exhibit the ability to cease T lymphocytes to proliferation and function because of the depletion of tryptophan surrounding the microenvironment. Furthermore, the tryptophan metabolites demonstrate the toxicity to lymphocytes, leading to the high IDO expressed tumor cells having the ability to be immune-escape.
Since the high levels of IDO-expressed tumor cells having the aptitude of immune escaping, we thus aimed at the preparation of the anti-IDO antibodies, including the polyclonal, monoclonal and scFv antibodies, hoping to neutralize the role of IDO in neoplasm and allow the patient's immune system to function. In this study, the IDO cDNA of human ovarian cancer cells (SKOV-3) was cloned into pET-28a plasmid. The human enzyme was over-expressed in Escherichia coli with hexahistidyl tag and purified with cobalt-agarose affinity chromatography. The anti-human IDO polyclonal antibody was obtained by immunizing mice with purified his-IDO protein and was used to detect the IDO presence in either SKOV-3 with high specificity. The monoclonal antibodies were also obtained with hybridoma technique. The cDNAs of variable regions of the heavy and light chain (VH and VL) of monoclonal antibody were obtained by RT-PCR of corresponding hybridoma cell. The DNA segment of the single chain fragment of variable region (scFv) antibody was also obtained by linking the VH and VL regions. Further studies, including scFv protein expression, bio-functional assays and animal model studies, are needed to proof the strategy being operational for anticancer therapy as supplemental medicine.
URI: http://hdl.handle.net/11455/24029
其他識別: U0005-0208201015575700
Appears in Collections:生物化學研究所

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