本文關(guān)鍵詞： 紅系發(fā)育相關(guān)基因 原核表達(dá) 單克隆抗體　出處：《安徽醫(yī)科大學(xué)》2007年碩士論文　論文類型：學(xué)位論文

【摘要】： 紅系發(fā)育相關(guān)基因(erythoid develop associated gene,EDAG-1)是一種特異表達(dá)于造血組織和細(xì)胞的新基因(中國(guó)專利申請(qǐng)?zhí)?01118268.8公開號(hào):CN1388130A),它在造血發(fā)育與分化調(diào)控過(guò)程中發(fā)揮重要作用。為進(jìn)一步研究EDAG-1調(diào)控細(xì)胞增殖、分化及凋亡的分子機(jī)制,及探討其臨床潛在應(yīng)用前景,研制EDAG-1的單克隆抗體是十分必要的。本實(shí)驗(yàn)用原核表達(dá)的方式獲得足夠的純化EDAG-1重組蛋白為抗原,通過(guò)細(xì)胞融合技術(shù)制備EDAG-1單克隆抗體,為EDAG-1的深入研究提供可能。 本實(shí)驗(yàn)選用pGEX-4T-3載體,成功構(gòu)建了含GST標(biāo)簽的pGEX-4T-3-EDAG-1原核表達(dá)載體,經(jīng)過(guò)對(duì)表達(dá)體系進(jìn)行優(yōu)化,在37℃培養(yǎng),OD600為1.4-1.5時(shí)加0.1mM IPTG誘導(dǎo)4小時(shí),可以獲得較高效率的GST-EDAG-1融合蛋白表達(dá),融合蛋白占菌體可溶蛋白的30%以上,并且表達(dá)的蛋白以可溶性形式存在于菌體中。通過(guò)對(duì)誘導(dǎo)蛋白的純化,可以獲得較高純度的EDAG-1蛋白。 利用原核表達(dá)獲得的GST-EDAG-1融合蛋白對(duì)BALB/C小鼠進(jìn)行免疫,將免疫后小鼠的脾臟細(xì)胞和來(lái)源于同種系小鼠的SP2/0骨髓瘤細(xì)胞(次黃嘌呤鳥嘌呤磷酸核糖轉(zhuǎn)移酶缺陷型和胸腺核苷激酶缺陷型)采用PEG介導(dǎo)的融合方式進(jìn)行細(xì)胞融合實(shí)驗(yàn),通過(guò)HAT培養(yǎng)基對(duì)融合細(xì)胞進(jìn)行初步篩選,進(jìn)而通過(guò)ELISA實(shí)驗(yàn)對(duì)所獲得雜交瘤細(xì)胞進(jìn)行抗體表達(dá)驗(yàn)證,獲得表達(dá)EDAG-1抗體的單克隆雜交瘤細(xì)胞。通過(guò)對(duì)單克隆抗體的初步實(shí)驗(yàn)表明,所獲單克隆抗體具有很好的特異性,較低的背景和較好的穩(wěn)定性。本實(shí)驗(yàn)制備的抗EDAG-1的單克隆抗體可用來(lái)檢測(cè)臨床白血病細(xì)胞中的表達(dá)產(chǎn)物,并為將來(lái)應(yīng)用于臨床白血病等腫瘤患者的診斷和治療提供可能,也對(duì)深入研究EDAG-1調(diào)控造血的分子機(jī)制等生物學(xué)功能具有重要意義。
[Abstract]:Erythroid develop associated gene EDAG-1 is a novel gene specifically expressed in hematopoietic tissues and cells (China Patent Application No.: 01118268.8 Open No.: CN1388130A1.It plays an important role in the regulation of hematopoietic development and differentiation. EDAG-1 regulates cell proliferation. The molecular mechanism of differentiation and apoptosis and its potential clinical application are discussed. It is necessary to develop monoclonal antibodies against EDAG-1. In this study, sufficient purified recombinant EDAG-1 protein was obtained by prokaryotic expression. EDAG-1 monoclonal antibody was prepared by cell fusion technique, which provides the possibility for the further study of EDAG-1. In this experiment, pGEX-4T-3 vector was used to construct pGEX-4T-3-EDAG-1 prokaryotic expression vector with GST tag successfully. After optimizing the expression system, the expression of GST-EDAG-1 fusion protein could be obtained by adding 0.1 mm IPTG for 4 hours at 37 鈩，

本文編號(hào)：1554570