一种新型造血相关转录调节因子EDAG-1的原核表达及单克隆抗体的制备

发布时间：2018-03-02 02:12

  本文关键词： 红系发育相关基因 原核表达 单克隆抗体　出处：《安徽医科大学》2007年硕士论文　论文类型：学位论文

【摘要】： 红系发育相关基因(erythoid develop associated gene,EDAG-1)是一种特异表达于造血组织和细胞的新基因(中国专利申请号:01118268.8公开号:CN1388130A),它在造血发育与分化调控过程中发挥重要作用。为进一步研究EDAG-1调控细胞增殖、分化及凋亡的分子机制,及探讨其临床潜在应用前景,研制EDAG-1的单克隆抗体是十分必要的。本实验用原核表达的方式获得足够的纯化EDAG-1重组蛋白为抗原,通过细胞融合技术制备EDAG-1单克隆抗体,为EDAG-1的深入研究提供可能。 本实验选用pGEX-4T-3载体,成功构建了含GST标签的pGEX-4T-3-EDAG-1原核表达载体,经过对表达体系进行优化,在37℃培养,OD600为1.4-1.5时加0.1mM IPTG诱导4小时,可以获得较高效率的GST-EDAG-1融合蛋白表达,融合蛋白占菌体可溶蛋白的30%以上,并且表达的蛋白以可溶性形式存在于菌体中。通过对诱导蛋白的纯化,可以获得较高纯度的EDAG-1蛋白。 利用原核表达获得的GST-EDAG-1融合蛋白对BALB/C小鼠进行免疫,将免疫后小鼠的脾脏细胞和来源于同种系小鼠的SP2/0骨髓瘤细胞(次黄嘌呤鸟嘌呤磷酸核糖转移酶缺陷型和胸腺核苷激酶缺陷型)采用PEG介导的融合方式进行细胞融合实验,通过HAT培养基对融合细胞进行初步筛选,进而通过ELISA实验对所获得杂交瘤细胞进行抗体表达验证,获得表达EDAG-1抗体的单克隆杂交瘤细胞。通过对单克隆抗体的初步实验表明,所获单克隆抗体具有很好的特异性,较低的背景和较好的稳定性。本实验制备的抗EDAG-1的单克隆抗体可用来检测临床白血病细胞中的表达产物,并为将来应用于临床白血病等肿瘤患者的诊断和治疗提供可能,也对深入研究EDAG-1调控造血的分子机制等生物学功能具有重要意义。
[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，

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