EDAG-1的原核表达及单克隆抗体的制备

Prokaryotic expression and monoclonal antibody preparalion of EDAG-1

目的构建红系发育相关基因(erythoid develop associated gene,EDAG-1)原核表达系统并制备其单克隆抗体,以进一步研究 EDAG-1调控细胞增殖、分化及凋亡的分子机制,探讨其临床应用前景。方法本实验选用 pGEX-4T-3载体,构建含 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,IPTG 诱导,获得较高效率的 GST-EDAG-1融合蛋白表达,纯化该蛋白,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。结果成功构建了含 GST 标签的pGEX-4T-3-EDAG-1原核表达载体,并表达出较高纯度的 EDAG-1蛋白,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。结论通过原核表达途径制备 EDAG-1单克隆抗体是可行的,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。

Objective The monoclonal antibody of EDAG-1 was prepared to study the mechanisms of EDAG-1 in the regulation of proliferation, differentiation and apoptosis of haematopoietic ceils. Methods The EDAG-1 cDNA was cloned from human fetal liver eDNA library and inserted into the pGEX-4T-3 vecror marked by GST, Highly-pure fusion protein of GST-EDAG-1 was obtained by induction of IPTG and purification. The fusion protein GST-EDAG-1 was used to immunize the mouse BALB/C. The spleen cells from immunized mouse and the SP2/0 myeloma cells of the mouse from the, same class as the imnmnized mouse were fused with PEG. A preliminary selection was performed among fused cells cultured in HAT base. And ELISA on hybridoma cells were done to judge whether the related antibody existed. Results pGEX-4T-3-EDAG-1 expressed vector marked by （;ST was construcred and highly-pure protein of EDAG-1 was obtained. By immunizing the mouse BALB/C,mono-clone hybridoma ceils which can express EDAG-1 antibody was obtained. From the results of the preliminary experiment, it was found that the monoclonal antibody had good specificity and stability and low background. Conclusion It is feasible to prepare the monoclonal antibody of EDAG-1 by prokaryotic expression system,and we found that the monoclonal antibody had good specificity and stability and low background.