Objective This work aims at preparation of mouse anti human IgE monoclonal antibody, for research and diagnosis of allergic diseases. Methods In this study, we first amplified the gene of IgE Fcε3-4 from the cDNA of CRL8033 cell line and cloned into pET-32a(+) expression vector. This recombinant vector was transformed into BL21(DE3)E.coli and the recombinant protein expression was induced by IPTG. The antigen obtained from the inclusion body was renatured and was purified by Ni2+ affinity chromatography. The purity of the protein was assessed by SDS-PAGE. Finally we used the purified protein as an antigen to immunize BABL/c mice and the spleen cells were fused with SP2/0 after the third immunization. The hybridomas secreting monoclonal antibodies that could recognize human IgE was identified by ELISA and Western blot. Results We successfully constructed the pET-32a(+)-IgE Fcε3-4 expression vector, purified the antigen from inclusion body, and used the purified protein as an antigen to immunize mice. Successfully prepared hybridoma cell lines secretes monoclonal antibodies capable of specifically recognize human IgE, which can be used for ELISA and Western blot to detect human IgE. Conclusion We successfully obtain a monoclonal antibody which can be used to specifically detect human IgE protein, laying the foundation for diagnosis and treatment of asthma and other allergic diseases.%目的：通过制备小鼠抗人 IgE单克隆抗体，为过敏性疾病的研究及诊断打下基础。方法以 CRL8033细胞的 cDNA为模板扩增人 IgE Fcε3-4段基因序列并将其连接到 pET-32a(+)载体中，转化 BL21(DE3)大肠杆菌并进行表达。然后将表达所得包涵体进行变复性，把复性所得蛋白进行亲和纯化，通过 SDS-PAGE验证纯化效果。最后将纯化的蛋白作为抗原免疫 BALB/c小鼠，经三次免疫后取小鼠脾脏细胞与 SP2/0细胞融合。用 ELISA法筛选出能结合人 IgE蛋白的阳性克隆，并应用 ELISA、Western blot等手段对杂交瘤所分泌单克隆抗体的特异性进行鉴定。结果成功构建 pET-32a(+)-IgE Fcε3-4载体并纯化 IgE Fcε3-4蛋白。用该蛋白作为免疫原免疫小鼠，成功制备了杂交瘤细胞株，其分泌的单克隆抗体能够特异性结合人 IgE蛋白，可用于 ELISA、Western blot等手段对人 IgE的检测。结论成功获得了可用于特异性检测人 IgE蛋白的单克隆抗体，为人 IgE的检测及哮喘等过敏性疾病的研究、诊断及治疗奠定了基础。
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