目的通过基因重组的方法表达IgE Cε3-Cε4区,鉴定重组蛋白与天然细胞表面受体FcεRⅠ之间的相互作用,并用重组蛋白免疫小鼠制备血清。方法用RT-PCR方法从过敏性疾病病人外周血淋巴细胞调取IgE Cε3-Cε4cDNA片段,克隆至pET28a(+)构建原核表达载体IgE Cε3-Cε4-pET28a(+),将重组质粒转化到大肠杆菌BL21(DE3),诱导表达IgE Cε3-Cε4区蛋白,通过Ni-NTA亲和层析纯化融合蛋白后,用免疫荧光方法鉴定重组蛋白与天然细胞表面受体FcεRⅠ之间的结合能力,并用UniCAP 100全自动分析检测仪对重组抗原进行定量检测与鉴定;用表达蛋白免疫BALB/c小鼠,制备抗鼠血清,用Western-blot法对多克隆抗体进行鉴定。结果成功调取的人IgE Cε3-Cε4区基因与已报道的序列相一致;获得的IgECε3-Cε4区蛋白相对分子质量(Mr)同预期的结果相一致;IgE Cε3-Cε4能特异性结合人嗜碱性粒细胞表面的FcεRⅠ受体;通过UniCAP 100全自动分析检测仪能够检测到重组抗原并精确到国际单位;免疫鼠血清多抗能特异性结合天然人血清IgE。结论成功构建了IgE Cε3-Cε4-pET28a(+)表达载体,获得了能被人嗜碱性粒细胞表面的FcεRⅠα亚基特异性识别的IgE Cε3-Cε4区蛋白,并用天然人血清IgE鉴定了多克隆抗体,为下一步单克隆抗体的制备打下了基础。 Objective To express the IgE Cε3-Cε4 region by gene recombination, identify the interaction between the recombinant protein and the natural cell surface receptor FcεRI, and immunize mice with the recombinant protein to prepare the serum. Methods The IgE Cε3-Cε4 cDNA fragment was extracted from the peripheral blood lymphocytes of patients with allergic diseases by RT-PCR and cloned into pET28a (+) to construct the prokaryotic expression vector IgE Cε3-Cε4-pET28a (+). The recombinant plasmid was transformed into Escherichia coli After the fusion protein was purified by Ni-NTA affinity chromatography, the binding capacity between the recombinant protein and the natural cell surface receptor FcεRI was identified by immunofluorescence. The expression of IgE Cε3-Cε4 protein was detected with UniCAP 100 The anti-rat serum was prepared by immunizing BALB / c mice with the expressed protein and the polyclonal antibody was identified by Western-blot. RESULTS: The IgE Cε3-Cε4 region of human IgE was successfully sequenced. The relative molecular mass (Mr) of the obtained IgECε3-Cε4 region was consistent with the expected results. IgE Cε3-Cε4 specifically bound to human FcεRI receptors on the surface of basic granulocytes; detection of recombinant antigens by International UniCAP 100 auto-assay detector and accurate to international units; immunized murine polyclonal antibodies specifically bind to native human serum IgE. Conclusion The IgE Cε3-Cε4-pET28a (+) expression vector was successfully constructed and the IgE Cε3-Cε4 region protein specifically recognized by the FcεRⅠα subunit on the surface of human basophils was obtained. Cloning of antibodies for the next step in the preparation of monoclonal antibodies laid the foundation.