人IgE重链3-4区蛋白的表达、纯化及功能分析

Prokaryotic Expression,Purification and Funcational Analysis of Human Recombinant IgE Cε3-Cε4

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 produce recombinant IgE Cε3-Cε4, identify the interreaction beteewn IgE Cε3-Cε4 and FeεR Ⅰ obtained from basophil, and prepare anti-human IgE Cε3-Cε4 polyelonal antibodies. Method The human IgE Cε3-Cε4 eDNA was obtained by RT-PCR using peripheral blood lymphocyte RNA of an allergic disease patient. The PCR fragment was ligated into the vector pET28a（＋） and eonstrueted an expression veetor IgE Cε3-Cε4-pET28a（＋）. The recombinant plasmid was transformed into E.coli BL21 （DE3）. The recombinant protein was expressed in BL21 （DE3）. The fusion protein was purified by Ni Sepharose 6 Fast Flow affinity chromatography. The interreaction beteewn IgE Cε3-Cε4 and Fear Ⅰ was identified by cell immunofluoreseenee. The fusion protein was quantified using automatic analysis detecting instrument UniCAP 100. BALB/e mice were immunized with recombinant IgE Cε3-Cε4. The polyelonal antibodies were prepared and identified by Western-blot. Result The human IgE Cε3-Cε4 eDNA clone was confirmed by sequence analysis and the DNA sequence was deposited in GenBank database. The fusion protein was analysed by SDS-PAGE and the relative molecular mass was as predieted. Cell immunofluorescence experiments confirmed that IgE Cε3-Cε4 could specifically bind to FcεR Ⅰ obtained from human basophils. Through the analysis of automatic detecting equipment UniCAP 100, the reeombinant protein could be detected to precision of the international unit. Western-blot confirmed the mouse polyelonal antibodies can recognize human IgE. Conclusion The IgE Cε3-Cε4-pET28a （＋） expression vector was constructed and the reeombinant IgE Cε3-Cε4 protein could be specifically identified by Fear Ⅰ on the surface of basophils and arose the production of specific polyelonal antibodies in mouse. This has laid the foundation for the next phase of our work.