【摘要】 目的利用基因重组的方法建立IgE低亲和力受体(FcεRⅡ)基因的原核表达系统。方法用RT-PCR方法从过敏性疾病病人外周血淋巴细胞扩增FcεRⅡcDNA基因,并将其克隆到原核表达载体pGEX-4T-1上,构建原核表达载体FcεRⅡ-pGEX-4T-1,将重组质粒转化到大肠杆菌Rosetta,诱导表达FcεRⅡ蛋白,通过割胶回收纯化,并用Western blot检测FcεRⅡ的表达。结果测序表明所扩增FcεRⅡcDNA基因,与已报道的序列一致。获得的重组蛋白经Western blot鉴定可以和人IgE特异性结合。结论成功构建了FcεRⅡ-pGEX-4T-1表达载体,获得了能和人IgE特异性结合的重组蛋白,为下一步FcεRⅡ单克隆抗体的制备及应用研究打下了基础。更多还原

【Abstract】 Objective To express the IgE low-affinity receptor (FcεRⅡ ) in prokaryotic expression system. Methods The FcεRⅡ cDNA was amplified from peripheral blood lymphocytes of a patient with allergic disease by RT-PCR and then cloned into the vector pGEX-4T-1. The protein was expressed in E.coli Rosette and identified by Western blot. Results Sequencing of PCR product showed that the cDNA sequence was consistent with GenBank data and restriction digest analysis showed that the FcεRⅡ cDNA was successfully cloned into pGEX-4T-1 vector. The recombinant protein was detected by SDS-PAGE and recognized by human IgE in Western blot. Conclusion The FcεRⅡ-pGEX-4T-1 expression vector was successfully constructed and the recombinant FcεRⅡ protein specifically recognized by human IgE was obtained. This study may suggest its application on next preparation of monocional antibodies against FcεRⅡ.更多还原