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目的构建小鼠CD25分子胞外段的原核表达载体,并在大肠杆菌中进行表达。方法从BALB/c小鼠脾细胞中提取总RNA,利用RT-PCR方法获得CD25分子胞外段序列;将其亚克隆入原核表达载体PET-32a,构建好PET-32a-CD25胞外段,并进行酶切及测序鉴定;将PET-32a-CD25胞外段转染大肠杆菌BL21进行表达,并对表达产物进行纯化和鉴定。结果RT-PCR扩增出708 bp的CD25胞外段的基因片段;pET32a-CD25胞外段/BL21经IPTG诱导后,SDS-PAGE显示有新生的蛋白表达条带,相对分子质量约为47×103,重组蛋白用镍树脂纯化后经Western blot检测显示具有良好的免疫反应性,并且可以在体外有效刺激自体T细胞疫苗免疫小鼠脾细胞的增殖并高分泌IL-4。结论CD25胞外段蛋白在原核细胞中成功表达,纯化后的蛋白保持良好的免疫反应性。
Objective To construct a prokaryotic expression vector for mouse CD25 extracellular domain and express it in E. coli. Methods Total RNA was extracted from splenocytes of BALB / c mice and the extracellular domain of CD25 was obtained by RT-PCR. The recombinant plasmid was subcloned into prokaryotic expression vector PET-32a to construct the extracellular domain of PET-32a-CD25. The recombinant plasmids were digested with restriction endonucleases and sequenced. The extracellular domain of PET-32a-CD25 was transfected into E. coli BL21 for expression and purification. Results The 708 bp fragment of extracellular CD25 was amplified by RT-PCR. The extracellular domain of pET32a-CD25 / BL21 was induced by IPTG and showed a new protein band by SDS-PAGE. The relative molecular mass was about 47 × 103. Recombinant protein purified by Nickel resin showed good immunoreactivity by Western blot and could effectively stimulate the proliferation of splenocytes from mice immunized with autologous T cell vaccine in vitro and high secretion of IL-4. Conclusion The extracellular domain of CD25 is successfully expressed in prokaryotic cells, and the purified protein retains good immunoreactivity.