小鼠可溶性IL-5受体α胞外区基因的克隆及原核表达

Cloning and Expression of Gene Encoding Extracellular Domain of Mouse Soluble IL-5 Receptor α

目的克隆并原核表达小鼠可溶性IL-5受体α(sIL-5Rα)胞外区基因。方法采用RT-PCR从BALB/c小鼠脾脏组织中分别扩增sIL-5Rα胞外区前后段基因片段,酶切后将两段基因连接,插入原核表达载体pPROEX中,构建重组表达质粒pPROEX/sIL-5Rα,转化大肠杆菌DH5α,IPTG诱导表达。采用SDS-PAGE和Western blot检测目的蛋白的表达。结果重组表达质粒经双酶切和DNA测序,证实构建正确。表达的sIL-5Rα胞外区蛋白相对分子质量约36100,表达量约占菌体总蛋白的30%,且可与兔抗小鼠sIL-5Rα单抗发生特异性免疫反应。结论已成功克隆并原核表达了小鼠sIL-5Rα胞外区基因,为进一步探讨sIL-5Rα对哮喘的治疗作用及开发新的哮喘治疗药物奠定了基础。

Objective To clone the gene encoding extracellular domain of mouse soluble IL-5 receptor α（sIL-5Rα）and ex-press in prokaryotic cells. Methods The anterior and posterior segments of gene encoding extracellular domain of sIL-5Rα were am-plified from the spleen tissues of BALB / c mice by RT-PCR,digested with restriction endonuclease,then linked and inserted into prokaryotic expression vector pPRO EX. The constructed recombinant plasmid pPRO EX / sIL-5Rα was transformed to E. coli DH5α for expression under induction of IPTG. The expressed product was identified by SDS-PAGE and Western blot. Results Both restriction analysis and DNA sequencing proved that recombinant plasmid pPRO EX / sIL-5Rα was constructed correctly. The expressed product,with a relative molecular mass of about 36 100,contained about 30% of total somatic protein and showed specific immune reaction with rabbit anti-mouse sIL-5Rα McAb. Conclusion The gene encoding extracellular domain of mouse sIL-5Rα was successfully cloned and expressed in prokaryotic cells,which laid a foundation of further study on curative effect of sIL-5Rα on asthema as well as development of novel drugs for asthema.