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

作　　者：宋爱玲[1] 蔡累[2] 杨怡[1] 王伟华[2] 王增禄[2] 张英起[2] 李志奎[1] 

机构地区：[1]第四军医大学西京医院,西安710032 [2]第四军医大学生物技术中心,西安710032

出　　处：《中国生物制品学杂志》2009年第4期327-330,共4页Chinese Journal of Biologicals

基　　金：国家自然科学基金资助项目(30770926).

摘　　要：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.

关 键 词：可溶性IL-5受体α 胞外区 克隆 原核表达 

分 类 号：Q785[生物学—分子生物学] Q786