人白细胞介素-10在大肠杆菌中的克隆与表达

Cloning and Prokaryotic Expression of Human Interleukin-10

目的:构建人IL-10原核表达载体,并对表达产物进行鉴定。方法:应用RT-PCR技术从ConA诱导的单核细胞中扩增出IL-10成熟肽cDNA片段,将其克隆到PGEM-T后载体测序,双酶切回收目的片段构建IL-10原核表达载体PET28a-IL10。PET28a-IL10转化大肠杆菌BL21(DE3),经IPTG诱导表达后对其表达产物进行SDS-Page和Western Blot分析。结果:SDS-Page电泳显示IL-10在大肠杆菌中得到了表达,分子量为21000,Western Blot分析显示表达产物可于IL-10多抗特异反应。结论:IL-10在大肠杆菌中成功表达,具有免疫原性。为其进一步生物学研究奠定了基础。

Objective：To construct Prokaryotic expression vector of human IL-10 and identify the expressed production.Methods：a cDNA encoding human interleukin-10（IL-10） was amplified by RT-PCR from lymphocyte actived by ConA.the IL-10 cDNA was cloned to vector PGEM-T.After confirming the sequence,IL-10 cDNA was extracted and construct the Prokaryotic expression vector（PET28a-IL10）.The E.coli BL21（DE3） was transfected with the constructed PET28a-IL10,and the expressed IL-10 protein was assayed by SDS-PAGE and Western-blot.Results： SDS - PAGE and Western - blot show that the IL- 10 protein, with a relative molecular weight of 21000, was successfully expresed and can specifically combine with the rabbit anti - human IL - 10 antibody. Conclusion： IL- 10 protein was expressed in E. coli and showed good specificity, which laid a foundation of IL - 10 further biololgy rsearch.