IL-18在大肠杆菌中的可溶性表达和纯化

目的：研究人IL-18成熟蛋白（mhIL-18）在大肠杆菌中的高效的非包涵体表达以及纯化方案。方法：利用PCR方法扩增出mhIL-18基因，构建融合型表达载体pPTYB1-IL18，转化入大肠杆菌ER2566中，IPTG诱导表达，优化表达条件，超声裂解细胞，采用SDS-PMGE和Weslern blot分析重组蛋白的表达，亲和层析后用MTT法检测表达mhIL-18的活性。结果：成功构建载体并转化入ER2566后，经IPTG诱导，表达量可占菌体总蛋白的26％以上，其中80％以上的mhIL-18融合蛋白以可溶、有活性的形式存在。亲和层析后的mhIL-18纯度可达95％，具有显著的IL-18的生物学活性。结论：成功的在大肠杆菌中的高效以非包涵体表达mhIL-18，并具有显著的IL-18的生物学活性。

High lever soluble expression and purification of recombinant human IL-18 in E.coli

Objective: Objective To study high-level expression and purification of recombinant mature human IL-18(mhIL-18)in E.coli with soluble form.Methods: The gene of mhIL-18 was amplified with PCR.Then,the recombinant expression plasmid pTYB1IL-18 was constructed by cloning mhIL-18 into pTYB1 vector and transformed to E.coli ER2566.IPTG was added into the E.coli ER2566 culture to express recombinant fusion protein.After the harvested bacteria dispersed with ultrasound,SDS-PAGE and Western blot were used to analyze the expression of recombinant fusion protein.The activity of mhIL-18 which had been purified by affinity chromatography was tested by MTT.Results:The cloned sequence of mhIL-18 was identical with the sequence in GenBank.After the induction by IPTG inducing,the fusion protein of mhIL-18 covered over 26% total bacterial proteins,which included 80% active fusion protein.Purification of recombinant expressed mhIL-18 purity was 95%,which had the activity of the mhIL-18.Conclusion: High level expression and purification of recombinant human IL-18 is succeeded which has the activity.