IL-18在大肠杆菌中的可溶性表达和纯化

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

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-PAGE和Western 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。

Objective： Objective To study high-level expression and purification of recombinant mature human IL-18 （ mhIL-18 ）in E. colt with soluble form. Methods： The gene of mhIL-18 was amplified with PCR. Then, the recombinant expression plasmid pTYBIIL-18 was constructed by cloning mhIL-18 into pTYBI vector and transformed to E.coli ER2566.IPTG was added into the E.coli ER2566 culture to express recombinanl 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 filsion 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.