SUMO表达系统高效、可溶性表达重组鼠源IL-1β  

Efficient expression of the soluble murine IL-1β with SUMO expression system

作　　者：郝建权[1] 任桂萍[1] 刘生伟[1] 傅俊华[1] 谷学佳[1] 姚文兵[1] 李宁[1] 李德山[1] 

机构地区：[1]东北农业大学生命科学学院生物制药教研室,哈尔滨150030

出　　处：《中国免疫学杂志》2010年第8期684-688,共5页Chinese Journal of Immunology

基　　金：哈尔滨市科技创新人才发展计划(2006RFXXS002);东北农业大学创新团队发展计划资助(2007年)

摘　　要：目的:从LPS刺激过的小鼠胸腺细胞中克隆IL-1β基因,通过原核表达,获得具有生物活性的可溶性鼠源IL-1β蛋白,为深入研究和利用IL-1β基因奠定基础。方法:提取LPS刺激的小鼠胸腺细胞总RNA,反转录成cDNA,以此为模板,根据GenBank报道的mIL-1β序列设计引物,进行巢式PCR,得到成熟mIL-1β的编码序列基因,并插入到原核表达载体pHisSUMO ex-press中SUMO标签的下游,构建重组表达载体pHisSUMO express-mIL-1β。将该载体转化大肠杆菌Rosetta(DE3),IPTG诱导表达mIL-1β/SUMO融合蛋白,Ni-NTA Agarose纯化后,表达产物经SDS-PAGE和Western blot进行鉴定,用SUMO protease-1切去SUMO标签,经纯化并切去融合标签后获得成熟mIL-1β蛋白。用MTT方法检测目的蛋白对L929细胞的生物学活性。结果:DNA测序证明所克隆基因序列与GenBank报道的完全一致,SDS-PAGE分析表明融合蛋白的相对分子质量为37kD,切割后的成熟蛋白为17kD,与理论值相符。且蛋白表达量高,主要以可溶形式存在。Western blot证实该蛋白为mIL-1β。成熟蛋白纯化产物纯度超过95%以上,通过MTT的方法检测证明其具有使L929细胞增殖的作用,从而说明其具有生物学活性。结论:利用大肠杆菌表达系统可高效可溶性表达高纯度的具有生物活性的mIL-1β蛋白。Objective：To clone mouse interleukin-1β from thymus that stimulated by LPS,and obtain the soluble and biologically active mouse IL-1β protein by prokaryotic expression for further study of the IL-1β protein.Methods：Total RNA was isolated from mouse thymus that stimulated by LPS,and reverse-transcripted into cDNA,which was used as the template for PCR reaction.The nested PCR primers for cloning mIL-1β were designed according to the published sequence.The cDNA coding for mIL-1β was cloned and constructed into the prokaryotic expressing vector pHisSUMO express-mIL-1β.The vector was transformed into E.coli Rosetta （DE3）,the mIL-1β/SUMO fusion protein was induced by IPTG and purified by Ni-NTA Agarose.The expressing product was evaluated by SDS-PAGE and Western blot.After purification,the SUMO tag was removed by protease-1 cleavage to obtain the mature mIL-1β protein.The biological activity of the target protein was determined by the MTT method in L929 cells.Results：The mIL-1β cDNA was confirmed by DNA sequencing.The recombinant protein was mainly expressed in soluble form and the molecular weight of the fusion protein was 37 kD,the untagged protein was 17 kD in SDS-PAGE gel,consistent with the theoretical value.The mIL-1β protein was verified by Western blot analysis.The purity of the untagged mature protein is more than 95% after purification procedure.The mature mIL-1β protein could stimulate proliferation of L929 cells.Conclusion：Sumoylation enhances the expression level and solubility of the recombinant protein expressed from the E.coli system,and the recombinant protein is bioactive.

关 键 词：mIL-1β SUMO 融合蛋白 原核表达 MTT 活性检测 

分 类 号：Q78[生物学—分子生物学]