SUMO融合系统表达人白细胞介素17及其活性的研究  

Utilization of SUMO expressing vector to express human interleukin-17 and studying of its bioactivity

作　　者：朱慧萌[1] 谷学佳[1] 任桂萍[1] 孙阳[1] 彭中宜[2] 徐彤[2] 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 [2]黑龙江省医院,哈尔滨150036

出　　处：《中国免疫学杂志》2010年第7期595-600,605,共7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

摘　　要：目的:克隆、表达和纯化人白细胞介素17(hIL-17),并对其生物学活性进行研究。方法:采用RT-PCR的方法从活化的人T细胞中克隆获得hIL-17cDNA。构建了含羟胺切割位点的重组载体pHisSUMO-hIL-17,导入宿主菌Rosetta中,经IPTG诱导后得到表达。用Ni-NTA琼脂糖颗粒层析纯化融合蛋白,经羟胺切割,复性,通过Western blot实验进行确认,并从核酸水平和蛋白水平鉴定其活性。结果:克隆表达出了hIL-17,利用Ni-NTA琼脂糖颗粒层析纯化得到的纯品蛋白经体外活性实验表明,该蛋白具有刺激3T3-L1细胞和HeLa细胞分泌IL-6、IL-8、G-CSF的作用。结论:制备了具有生物学活性的成熟hIL-17蛋白,为进一步研究该分子在疾病中的作用奠定了基础。Objective：The aim of the paper is to clone,express,purify and characterize human interleukin-17（hIL-17）.Methods：The hIL-17 cDNA was cloned from activated human T cells by RT-PCR.Recombinant vector pHisSUMO-hIL-17 with a hydroxylamine cleavage site was constructed and transformed into the host bacteria Rosetta.The fusion protein was expressed by IPTG induction.Ni-NTA affinity chromatography purification column was utilized to purify His-SUMO-hIL-17 fusion protein.The recombinant protein was confirmed by Western blot.Cytokines induction by the hIL-17 were analyzed at both RNA and protein levels.Results：The IL-17 was cloned from human T cells,expressed in prokaryotic cells and purified by Ni-NTA affinity column.The hIL-17 could stimulate 3T3-L1 and Hela cell to express IL-6,IL-8,G-CSF at both RNA and protein levels.Conclusion：The hIL-17 is cloned and expressed in prokaryotic cells.The purified and bioactive hIL-17 protein was obtained for further studies.

关 键 词：hIL-17 表达 纯化 活性试验 

分 类 号：Q78[生物学—分子生物学]