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

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.