人白细胞介素-17基因的克隆、表达及其表达产物的分离纯化

利用ＲＴ－ＰＣＲ方法成功地从ＰＩ（ＰＭＡ，Ｉｏｎｏｍｙｃｉｎ）、ＰＨＡ活化的人Ｔ细胞中扩增出ｈＩＬ－１７编码区ｃＤＮＡ（４６８ｂｐ），克隆测序证实得到该基因．用特殊设计的引物扩增ｈＩＬ－１７成熟肽编码区（４１４ｂｐ），接入表达载体ｐＥＴ３０ａ质粒中．ｐＥＴ３０ａ－ｍｈＩＬ－１７在大肠杆菌ＢＬ２１（ＤＥ３）中获得高效表达，目的蛋白占总菌体蛋白５０％左右．经凝胶过滤和阴离子交换层析两步分离纯化和复性，得到单体型ｒｍｈＩＬ－１７，纯度达９８％以上，Ｎ端１６个氨基酸序列分析，结果与文献报道一致．ＳＤＳ－ＰＡＧＥ法测定分子量为１６ｋＤ，薄层丙烯酰胺凝胶等电聚焦分析该蛋白等电点为ｐＨ８．８～８．９，３ＨＴｄＲ参入法测定ｒｍｈＩＬ－１７单体的体外活性，实验结果表明ｒｍｈＩＬ－１７对ＰＨＡ活化人的ＰＢＭＣ细胞具有抑制作用，结果同可溶型ＩＬ－１７Ｒ性质相似

Molecular Cloning,Expressing and Purifying of Human Interleukin 17 Gene

Human interleukin 17(hIL 17) cDNA(468 bp) was successfully amplified from human T cells activated by PI(PMA,Ionomycin)and PHA with RT PCR method.The results of cloning and sequencing proved this cDNA was the hIL 17 gene full length coding sequence.The cDNA(414 bp) coding mature hIL 17 was amplified with special designed primers.The cDNA fragments were cloned into expression vectors:pET30a.The pET30a mhIL 17 could highly express in E.coli BL21 (DE3),the recombinant hIL 17 was about 50% of total proteins.Monomer recombinant mhIL 17(98% pure) could be obtained with two steps of purification:gel filtration and ion exchange chromatography and renaturation.N terminal sequencing of rmhIL 17 showed the first 16 amino acids were the same as the sequence reported.Molecular weight and pI of rmhIL 17 were 16 kD and pH 8 8～8 9,measured with SDS PAGE and IEF respectively. In vitro activity assay found monomer rmhIL 17 could suppress the activation of human PBMC by PHA.It suggested that monomer rmhIL 17 had the similar activity with soluble IL 17 receptor.