人可溶性IL-6R基因在COS7细胞中的表达及活性分析

目的在ＣＯＳ７细胞中表达具有生物学功能的人可溶性ＩＬ－６Ｒ（ｓＩＬ－６Ｒ），作为研究ｓＩＬ－６Ｒ结构与功能关系的基础。方法首先利用ＰＣＲ技术扩增出人可溶性ＩＬ－６Ｒ（ｈｓＩＬ－６Ｒ）编码基因片段，并重组入克隆载体ｐＡＬＴＥＲ－１。通过基因序列分析确定了目的基因的核苷酸序列，并进一步构建了由ＳＶ４０晚期启动子和ＨＣＭＶ早期启动子控制的表达质粒ｐＳＶＬ６Ｒ和ｐＣＭＶ６Ｒ。用脂质体介导的方法将表达质粒转染ＣＯＳ７细胞，并分别在ｍＲＮＡ水平（斑点杂交）和蛋白水平（ＥＬＩＳＡ和Ｗｅｓｔｅｒｎ－ｂｌｏｔ）检测ｓＩＬ－６Ｒ基因在ＣＯＳ７细胞中的表达。在７ＴＤ１，ＬＴ１２两种ＩＬ－６反应细胞系上检测转染细胞上清（含ｓＩＬ－６Ｒ）的生物学活性。结果在ｍＲＮＡ水平和蛋白水平分别检测到ｓＩＬ－６Ｒ基因在ＣＯＳ７细胞中的表达，表达产物分子量约为５００００。表达产物在７ＴＤ１，ＬＴ１２细胞系上检测到明显的生物学活性。结论天然ｓＩＬ－６Ｒ基因在ＣＯＳ７细胞中的成功表达为进一步制备ｓＩＬ－６Ｒ突变体及其结构与功能关系的研究奠定了基础

Expression and biological activity analysis of human soluble IL 6R

Objective To express the soluble IL 6R gene in the COS7 cells and lay a foundation for investigating the structure function relationship of hsIL 6R. Methods The fragment of hsIL 6R gene obtained by PCR was inserted into clonal vector pALTER 1 and sequenced.The expression plasmids pSVL6R and pCMV6R,constructed by inserting sIL 6R DNA separately into the downstream of SV40 late promotor of eukaryotic expression vector pSVL and CMV early promotor of pCMV4,were introduced into COS7 cells by Lipofectin.Dot blot hybridization ELISA and Western blot were used to detect the expression of sIL 6R gene in COS7 cells,and the IL 6 responsive cells 7TD1 and LT12 were used to analyze the biological activity of the expressed sIL 6R. Results The sIL 6R gene was successfully expressed in the COS7 cells and the molecular weight of the expressed product was about 50kD.The expressed sIL 6R had obvious biological activities on the 7TD1 and LT12 cells. Conclusions The successful expression of sIL 6R gene in the COS7 cell has laid a foundation to prepare the IL 6R mutant and analyze the structure function relationship of it.