人可溶性IL-6R及其突变体基因在COS7细胞中的表达及活性分析

Expression and biological activity analysis of human soluble IL 6R and its mutant

目的：研究人可溶性ＩＬ６Ｒ（ｈｓＩＬ６Ｒ）的空间构效关系，为小分子拮抗剂设计提供理论依据。方法：首先利用ＰＣＲ技术扩增天然人ｓＩＬ６Ｒ基因片段，然后将第２８０位Ｈｉｓ残基突变成Ｉｌｅ。天然基因和突变体基因分别转染ＣＯＳ７细胞，经ＥＬＩＳＡ和Ｗｅｓｔｅｒｎｂｌｏｔ检测证实转染细胞上清中的表达产物，并利用ＢｉｎｄｉｎｇａｓａｙＥＬＩＳＡ和生物学功能分析法检测表达产物与ＩＬ６的结合能力以及它们所介导的ＩＬ６信号转导功能的差异。结果：突变体Ｈ２８０Ｉ比天然ｓＩＬ６Ｒ具有更高的ＩＬ６结合能力，但拮抗ＩＬ６在两种效应细胞系上的信号传递功能。结论：第２８０位Ｈｉｓ残基对ｓＩＬ６Ｒ发挥正常生物学功能具有重要影响，是符合拮抗剂设计要求的一个很好的候选位点。

Objective:To study the structure function relationship of human soluble IL 6 receptor(hsIL 6R) and lay a foundation to design the antagonists.Methods:The sIL 6R gene was obtained by PCR and then histine 280 was mutanted into isolucine with site directed mutagenesis system.The expression plasmids of the two genes were introduced into COS7 cells and then the expressed products were confirmed by ELISA and Western blot.IL 6 binding abilities were measured by binding assay ELISA.The biological function analysis of the expressed products on the IL 6 responsive cells indicated their different abilities of signal transduction.Results:The mutant H280I had higher IL 6 binding ability than that of wtsIL 6R,but showed antagonistic function on IL 6 signal transdution.Conclusion:H280 played an important role in the function of sIL 6R and might be used as a target site for the antagonist design.