13种人I型α干扰素亚型的制备与特性分析
Production and characterization of thirteen human type-I interferon-α subtypes.
作者信息
Kuruganti Srilalitha, Accavitti-Loper Mary Ann, Walter Mark R
机构信息
Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
Department of Microbiology, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
出版信息
Protein Expr Purif. 2014 Nov;103:75-83. doi: 10.1016/j.pep.2014.08.010. Epub 2014 Aug 20.
Abstract
Thirteen human interferon-α (IFNα) subtypes were expressed in Escherichiacoli and purified using an N-terminal affinity tag from the prodomain of subtilisin. IFNα subtypes were expressed in soluble form and purified from cell lysates or refolded and purified from inclusion bodies. Proteins produced by either protocol exhibited biological activities equal to or greater than commercially prepared IFNα preparations. The IFNαs were used to produce an anti-IFNα16 antibody (MAb-1B12) that specifically neutralized the biological activity of IFNα16, but not the 12 other IFNαs. Using MAb-1B12, and a previously generated IFNAR1/IFNAR2-FChk heterodimer, an assay was developed to determine total type I IFN biological activity and IFNα16-derived biological activity in an unknown sample.
摘要
13种人干扰素-α（IFNα）亚型在大肠杆菌中表达，并使用来自枯草杆菌蛋白酶前结构域的N端亲和标签进行纯化。IFNα亚型以可溶形式表达，从细胞裂解物中纯化，或从包涵体中复性并纯化。两种方法产生的蛋白质均表现出等于或大于商业制备的IFNα制剂的生物活性。IFNα用于产生一种抗IFNα16抗体（单克隆抗体-1B12），该抗体可特异性中和IFNα16的生物活性，但不能中和其他12种IFNα。使用单克隆抗体-1B12和先前产生的IFNAR1/IFNAR2-FChk异二聚体，开发了一种测定方法，以确定未知样品中的总I型干扰素生物活性和IFNα16衍生的生物活性。
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