猫白介素31蛋白的真核细胞表达及应用

Eukaryotic Expression and Identification of Feline Interleukin 31 Protein

该文通过哺乳动物细胞表达系统表达猫白介素31(fIL-31),并鉴定其与受体的结合。首先,构建重组质粒。其次,将重组质粒转染至293T细胞,经蛋白质印迹实验(Western blot)鉴定重组蛋白fIL-31-hFc的表达后,大量转染通过Protein A亲和层析和分子筛凝胶层析的方式纯化目的蛋白。最后,利用细胞流式的方法检测fIL-31-hFc与其受体猫抑瘤素M受体β(fOSMRβ)的结合情况。结果表明,fIL-31-hFc以可溶性的形式表达在细胞的上清中,经分子筛层析纯化后,可获得聚集形式均一的目的蛋白,该蛋白可与表达在细胞表面的fOSMRβ受体结合。

In this study,feline IL-31(fIL-31)was expressed by mammalian expression system 293T cells and its binding to receptor was identified.First,the recombinant plasmid was constructed.Secondly,the recombinant plasmid was transfected into 293T cells,and after the expression of the recombinant protein fIL-31-hFc was identified by western blot assay,the target protein was purified by Protein A and Superdex 20010/300 GL.Finally,the binding of fIL-31-hFc to its receptor feline OSMRβ(fOSMRβ)was detected by flow cytometry.The results showed that fIL-31-hFc was expressed in soluble form in the supernatant of 293T cells,and after purification by Superdex 20010/300 GL,the target protein in uniform aggregation form was obtained,which could bind to the fOSMRβexpressed on the cell surface.