牛IgG Fc受体Ⅱ胞外区在大肠杆菌中的表达及特性鉴定

从牛肺巨噬细胞cDNA文库中扩增编码牛IgGFc受体Ⅱ(boFcγRⅡ)胞外区基因,PCR产物经EcoRⅠ和HindⅢ双酶切后,插入原核表达载体pET28a的T7启动子下游,构建重组表达质粒pETboRⅡ。以pETboRⅡ转化大肠杆菌BL21(DE3),IPTG诱导表达。SDS-PAGE分析诱导表达菌体可见分子量约为27kDa的蛋白条带,该表达蛋白在细胞质中主要以不溶性包涵体形式存在。Westernblotting和Dotblot检测表明,boFcγRⅡ重组蛋白在变性条件下可与牛IgG特异结合,提示牛FcγRⅡ胞外区可能存在IgG线性结合表位。

Expression and Characterization of the Extracellular Domain of Bovine IgG Fc Receptor Type Ⅱ in E. coli

The DNA fragment encoding the extracellular domain of bovine IgG Fc receptor type Ⅱ (boFcγRⅡ, CD32) was amplified from the cDNA library of bovine alvcolar macrophage. The PCR product digested by EcoR Ⅰ and Hind Ⅲ was inserted into an expression vector of pET28a under the control of T promoter, and the recombinant plasmid designated as pETboRⅡ. Sodium doecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) analysis showed that E.coli BL21(DE3) transformed with pETboRⅡ expressed a recombinant protein of approximate 27kDa. The protein predominantly existed as inclusion bodies in the cytoplasm. It was shown that the denatured protein of boFcγRⅡ was able to bind bovine IgG specifically as analyzed by western blotting and dot-blot, suggesting that there be linear epitopes on the protein responsible for binding of bovine IgG.