重组猪IgGⅡB类Fc受体蛋白的原核表达和抗体的制备

Prokaryotic expression of recombinant swFcγRⅡB protein and preparation of polyclonal antibody against swFcγRⅡB

根据GenBank中猪IgGⅡB类Fc受体剪接异构体cDNA(swFcγRⅡB)基因序列(FJ608551),利用Primer5.0软件设计合成了1对特异性引物,扩增swFcγRⅡB去掉胞内区和跨膜区的基因,PCR产物经KpnⅠ+EcoRⅠ双酶切后,将截短的swFcγRⅡB基因亚克隆到原核表达载体pET-32a中构建了重组表达质粒pET-swFcγRⅡB,在IPTG诱导下成功表达了分子质量约为40ku的融合蛋白swFcγRⅡB-His,该表达蛋白在细胞质中主要以不溶性包涵体的形式存在。原核表达蛋白经纯化免疫小鼠,制备鼠源swFcγRⅡB封闭抗体,ELISA检测抗体效价为1:5120,具有高度特异性。Western-blotting检测表明,制备的多克隆抗体可以与融合蛋白swFcγRⅡB-His特异性结合。

According to the cDNA sequence （FJ608551）of the porcine Fc gamma receptor Ⅱ B＇s （swFcγR Ⅱ B＇s） RNA transcript variant in GenBank,a pair of primers was designed using Primer5.0 soft-ware. The gene was amplified from swFcγR Ⅱ B abscised intracellular region and transmembrane domain. The PCR product was digested with Kpn Ⅰ and EcoR Ⅰ . The truncated swFcγR Ⅱ B gene was cloned into pET-32a vector to generate the recombinant plasmid pET-swFcγR Ⅱ B for expression. The swFcγR Ⅱ B-His fusion protein was successfully expressed with IPTG induction, and the protein predominantly existed as inclusion bodies in the cytoplasm. After expression in Escherichia coli and purification, the purified protein was used to immunize mice to prepare antisera. The results of ELISA and Western-blotting indicated that the prepared polyclonal antibodies had high titer and specificity.