牙鲆多聚免疫球蛋白受体基因的克隆、表达及鉴定

摘要

Polymeric immunoglobulin receptor (pIgR) is one of the most important mucosal factors mediating the transcytosis of polymeric immunoglobulins (pIgs) to protect the organism.In this paper,the full-length open reading frame (ORF) of pIgR gene was cloned and expressed by PCR and by constructing prokaryotic expression vector,and then identified by SDS-PAGE,western-blotting,and ELISA.The results show that PCR amplified an ORF of 1005bp.Using PCR and restriction digestion,the constructed pET-32(a)-pIgR recombinant plasmid was identified containing full-length ORF gene.SDS-PAGE shows that the target protein had relative molecular mass of 58 kDa,which is consistent with the theoretical value,and achieved stable expression when induced by IPTG for 6 h.After purified by affinity chromatography,recombinant protein was obtained in high purity with no other band in SDS-PAGE.Western-blotting shows that recombinant protein reacted specifically with anti-His-tag mouse monoclonal antibody,and ELISA results show that recombinant pIgR could bind with the IgM.The ORF was successfully expressed in Escherichia coli BL21 (DE3); and the purified recombinant protein displayed binding ability to IgM.Therefore,flounder pIgR may be involved in the pIgs transport,which provides a molecular basis for the research on the roles of fish pIgR in mucosal immunity.%采用PCR扩增、构建重组高效表达载体的方法,进行了牙鲆多聚免疫球蛋白受体(pIgR)基因的克隆、原核表达研究,并利用SDS-PAGE、western-blot及ELISA方法对纯化的重组蛋白特性进行了分析.结果表明,PCR扩增出pIgR开放阅读框(ORF)基因全长为1005 bp,所构建的pET-32(a)-pIgR重组质粒经PCR和双酶切鉴定含有ORF全长基因.SDS-PAGE结果表明,表达的目的蛋白相对分子质量为58 kDa,与理论预期值一致,IPTG诱导6h后表达量趋于稳定,经亲和层析纯化得到高纯度的重组蛋白.Western-blot结果表明,重组pIgR能够与鼠抗His-tag单克隆抗体发生特异性反应;ELISA结果表明重组pIgR能够与牙鲆IgM发生特异性结合.本研究获得了纯化的重组pIgR,证明其具有IgM结合活性,为下一步研究牙鲆pIgR的转运机制及在黏膜免疫防御中的作用机理提供了分子基础.