牙鲆多聚免疫球蛋白受体基因的克隆、表达及鉴定

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。

CLONING AND EXPRESSION OF POLYMERIC IMMUNOGLOBULIN RECEPTOR IN FLOUNDER PARALICHTHYS OLIVACEUS

The polymeric immunoglobulin receptor (pIgR) is one of the most important mucosal effectors mediating the transcytosis of polymeric immunoglobulins (pIgs) to protect the organisms. In this paper, the full-length open reading frame (ORF) of pIgR gene was cloned and expressed by PCR and constructing prokaryotic expression vector, and then identified by SDS-PAGE, Western-blot and ELISA. The results showed that PCR amplified an ORF of 1005 bp. Using PCR and restriction digestion, the constructed pET-32 (a) -pIgR recombinant plasmid was identified containing full-length ORF gene. SDS-PAGE showed that the target protein had the relative molecular mass of 58 kDa, consistent with the theoretical expected value, and achieved stable expression when induced by IPTG for 6 h. After purified by affinity chromatography, recombinant protein obtained high purity and no other band in SDS-PAGE. Western-blot showed that recombinant protein specifically reacted with anti-His-tag mouse monoclonal antibody and ELISA results showed that recombinant pIgR could bind with the IgM. In present study, the ORF was successfully expressed in Escherichia coli BL21 (DE3) and the purified recombinant protein displayed binding capability to IgM. These results indicated that flounder pIgR probably involved in the pIgs transport and provided molecular basis for the research on the roles of fish pIgR in the mucosal immunity.