| 摘要: |
| 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。 |
| 关键词: 牙鲆 多聚免疫球蛋白受体 基因克隆 原核表达 黏膜免疫 |
| DOI：10.11693/hyhz20130500065 |
| 分类号: |
| 基金项目:国家自然科学基金项目资助, 31072232 号, 31172429 号; 国家重点基础研究发展规划(973)项目资助, 2012CB114406 号 |
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| CLONING AND EXPRESSION OF POLYMERIC IMMUNOGLOBULIN RECEPTOR IN FLOUNDER PARALICHTHYS OLIVACEUS |
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XU Guo-jing,SHENG Xiu-zhen and ZHAN Wen-bin
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The key Laboratory of Mariculture,Ministry of Education,Ocean University of China,Qingdao,266003,The key Laboratory of Mariculture,Ministry of Education,Ocean University of China,Qingdao,266003,The key Laboratory of Mariculture,Ministry of Education,Ocean University of China,Qingdao,266003
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| Abstract: |
| 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. |
| Key words: Paralichthys olivaceus polymeric immunoglobulin receptor gene cloning prokaryotic expression mucosal immune |
