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尼罗罗非鱼PPARδ的原核表达及其多抗制备和纯化

潘传燕 冯鹏霏 张永德 罗洪林

潘传燕,冯鹏霏,张永德,等. 尼罗罗非鱼PPARδ的原核表达及其多抗制备和纯化 [J]. 福建农业学报,2019,34(9):1053−1058. doi: 10.19303/j.issn.1008-0384.2019.09.009
引用本文: 潘传燕,冯鹏霏,张永德,等. 尼罗罗非鱼PPARδ的原核表达及其多抗制备和纯化 [J]. 福建农业学报,2019,34(9):1053−1058. doi: 10.19303/j.issn.1008-0384.2019.09.009
PAN C Y, FENG P F, ZHANG Y D, et al. Prokaryotic Expression, Polyclonal Antibody Preparation and Purification of PPARδ Protein in Nile Tilapia [J]. Fujian Journal of Agricultural Sciences,2019,34(9):1053−1058. doi: 10.19303/j.issn.1008-0384.2019.09.009
Citation: PAN C Y, FENG P F, ZHANG Y D, et al. Prokaryotic Expression, Polyclonal Antibody Preparation and Purification of PPARδ Protein in Nile Tilapia [J]. Fujian Journal of Agricultural Sciences,2019,34(9):1053−1058. doi: 10.19303/j.issn.1008-0384.2019.09.009

尼罗罗非鱼PPARδ的原核表达及其多抗制备和纯化

doi: 10.19303/j.issn.1008-0384.2019.09.009
基金项目: 国家自然科学基金项目(31372553、31760765);广西重点研发计划项目(桂科AB16380074)
详细信息
    作者简介:

    潘传燕(1988−),女,硕士,工程师,研究方向:水生动物遗传育种(E-mail:723870955@qq.com

    通讯作者:

    罗洪林(1977−),男,博士,副研究员,研究方向:动物遗传育种(E-mail:541365548@qq.com

  • 中图分类号: S 917.4

Prokaryotic Expression, Polyclonal Antibody Preparation and Purification of PPARδ Protein in Nile Tilapia

  • 摘要:   目的  为研究尼罗罗非鱼过氧化物酶体增殖物激活受体δ(PPARδ)的表达情况,通过大肠杆菌表达系统表达PPARδ,纯化重组蛋白并获得多克隆抗体。  方法  对PPARδ进行生物信息学分析后设计特异性引物,扩增PPARδ,将其克隆至原核表达载体pET-B2m中,构建重组表达载体;重组质粒转入大肠杆菌B21并用IPTG进行诱导表达,用SDS-PAGE鉴定表达产物。用镍柱纯化重组蛋白后免疫日本大耳兔制备多克隆抗体,用间接ELISA技术检测抗体效价,Western Blot鉴定抗体的特异性。  结果  成功构建了原核表达载体pET-B2m-PPARδ,实现了重组蛋白的原核表达和纯化,重组蛋白以包涵体蛋白和可溶性蛋白2种形式存在,分子量约为90 kD;制备的多克隆抗体效价为1 2 048 000,Western Blot检测结果表明该抗体能特异性识别尼罗罗非鱼PPARδ。  结论  成功实现了尼罗罗非鱼PPARδ重组蛋白的融合表达,在大肠杆菌中高效表达后纯化重组蛋白,免疫日本大耳兔获得了效价高、特异性强的多克隆抗体,为尼罗罗非鱼PPARδ的功能研究奠定了基础。
  • 图  1  尼罗罗非鱼PPARδ抗原表位分析

    Figure  1.  Analysis on epitope of PPARδ in Nile tilapia

    图  2  PPARδ基因扩增结果

    注:M:DL2000;1:PPARδ

    Figure  2.  PCR amplification of PPARδ

    Note: M:DL2000;1:PPARδ

    图  3  重组蛋白的SDS-PAGE分析

    注:M:蛋白标准;1:表达菌沉淀;2:表达菌上清

    Figure  3.  SDS-PAGE analysis on recombinant protein

    Note: M: marker; 1: precipitate of expressed proteins; 2: supernatant of expressed proteins.

    图  4  重组蛋白纯化结果

    注:M:蛋白标准;1:PPARδ

    Figure  4.  Purification of recombinant protein

    Note: M:Marker;1:PPARδ

    图  5  Western blotting分析PPARδ多克隆抗体的特异性

    注:M:蛋白标准;1:25 ng PPARδ重组蛋白(G480);2:10 ng PPARδ重组蛋白(G480);3:25 ng PPARδ重组蛋白(G479);4:10 ng PPARδ重组蛋白(G479);5:阴性对照(G480);6:阴性对照(G479)。其中,G480和G479是日本大耳兔的实验编号。

    Figure  5.  Specificity of PPARδ polyclonal antibody by western blotting

    Note: M: marker; 1: 25 ng PPARδ recombinant protein (G480); 2: 10 ng PPARδ recombinant protein (G480); 3: 25 ng PPARδ recombinant protein (G479); 4: 10 ng PPARδ recombinant protein (G479); 5: negative control (G480); and, 6: negative control (G479). G480 and G479 are codes of immunized Japanese white rabbits.

    图  6  PPARδ抗体纯化结果

    注:M:蛋白标准;1:PPARδ抗体

    Figure  6.  Purification of PPARδ polyclonal antibody

    Note: M: Marker; 1: PPARδ polyclonal antibody

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  • 收稿日期:  2019-06-17
  • 修回日期:  2019-08-02
  • 刊出日期:  2019-09-01

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