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南方根结线虫MiPDCD6基因多克隆抗体的制备

陈晨 高永峰 王新荣 袁永强 蔡书静 刘松松 叶雯华 王燕

陈晨,高永峰,王新荣,等. 南方根结线虫MiPDCD6基因多克隆抗体的制备 [J]. 福建农业学报,2023,38(3):322−328 doi: 10.19303/j.issn.1008-0384.2023.03.008
引用本文: 陈晨,高永峰,王新荣,等. 南方根结线虫MiPDCD6基因多克隆抗体的制备 [J]. 福建农业学报,2023,38(3):322−328 doi: 10.19303/j.issn.1008-0384.2023.03.008
CHEN C, GAO Y F, WANG X R, et al. Preparation of Polyclonal Antibody against MiPDCD6 Protein in Meloidogyne incognita [J]. Fujian Journal of Agricultural Sciences,2023,38(3):322−328 doi: 10.19303/j.issn.1008-0384.2023.03.008
Citation: CHEN C, GAO Y F, WANG X R, et al. Preparation of Polyclonal Antibody against MiPDCD6 Protein in Meloidogyne incognita [J]. Fujian Journal of Agricultural Sciences,2023,38(3):322−328 doi: 10.19303/j.issn.1008-0384.2023.03.008

南方根结线虫MiPDCD6基因多克隆抗体的制备

doi: 10.19303/j.issn.1008-0384.2023.03.008
基金项目: 广东省基础与应用基础研究基金(2019A1515012080);国家自然科学基金项目(31171825、30771409)
详细信息
    作者简介:

    陈晨(1987−),女, 硕士, 研究方向: 分子植物病理学(E-mail:zifeng20032001@aliyun.com

    通讯作者:

    王新荣(1968−),女,博士,教授,研究方向: 分子植物病理学(E-mail:xinrongw@scau.edu.cn

  • 中图分类号: S435

Preparation of Polyclonal Antibody against MiPDCD6 Protein in Meloidogyne incognita

  • 摘要:   目的  制备针对南方根结线虫食道腺蛋白MiPDCD6多克隆抗体,为进一步研究南方根结线虫MiPDCD6蛋白的致病机制提供技术支持和材料准备。  方法  将扩增MiPDCD6基因的功能片段,与原核表达质粒pET-32a(+)构建重组质粒pET-32a-MiPDCD6,然后将重组质粒转化大肠杆菌 BL21 细胞进行诱导表达;将纯化的根结线虫MiPDCD6融合蛋白免疫雄性新西兰大白兔,获得高效价高纯度的多克隆抗体。  结果  构建的重组质粒pET-32a-MiPDCD6转化大肠杆菌 BL21细胞后,在诱导剂IPTG 浓度1.0 mmol·L−-1、摇床温度37 ℃、转速150 r·min−−1和 振荡培养 5 h 条件下,成功表达MiPDCD6融合蛋白。ELISA 和 SDS-PAGE 检测表明:将纯化的根结线虫MiPDCD6融合蛋白免疫雄性新西兰大白兔,获得高效价高纯度的多克隆抗体,效价约为1∶50 000。  结论  明确了 MiPDCD6基因原核表达条件;制备获得的根结线虫MiPDCD6蛋白的多克隆抗体效价和纯度较高,可用于后续研究MiPDCD6蛋白在南方根结线虫致病机理中发挥的功能。
  • 图  1  MiPDCD6表达质粒pET-32a-MiPDCD6的构建

    A:MiPDCD6用酶切引物扩增的目的DNA片段;M:DL 2000 DNA Marker;1~5:MiPDCD6用酶切引物PCR扩增的目的DNA片段。B:MiPDCD6加酶切位点的菌落PCR检测;M:DL 2000 DNA Marker;1~8:MiPDCD6加酶切位点的部分菌液PCR扩增产物。C:pET-32a载体、MiPDCD6酶切后的目的DNA片段; M:DL 2000 DNA Marker;1:pET-32a载体酶切后的目的DNA片段琼脂糖;2:MiPDCD6酶切后的目的片段。D:pET-32a-MiPDCD6原核表达质粒转化后菌落PCR扩增: M:L 2000 DNA Marker;1~8:pET-32a-MiPDCD6原核表达质粒转化后菌落PCR扩增的目的DNA片段。

    Figure  1.  Construction of expression plasmid pET-32a-MiPDCD6

    A- Agarose gel electrophoresis of MiPDCD6 amplification with digestion primers: M-DL 2000 DNA Marker; 1-5: MiPDCD6 amplification with digestion primers. B- Agarose gel electrophoresis of colony PCR with enzyme digestion site primers: M-DL 2000 DNA Marker; 1-8: positive colony samples. C- Agarose gel electrophoresis of pET-32a vector, and MiPDCD6 digestion: M-DL 2000 DNA Marker; 1-pET-32a vector digestion; 2- MiPDCD6 digested fragment. D- Agarose gel electrophoresis of pET-32a-MiPDCD6 colony PCR amplification: M-DL 2000 DNA Marker; 1-8: positive colony PCR of pET-32a-MiPDCD6 prokaryotic expression plasmids

    图  2  MiPDCD6基因的原核表达蛋白与特异性鉴定

    A:MiPDCD6原核表达蛋白的SDS-PAGE电泳图;M:Protein Marker;1:pET-32a(+)空载体的原核表达蛋白;2~4:MiPDCD6的原核表达蛋白。B:MiPDCD6原核表达蛋白的Western blot显影图;1~2:MiPDCD6原核表达蛋白的Western bolt杂交条带。

    Figure  2.  Expression and confirmation of MiPDCD6

    A: Expression of MiPDCD6 detected by SDS-PAGE; M: protein marker; 1: pET-32a (+) prokaryotic expression protein of empty vector; 2–4: expression protein of pET-32a -MiPDCD6. B: Expression of MiPDCD6 confirmed by Western blot; 1–2: western blot hybridization bands of MiPDCD6 prokaryotic expression protein.

    图  3  MiPDCD6融合蛋白兔抗血清的制备

    A-MiPDCD6融合蛋白纯化后的SDS-PAGE电泳图:M-Protein Marker;1-MiPDCD6融合蛋白上清样品;2-上样后流出液;3-洗杂液;4-洗脱液。B- 3529、3530号兔子血清效价,纵坐标为OD450

    Figure  3.  Preparation of rabbit antiserum of MiPDCD6 fusion protein

    A: SDS-PAGE electrophoresis of MiPDCD6 fusion protein purification; M: protein marker; 1: MiPDCD6 fusion protein supernatant sample; 2: effluent after loading; 3: detergent solution; 4: eluent. B: titers of No. 3529 and No. 3530 rabbit serums.

    图  4  抗MiPDCD6兔多克隆抗体的纯化与特异型鉴定

    A- 抗MiPDCD6兔多克隆抗体纯化后的Western blot显影图:1为抗体以1∶1000稀释时的Western blot显影条带;2为抗体以1∶5000稀释时的Western blot显影条带。B- 抗MiPDCD6兔多克隆抗体的效价。C-抗MiPDCD6兔多克隆抗体的Western blot显影图:1为抗体以1∶5000稀释时的Western blot显影条带;2为抗体以1∶10000稀释时的Western blot显影条带。

    Figure  4.  Purity and specificity of anti-MiPDCD6 rabbit polyclonal antibodies

    A: western blot detection of purified anti-MiPDCD6 rabbit polyclonal antibody; 1: western blot bands of anti-MiPDCD6 antibody diluted at 1∶1 000; 2: western blot bands of the anti-MiPDCD6 antibody diluted at 1∶5 000. B: titer of anti-MiPDCD6 rabbit polyclonal antibody. C: western blot detection of anti-MiPDCD6 rabbit polyclonal antibody; 1: western blot bands of antibody diluted at 1∶5 000; 2: western blot bands of antibody diluted at 1∶10 000.

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出版历程
  • 收稿日期:  2022-10-09
  • 修回日期:  2022-12-22
  • 网络出版日期:  2023-03-28
  • 刊出日期:  2023-03-28

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