南方根结线虫<i>MiPDCD6</i>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

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

华南农业大学植物保护学院/广东省微生物信号与作物病害防控重点实验室，广东　广州　510642

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
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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

Preparation of Polyclonal Antibody against MiPDCD6 Protein in Meloidogyne incognita

Guangdong Province Key Laboratory of Microbial Signals and Disease Control, College of Plant Protection, South China Agricultural University, Guangzhou, Guangdong　510642, China

摘要

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。
- MiPDCD6 /
- 多克隆抗体 /
- 南方根结线虫 /
- 效价
Abstract: Objective The polyclonal antibody against MiPDCD6 protein in the esophageal glands of Meloidogyne incognita was prepared to study the pathogenic mechanism of the root-knot disease on plants transmitted by the nematode. Method The functional fragment of MiPDCD6 was amplified by PCR to construct recombinant plasmid pET-32a-MiPDCD6 and transform it into Escherichia coli BL21 cells for MiPDCD6 fusion protein induction and expression. Polyclonal antibodies were prepared by immunizing male New Zealand white rabbits with purified MiPDCD6 expression protein. Titer and purification of the obtained antibody were verified using ELISA and SDS-PAGE techniques. Result Under IPTG concentration of 1.0 mmol·L⁻¹ at 37℃with constant rotation of 150 r·min⁻¹ for 5 h, MiPDCD6 transformed in the E. coli BL21 was clearly expressed. The secured polyclonal antibody was highly specific with a high titer of approximately 1∶50000 as shown by ELISA and SDS-PAGE. Conclusion The prokaryotic expression conditions of MiPDCD6 were determined. The polyclonal antibody obtained had a high titer and specification against MiPDCD6 and was considered adequate for studying the pathogenesis of the root knot disease on plants infected through M. incognita.
- MiPDCD6 /
- polyclonal antibody /
- Meloidogyne incognita /
- titer

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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

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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.

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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.

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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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