辣椒斑驳病毒CP蛋白多克隆抗体制备与应用

王鑫; 叶倩; 吕小园; 田培洁; 张松柏; 张宇; 张德咏; 罗香文

doi:10.19303/j.issn.1008-0384.2022.012.011

辣椒斑驳病毒CP蛋白多克隆抗体制备与应用

doi: 10.19303/j.issn.1008-0384.2022.012.011

王鑫^1,,
叶倩²,
吕小园³,
田培洁²,
张松柏^{1, 2},
张宇²,
张德咏^{1, 2},
罗香文^{1, 2, ,}

1.
湖南大学生物学院隆平分院，湖南　长沙　410125
2.
湖南省农业科学院植物保护研究所，湖南　长沙　410125
3.
长沙海关技术中心，湖南　长沙　410300

基金项目: 国家自然科学基金项目（31972242）；国家现代农业产业技术体系建设专项（CARS-23-D-02）；湖南省自然科学基金项目（2021JJ30415）；湖南省农业科学院创新项目（2021CX83）

详细信息

作者简介:
王鑫（2000−），男，硕士研究生，主要从事植物病毒致病分子机制研究（E-mail：wangxin221@hnu.edu.cn）

通讯作者:
罗香文（1977−），女，博士，副研究员，主要从事蔬菜病害绿色综合防控技术及农田环境治理研究（E-mail：luoxwxihe@126.com）

中图分类号: S 436
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出版历程
- 收稿日期: 2022-09-01
- 修回日期: 2022-09-16
- 网络出版日期: 2022-12-28
- 刊出日期: 2022-03-28

Preparation and Application of Polyclonal Antibodies Recognizing CP Protein in Pepper Mottle Virus

WANG Xin^1
,,
YE Qian²,
LYU Xiaoyuan³,
TIAN Peijie²,
ZHANG Songbai^{1, 2},
ZHANG Yu²,
ZHANG Deyong^{1, 2},
LUO Xiangwen^{1, 2
, ,}

1.
Longping Branch, Biology College, Hunan University, Changsha, Hunan　410125, China
2.
Plant Protection Institute, Hunan Academy of Agricultural Sciences, Changsha, Hunan　410125, China
3.
Technical Center of Changsha Customs, Changsha, Hunan　410300, China

摘要

摘要: 目的辣椒斑驳病毒（Pepper mottle virus，PepMoV）是近年来生产上主要侵染辣椒的新发病毒之一，目前有在我国快速扩展的趋势，因此，亟需开展该病毒的特异性快速检测技术，为明确该病毒在我国辣椒主产区的分布、发生致害规律及机制等研究提供科学手段。本研究以PepMoV编码的外壳蛋白CP为免疫源，制备特异性多克隆抗体，建立PepMoV的特异性快速检测方法，为PepMoV的分布和发生致害规律等研究奠定基础。方法采用特异性RT-PCR技术，从感染PepMoV辣椒的cDNA中扩增获得片段大小为822 bp的CP基因，并克隆到原核表达载体pET28α，转化E coli DH5α中进行诱导表达，采用Ni-NTA柱层析纯化。以纯化的重组CP蛋白作为抗原免疫新西兰白兔制备特异性多克隆抗体。制备的多克隆抗体采用ID-ELISA和Western blotting检测。结果获得原核表达的PepMoV重组CP蛋白，SDS-PAGE结果表明纯化蛋白为分子量约为37 kDa的单一条带。Western blotting和ID-ELISA检测结果表明，制备的多克隆抗体特异性高，仅识别PepMoV CP蛋白，不识别寄主蛋白和其他选择的马铃薯Y病毒；田间样本检测结果表明，PepMoV在湖南和贵州辣椒上的检出率为20.00%和43.33%。结论基于PepMoV CP蛋白特异性多克隆抗体建立的PepMoV快速检测方法，可为进一步深入研究该病毒在我国的分布和发生规律提供科学手段，也为该病毒CP蛋白的功能研究奠定基础。
- 辣椒斑驳病毒 /
- CP基因 /
- 多克隆抗体 /
- 间接ELISA /
- 快速检测
Abstract: Objective A specific, rapid method based on the polyclonal antibody prepared using purified recombinant CP protein for detecting a typical Potyvirus, pepper mottle virus (PepMoV), on Capsicum annuum L. was developed to assess the distribution, occurrence ratio, and pathogenicity of the disease in China. Methods The 822 bp of CP was amplified by specific RT-PCR using the total RNA of PepMoV-infected chili peppers. It was cloned into prokaryotic expressing plasmid pET28α and expressed in E. coli DH5α. The recombinant CP protein was purified by Ni-NTA chromatography and used as the antigen to prepare the polyclonal antibodies to be verified by ID-ELISA and western blotting. Results The purified recombinant CP protein was approximately 37 kDa, and the prepared polyclonal antibody verified to specifically recognize PepMoV CP protein. The ID-ELISA method detected 20.00% PepMoV infection on field chili pepper specimens in Hunan and 43.33% in Guizhou. Conclusion The established specific and rapid detection method based on the polyclonal antibody against CP protein of PepMoV was applied to survey the disease spreading on chili peppers in China. It provided a tool for further studies on the CP protein.
- Pepper mottle virus /
- CP gene /
- polyclonal antibody /
- ID-ELISA /
- Rapid detection

HTML全文

图 1 PepMoV CP基因原核表达载体构建PCR验证

M：DNA分子量标准DL5000；1：PepMoV CP基因原核表达载体PCR扩增产物；2：阴性对照。

Figure 1. Electrophoresis of PCR amplified product of PepMoV CP recombinant prokaryotic expressing plasmid

M: DNA ladder DL5000; 1: PCR products of recombinant prokaryotic expressing plasmid of PepMoV CP; 2: CK.

下载: 全尺寸图片幻灯片

图 2 PepMoV CP蛋白诱导表达纯化产物SDS-PAGE分析

M：蛋白分子量标准；1：未诱导总蛋白；2：诱导总蛋白；3：诱导上清液；4：诱导沉淀；5：纯化CP重组蛋白；6：BSA。

Figure 2. SDS-PAGE on purified PepMoV CP -induced expressing products

M: Protein ladder; 1: non-induced total protein; 2: induced total protein; 3: supernatant of induced total protein; 4: precipitate of induced total protein; 5: purified CP recombinant protein; 6: BSA.

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图 3 PepMoV CP蛋白多克隆抗体灵敏度检测

Figure 3. Sensitivity of PepMoV CP protein polyclonal antibody

下载: 全尺寸图片幻灯片

图 4 PepMoV CP蛋白多克隆抗体特异性检测

A：Western blotting检测CP蛋白特异性检测；1：PVY；2：ChiRSV；3：PepMoV；4：纯化PepMoV CP重组蛋白；5：健康辣椒。B：ID-ELISA检测CP蛋白特异性；1：PVY；2：ChiRSV；3：PepMoV；4：阴性对照。

Figure 4. Specificity of PepMoV CP protein polyclonal antibody

A: Specificity of CP protein polyclonal antibody by western blotting; 1: PVY; 2: ChiRSV; 3: PepMoV; 4: purified recombinant CP protein. B: Specificity of CP protein polyclonal antibody by ID-ELISA; 1: PVY; 2: ChiRSV; 3: PepMoV; 4: negative control.

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表 1 湖南和贵州辣椒PepMoV检出率

Table 1. Rates of positive detection of PepMoV on chili peppers in Hunan and Guizhou

采样地点 Sampling site	样本数 Number of samples	ID-ELISA阳性样本数 Number of positive samples by ID-ELISA	RT-PCR阳性样本数 Number of positive samples by RT-PCR	PepMoV检出率 Ratio of PepMoV/%
湖南 Hunan	20	4	4	20.00
贵州 Guizhou	30	13	13	43.33

下载: 导出CSV

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