辣椒脉黄病毒P4蛋白多克隆抗体制备与应用

卜姗; 罗香文; 张德咏; 张松柏; 张宇; 刘勇

doi:10.19303/j.issn.1008-0384.2022.01.010

辣椒脉黄病毒P4蛋白多克隆抗体制备与应用

doi: 10.19303/j.issn.1008-0384.2022.01.010

卜姗^1,,
罗香文²,
张德咏^{1, 2},
张松柏^{1, 2},
张宇²,
刘勇^{1, 2, ,}

1.
湖南大学研究生院隆平分院，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

基金项目: 国家自然科学基金（31772133）；国家现代农业产业技术体系建设专项（CARS-23-D-02）；湖南省农业科学院创新项目（2021CX89）

详细信息

作者简介:
卜姗（1998−），女，硕士研究生，主要从事植物病毒致病分子机制研究（E-mail：bushan@hnu.edu.cn）

通讯作者:
刘勇（1966−），男，博士，研究员, 主要从事蔬菜病害致害机制及绿色综合防控技术研究（E-mail: haoasliu@hunaas.cn）

中图分类号: S 436
计量
- 文章访问数: 563
- HTML全文浏览量: 209
- PDF下载量: 46
- 被引次数: 0
出版历程
- 收稿日期: 2021-08-31
- 修回日期: 2021-12-06
- 网络出版日期: 2022-01-21
- 刊出日期: 2022-01-28

Preparation and Application of Polyclonal Antibody against Vein Yellows Virus P4 on Chili Pepper Plants

BU Shan^1
,,
LUO Xiangwen²,
ZHANG Deyong^{1, 2},
ZHANG Songbai^{1, 2},
ZHANG Yu²,
LIU Yong^{1, 2
, ,}

1.
Longping Branch, Graduate School, Hunan University, Changsha, Hunan　410125, China
2.
Plant Protection Institute, Hunan Academy of Agricultural Sciences, Changsha, Hunan　410125, China

摘要

摘要: 目的辣椒脉黄病毒（Pepper vein yellows virus， PeVYV）属马铃薯卷叶病毒属（Polerovirus），生产上主要侵染辣椒，目前在我国呈现快速扩展态势，因此，亟需开展PeVYV致病性研究，为分析对辣椒的潜在威胁提供依据。以PeVYV编码运动蛋白P4为免疫源，制备多克隆抗体，建立PeVYV P4蛋白特异性检测方法，为PeVYV P4蛋白功能研究奠定基础。方法采用RT-PCR技术，从感染PeVYV辣椒cDNA中扩增获得片段大小为471 bp的PeVYV P4基因，并克隆到原核表达载体pDEST17，转化E. coli Rosetta中进行诱导表达，采用Ni-NTA柱层析纯化。以纯化P4蛋白作为抗原免疫新西兰白兔制备多克隆抗体。制备的多克隆抗体采用Western blotting检测。结果原核表达的PeVYV P4蛋白，SDS-PAGE表明纯化蛋白为一分子量约为25 kDa的单一条带。Western blotting检测表明，制备的多克隆抗体，特异性的结合P4蛋白。PeVYV侵染辣椒样本检测表明，制备的P4多克隆抗体能特异性检测PeVYV。结论制备的PeVYV P4多克隆抗体，可用于特异性检测PeVYV P4蛋白，为进一步深入研究P4蛋白的功能奠定基础。
- 辣椒脉黄病毒 /
- P4基因 /
- 原核表达 /
- 蛋白纯化 /
- 多克隆抗体
Abstract: Objective Pathogenicity of the pepper vein yellows virus (PeVYV) of genus Polerovirus that caused epidemic infection on chili peppers, Capsicum annuum L., in China was studied to assess the potential threat to the crop, and the polyclonal antibody against the virus prepared. Methods Polyclonal antibody of PeVYV was prepared using the purified recombinant P4 protein. A detection method based on the antibody was established to determine the functions and characteristics of P4. The 471 bp of P4 gene was amplified by RT-PCR using the total RNA of infected chili peppers and cloned into prokaryotic expressing plasmid pDEST17 expressed in E. coli Rosetta by arabinose induction. The recombinant P4 was purified by Ni-NTA chromatography for the antibody preparation and verified by western blotting. Results The recombinant P4 protein was soluble with a molecular weight of approximately 25 kDa. The prepared polyclonal antibody was confirmed by western blot to specifically recognize the protein. Conclusions The polyclonal antibody prepared in this study specifically recognized P4. It could be used to characterize and determine the functions of the protein.
- Pepper vein yellows virus /
- P4 gene /
- prokaryotic expression /
- protein purification /
- polyclonal antibody

HTML全文

图 1 PeVYV P4基因RT-PCR克隆及原核表达载体构建PCR验证

注：A, PeVYV P4基因PCR扩增产物；M, DNA分子量标准DL2000；1, PeVYV-P4基因PCR扩增产物； 2,阴性对照。B, 重组pDEST17-P4菌落PCR产物；1, 重组载体pDEST17-P4，2, 阴性对照。

Figure 1. Electrophoresis of PeVYV P4 cloned by RT-PCR and using pDEST17-P4

Note: A, PCR products of PeVYV P4; M, DNA ladder DL2000; 1, PCR products of PeVYV P4; 2, CK. B, Colony PCR of recombinant plasmid pDEST17-P4; 1, Recombinant plasmid pDEST17-P4; 2, CK.

下载: 全尺寸图片幻灯片

图 2 P4蛋白诱导表达纯化产物SDS-PAGE分析

注：M，蛋白分子量标准；1，纯化P4重组蛋白；2，诱导总蛋白。

Figure 2. SDS-PAGE analysis on purified P4 protein-induced expression products

Note: M, Protein ladder; 1, Purified P4 recombinant protein; 2, Entire induced protein.

下载: 全尺寸图片幻灯片

图 3 PeVYV P4蛋白多克隆抗体灵敏度检测

Figure 3. Sensitivity of PeVYV P4 polyclonal antibody

下载: 全尺寸图片幻灯片

图 4 P4蛋白多克隆抗体特异性检测

注：A，P4蛋白特异性检测；1，健康辣椒；2，PeVYV侵染辣椒；3，纯化的P4重组蛋白。B，P4蛋白检测方法优化；1，P4蛋白多克隆抗体稀释100倍；2，P4蛋白多克隆抗体稀释200倍；3，空白对照。

Figure 4. Specificity of PeVYV P4 polyclonal antibody

Note: A, Specificity of P4 polyclonal antibody; 1, Healthy chili pepper; 2, PeVYV-infected chili pepper; 3, Purified recombinant P4 protein. B, Optimization of P4 polyclonal antibody; 1, 100x dilution; 2, 200x dilution; 3, Blank control.

下载: 全尺寸图片幻灯片

参考文献(20)

[1]	PANNO S, BIONDO C, PIAZZA V, et al. First outbreak of Pepper vein yellows virus infecting sweet pepper in Italy [J]. New Disease Reports, 2016, 34: 3. doi: 10.5197/j.2044-0588.2016.034.003
[2]	VILLANUEVA F, CASTILLO P, FONT M I, et al. First report of Pepper vein yellows virus infecting sweet pepper in Spain [J]. Plant Disease, 2013, 97(9): 1261.
[3]	ALFARO-FERNÁNDEZ A, ELSHAFIE E E, ALI M A, et al. First report of Pepper vein yellows virus infecting hot pepper in Sudan [J]. Plant Disease, 2014, 98(10): 1446.
[4]	YONAHA T, TOYOSATO T, KAWANO S, et al. Pepper vein yellows virus, a novel luteovirus from bell pepper plants in Japan [J]. Japanese Journal of Phytopathology, 1995, 61(3): 178−184. doi: 10.3186/jjphytopath.61.178
[5]	ZHANG S B, ZHAO Z B, ZHANG D Y, et al. First report of Pepper vein yellows virus infecting red pepper in mainland China [J]. Plant Disease, 2015, 99(8): 1190.
[6]	汤亚飞, 何自福, 佘小漫, 等. 辣椒黄脉病毒RT-LAMP快速检测方法的建立 [J]. 植物保护, 2016, 42(6):100−104. TANG Y F, HE Z F, SHE X M, et al. Developmet of RT-LAMP for rapid detection of Pepper vein yellows virus [J]. Plant Protection, 2016, 42(6): 100−104.(in Chinese)
[7]	谭伟萍. 山东省辣椒病毒病病原分子鉴定及辣椒脉黄化病毒全基因组序列分析[D]. 济南: 山东农业大学, 2016. TAN W P. Molecular identification of the pathogen of pepper virus disease in Shandong and complete sequences analysis of Pepper vein yellows virus [D]. Jinan: Shandong Agricultural University, 2016. (in Chinese)
[8]	冯耿, 辛敏, 曹孟籍, 等. 深度测序发现贵阳发生的辣椒病毒病由多种病毒复合侵染所致 [J]. 植物病理学报, 2017, 47(5):591−597. FENG G, XIN M, CAO M J, et al. Identification of multiple viruses infecting hot pepper in Guiyang by deep sequencing [J]. Acta Phytopathologica Sinica, 2017, 47(5): 591−597.(in Chinese)
[9]	刘勇, 李凡, 李月月, 等. 侵染我国主要蔬菜作物的病毒种类、分布与发生趋势 [J]. 中国农业科学, 2019, 52(2):239−261. doi: 10.3864/j.issn.0578-1752.2019.02.005 LIU Y, LI F, LI Y Y, et al. Identification, distribution and occurrence of viruses in the main vegetables of China [J]. Scientia Agricultura Sinica, 2019, 52(2): 239−261.(in Chinese) doi: 10.3864/j.issn.0578-1752.2019.02.005
[10]	王莉爽, 李淳, 杨学辉, 等. 贵州辣椒叶脉黄化病毒分离物检测及其P0、CP、MP 基因进化分析 [J]. 园艺学报, 2020, 47(12):2415−2426. WANG L S, LI C, YANG X H, et al. Identification of Pepper vein yellows virus and evolution analysis of P0, CP and MP genes [J]. Acta Horticulturae Sinica, 2020, 47(12): 2415−2426.(in Chinese)
[11]	李桑桑. 辣椒脉黄病毒P4蛋白介导病毒胞间运动功能研究[D]. 长沙: 湖南大学, 2020. LI S S. P4 protein encoded by Pepper vein yellows virus facilitates virus cell-to-cell movement[D]. Changsha: Hunan University, 2020. (in Chinese)
[12]	PEREHINEC T M, QAZI S N A, GADDIPATI S R, et al. Construction and evaluation of multisite recombinatorial (Gateway) cloning vectors for Gram-positive bacteria [J]. BMC Molecular Biology, 2007, 8: 80. doi: 10.1186/1471-2199-8-80
[13]	赵忠豪, 潘慧, 刘勇, 等. SRBSDV P8 蛋白的多克隆抗体制备及其应用 [J]. 杂交水稻, 2020, 35(3):71−75. ZHAO Z H, PAN H, LIU Y, et al. Preparation and application of polyclonal antibodies against the small core protein P8 encoded by SRBSDV [J]. Hybrid Rice, 2020, 35(3): 71−75.(in Chinese)
[14]	MARZOCCHI E, GRILLIA S, CIANA L D. Chemiluminescent detection systems of horseradish peroxidase employing nucleophilic acylation catalysts [J]. Analytical Biochemistry, 2008, 377(2): 189−194. doi: 10.1016/j.ab.2008.03.020
[15]	PENG J J, BU S, YIN Y Y, et al. Biological and genetic characterization of Pod pepper vein yellows virus-associated RNA from Capsicum frutescens in Wenshan, China [J]. Frontiers in Microbiology, 2021, 12: 662352. doi: 10.3389/fmicb.2021.662352
[16]	WANG L S, TIAN P J, YANG X L, et al. Key amino acids for Pepper vein yellows virus P0 protein pathogenicity, gene silencing, and subcellular localization [J]. Frontiers in Microbiology, 2021, 12: 680658. doi: 10.3389/fmicb.2021.680658
[17]	LI S S, SU X Y, LUO X W, et al. First evidence showing that Pepper vein yellows virus P4 protein is a movement protein [J]. BMC Microbiology, 2020, 20(1): 72. doi: 10.1186/s12866-020-01758-y
[18]	MOSHE A, GOROVITS R. Virus-induced aggregates in infected cells [J]. Viruses, 2012, 4(10): 2218−2232. doi: 10.3390/v4102218
[19]	KAIDO M, ABE K, MINE A, et al. GAPDH: a recruits a plant virus movement protein to cortical virus replication complexes to facilitate viral cell-to-cell movement [J]. PLoS Pathogens, 2014, 10(11): e1004505. doi: 10.1371/journal.ppat.1004505
[20]	贺振, 甘海锋, 陈雯, 等. 雀麦花叶病毒外壳蛋白基因原核表达及抗血清制备 [J]. 植物保护学报, 2020, 47(2):455−456. HE Z, GAN H F, CHEN W, et al. Prokaryotic expression of Brome mosaic virus coat protein gene and preparation of antiserum [J]. Journal of Plant Protection, 2020, 47(2): 455−456.(in Chinese)