西瓜花叶病毒（WMV）单克隆抗体的制备及其应用

doi:10.3864/j.issn.0578-1752.2016.14.006

摘要/Abstract

摘要： 【目的】制备抗西瓜花叶病毒（Watermelon mosaic virus, WMV）的特异性单克隆抗体，并以其为核心建立能快速有效地检测WMV的血清学方法，从而为中国田间西瓜花叶病毒病的诊断和检测、预测预警及科学防控体系的建立提供物质和技术支撑。【方法】用提纯的WMV病毒粒子免疫BALB/c小鼠，经细胞融合和细胞培养、抗体筛选和细胞克隆等杂交瘤细胞技术，获得能稳定分泌抗WMV单克隆抗体的杂交瘤细胞株，将杂交瘤细胞注射入BALB/c小鼠腹腔制备其单抗腹水，并以制备的单抗为核心建立能准确、特异、灵敏地检测田间植物中WMV的ACP-ELISA、DAS-ELISA、dot-ELISA、Tissue blot-ELISA和IC-RT-PCR方法，以及能检测单头传毒介体蚜虫体内WMV的dot-ELISA方法。【结果】3株能稳定分泌WMV单克隆抗体的杂交瘤细胞株（2C8、15A8和16C12）及其单抗腹水被制备，3株杂交瘤细胞分泌的单抗腹水的间接ELISA效价均达到了10-6以上，抗体类型及亚类均为IgG1、kappa轻链。Western blot分析发现，这3个单抗均与WMV的外壳蛋白亚基有特异性反应。灵敏度分析结果表明，ACP-ELISA、DAS-ELISA、dot-ELISA和IC-RT-PCR方法检测WMV病叶的灵敏度分别达到1﹕163 840、1﹕327 680、1﹕5 120和1﹕1 310 720倍稀释（w/v，g/mL）。特异性分析结果表明，以这3个单抗为核心建立的ACP-ELISA、DAS-ELISA、dot-ELISA、Tissue blot-ELISA和IC-RT-PCR 5种血清学检测方法均能与感染WMV的病叶发生特异性免疫反应，而与感染小西葫芦黄花叶病毒(Zucchini yellow mosaic virus，ZYMV)、黄瓜花叶病毒(Cucumber mosaic virus，CMV)、黄瓜绿斑驳花叶病毒(Cucumber green mottle mosaic virus，CGMMV)、马铃薯Y病毒(Potato virus Y，PVY)的植物样品、健康白南瓜、西瓜、葫芦和烟草的植物组织均呈阴性反应，且dot-ELISA方法还能特异性地检测单头蚜虫体内的WMV，而在检测无毒蚜虫时呈阴性反应。利用建立的血清学检测方法对采自浙江省、江苏省、山东省和海南省的275株葫芦科疑似病株进行检测，结果发现187株植物感染WMV，发病率达68%，说明WMV在中国田间葫芦科植物中广泛流行发生，且血清学方法的检测结果与RT-PCR方法的检测结果完全一致，将部分PCR产物进行核酸测序和序列比对分析，结果表明这些PCR产物是WMV CP基因片段，证明血清学方法检测阳性的样品确实感染WMV。【结论】获得了3株特异、灵敏的WMV单克隆抗体，以其为核心建立的5种血清学方法能准确、灵敏、可靠地应用于田间样品中WMV的检测，从而为中国WMV田间样品的大规模快速检测和诊断、该病害预报预警和科学防控提供物质和技术支撑。

关键词: 西瓜花叶病毒（WMV）, 单克隆抗体, ACP-ELISA, DAS-ELISA, dot-ELISA, Tissue blot-ELISA, IC-RT-PCR

Abstract: 【Objective】The aim of this study is to prepare monoclonal antibodies (MAbs) against Watermelon mosaic virus (WMV) and develop effective serological assays for rapid and reliable virus detection, and to provide technology and materiel for diagnosis and detection, forecast and early warning and establishment of a scientific prevention and control system of the WMV disease.【Method】Using the purified WMV particles as an immunogen, hybridoma lines secreting MAbs specific for WMV were obtained via cell fusion, cell culture, antibody detection and cell cloning. The hybridomas were injected intraperitoneally into BALB/c mice to produce MAb-containing ascitic fluids. Based on the prepared MAbs, five detection assays, ACP-ELISA, DAS-ELISA, dot-ELISA, Tissue blot-ELISA and IC-RT-PCR were developed for accurately, sensitively and specifically detecting WMV in field plant samples. Besides, a dot-ELISA for specifically detecting WMV in individual viruliferous aphid was established.【Result】Three hybridoma lines (2C8, 15A8 and 16C12) steadily secreting MAbs specific for WMV and their MAb-containing ascitic fluids were produced. The titers of ascitic fluids of MAbs were up to 10-6 by indirect-ELISA. All these MAbs belong to IgG1 isotype, κ light chain. Western blot analyses indicated that all these three MAbs could specifically react with the coat protein of WMV. The ACP-ELISA, DAS-ELISA, dot-ELISA and IC-RT-PCR could detect WMV in infected plant crude extracts diluted up to 1﹕163 840, 1﹕327 680, 1﹕5 120 and 1﹕1 310 720 (w/v, g/mL), respectively. And the specificity analyses demonstrated that the developed ACP-ELISA, DAS-ELISA, dot-ELISA, Tissue blot-ELISA and IC-RT-PCR had strongly positive immune reactions with WMV-infected plant tissues, but had negative reactions with healthy, ZYMV-, CMV-, CGMMV-infected cucurbitaceous plant tissues or PVY-infected tobacco plant tissues. Besides, the developed dot-ELISA for detecting vector sample had a strongly positive immune reaction with individual viruliferous aphid, and negative reactions with non-viruliferous aphids. A total of 275 cucurbitaceous plant samples showing virus-like symptoms from Zhejiang, Jiangsu, Shandong and Hainan provinces in China were screened for the presence of WMV using the developed assays, and the detection results showed that 187 of the 275 plant samples were infected by WMV and the incidence rate was up to 68%, demonstrating that WMV is prevalent in field cucurbitaceous plants in China. And the detection results of serological assays were in agreement with those of RT-PCR. The sequences of PCR-amplified products were sequenced and compared with the WMV CP sequences. The results indicated that the nucleotide sequences of the PCR-amplified products were WMV CP gene segment, demonstrating that the positive samples tested by serological assays were really infected with WMV.【Conclusion】Three specific and sensitive MAbs against WMV and the five developed assays based on prepared MAbs in this study could accurately, sensitively and reliably detect WMV in field plant or vector samples, which would provide technology and materiel for rapid detection and diagnoses of field large-scale samples, forecast and early warning, scientific prevention and control of WMV disease in China.

Key words: Watermelon mosaic virus (WMV), monoclonal antibody, ACP-ELISA, DAS-ELISA, dot-ELISA, Tissue blot- ELISA, IC-RT-PCR

陈浙，宋革，周雪平，吴建祥. 西瓜花叶病毒（WMV）单克隆抗体的制备及其应用[J]. 中国农业科学, 2016, 49(14): 2711-2724.

CHEN Zhe, SONG Ge, ZHOU Xue-ping, WU Jian-xiang. Preparation and Application of Monoclonal Antibodies Against Watermelon mosaic virus (WMV)[J]. Scientia Agricultura Sinica, 2016, 49(14): 2711-2724.

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