间座壳菌属真菌<em>Diaporthe eres</em>的单克隆抗体制备及检测试剂盒研发

doi:10.3969/j.issn.1674-7968.2024.12.021

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摘要浙贝母(Fritillaria thunbergii)为百合科贝母属植物,鳞茎为药用部位,具有清热散结之功效。叶斑病是浙贝母的主要真菌性病害之一,间座壳菌属真菌Diaporthe eres为其病原真菌。目前,植保人员和种植户主要通过经典的症状观察法判断浙贝母叶斑病的发生,无法在其发病初期实现快速准确诊断。本研究旨在制备针对D. eres的单克隆抗体(单抗)及免疫学检测试剂盒,为田间叶斑病的快速准确鉴定提供技术支撑。首先,以D. eres菌丝蛋白提取液免疫Bal b/c小鼠(Mus musculus),然后采用杂交瘤技术得到1种针对D. eres的单抗及其杂交瘤细胞株PoF7。间接ELISA法和Western blot分析表明,该单抗对D. eres具有高度特异性,检测灵敏度为11 ng/mL,效价≥2.56×10⁴,抗体类型为IgM,结合蛋白分子量为107 kD。基于PoF7的ELISA试剂盒的检测灵敏度为44 ng/mL,具有良好的重复性和特异性。该试剂盒使用操作简便,田间浙贝母病株只要经过简单的研磨,即可取汁液进行检测,适用于田间样品的大规模检测。

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	刘强
	杭群莺
	胡光锦
	温思思
	赵伟春

关键词 ：浙贝母, 叶斑病, Diaporthe eres, 单克隆抗体(单抗), ELISA检测试剂盒

Abstract：Fritillaria thunbergii blongs to the genus Fritillaria in the Liliaceae family. The bulb is the main medicinal part, it has the function of clearing heat and dispersing clumps. Leaf spot is one of the main fungal diseases of F. thunbergii, which caused by Diaporthe eres. At present, plant protection personnel and growers mainly rely on classic symptom observation methods to determine the occurrence of leaf spot disease in F. thunbergii, which cannot achieve rapid and accurate diagnosis in its early stages. The purpose of this study was to prepare monoclonal antibodies (McAbs) against D. eres and the immunological detection kits to provide technical support for the rapid and accurate identification of leaf spot in the field. Initially, the protein extract of the hyphae of D. eres was used to immunize Bal b/c mice (Mus musculus). Subsequently, a McAb against D. eres and its hybridoma cell line PoF7 was prepared using hybridoma technology. Indirect ELISA and Western blot analysis showed that the McAb had high specificity against D. eres, with a detection sensitivity of 11 ng/mL. The titer of McAb was ≥ 2.56×10⁴-fold dilution. The antibody type was IgM. It binded to a protein with a molecular weight of 107 kD. The detection sensitivity of the ELISA test kit based on PoF7 was 44 ng/mL, demonstrating good repeatability and specificity. The kit is easy to operate and can be used for large-scale detection of F. thunbergii leaf spot disease in the field after a simple grinding.

Key words： Fritillaria thunbergii Leaf spot disease Diaporthe eres Monoclonal antibody (McAb) ELISA detection kit

收稿日期: 2024-01-31

中图分类号： S41-30

基金资助:浙江省基础公益研究计划(LGN21C140004); 浙江省大学生科技创新项目(2022R410A037); 国家级大学生创新创业训练计划项目(202310344077)

通讯作者: *weichunzhao@zcmu.edu.cn

引用本文:

刘强, 杭群莺, 胡光锦, 温思思, 赵伟春. 间座壳菌属真菌Diaporthe eres的单克隆抗体制备及检测试剂盒研发[J]. 农业生物技术学报, 2024, 32(12): 2926-2934.
LIU Qiang, HANG Qun-Ying, HU Guang-Jin, WEN Si-Si, ZHAO Wei-Chun. Preparation of Monoclonal Antibodies Against Diaporthe eres Fungus and Development of Detection Kit. 农业生物技术学报, 2024, 32(12): 2926-2934.

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https://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2024.12.021 或 https://journal05.magtech.org.cn/Jwk_ny/CN/Y2024/V32/I12/2926

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