|
|
|
| Preparation of Monoclonal Antibodies Against Diaporthe eres Fungus and Development of Detection Kit |
| LIU Qiang, HANG Qun-Ying, HU Guang-Jin, WEN Si-Si, ZHAO Wei-Chun* |
| School of Life Sciences, Zhejiang Chinese Medical University, Hangzhou 310053, China |
|
|
|
|
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.
|
|
Received: 31 January 2024
|
|
|
|
Corresponding Authors:
*weichunzhao@zcmu.edu.cn
|
|
|
|
[1] 李吉二, 温思思, 张羽加, 等. 2022. 浙贝母灰霉病病原真菌的分子鉴定[J]. 植物保护, 48(02): 151-156.
(Li J E, Wen S S, Zhang Y Jet al.2022. Identification of pathogenic fungi species of gray mold disease of Fritillaria thunbergii[J]. Plant Protection, 48(02): 151-156.)
[2] 梁新苗, 边勇, 李建光, 等. 2013. 4种葫芦科植物病毒复合胶体金免疫层析试纸条的研制[J]. 植物检疫, 27(01): 39-42.
(Liang X M, Bian Y, Li J G, et al.2013. Gold immunochromatography assay for rapid detection of four cucurbitaceous viruses[J]. Plant Quarantine, 27(01): 39-42.)
[3] 卢诗雨, 温思思, 杭群莺, 等. 2023. 浙贝母叶斑病病原真菌的分子鉴定及杀菌剂筛选[J]. 浙江中医药大学学报, 47(6): 836-844.
(Lu S Y, Wen S S, Hang Q Y, et al.2023. Molecular identification and indoor fungicide screening of pathogenic fungi of leaf spot disease of Fritillaria thunbergii Miq[J]. Journal of Zhejiang Chinese Medical University, 47(6): 836-844.)
[4] 牛晓庆, 余凤玉, 符海泉, 等. 2015. 椰子茎泻血病菌人工接种方法的研究及抗病品种的筛选[J]. 中国南方果树, 44(01): 44-46.
(Niu X Q, Yu F Y, Fu H Q, et al.2015. Study on the method of artificial inoculation of stem bleeding bacteria from coconut stem and selection of resistant varieties[J]. South China Fruits, 44(01): 44-46.)
[5] 汪琨, 赵福运, 徐云飞, 等. 2022. 茄病镰刀菌单克隆抗体的制备及胶体金免疫层析试纸条的研发[J]. 中国生物工程杂志, 42(07): 54-61.
(Wang K, Zhao F Y, Xu Y F, et al.2022. Preparation of monoclonal antibody against Fusarium aolani and development of its colloidal gold immunochromatographic strip[J]. China Biotechnology, 32(01): 61-73.)
[6] 温思思, 赵福运, 郭飘逸, 等. 2022. 引起浙贝母黑斑病的交链格孢单抗及检测试纸条的制备[J]. 植物保护, 48(05): 227-233.
(Wen S S, Zhao F Y, Guo P M, et al.2022. Preparation of monoclonal antibody and test strips for Alternaria alternate causing black spot disease of Fritillaria thunbeigii[J]. Plant Protection, 48(05): 227-233.)
[7] 徐朗希, 范琳资, 姜爽, 等. 2022. 贝母属植物的化学成分和药理作用研究进展[J]. 中国药物化学杂志, 32(01): 61-73.
(Xu L X, Fan L Z, Jiang S, et al.2022. Recent progress of chemical constituents and pharmacological effects of Frotillaria[J]. Chinese Journal of Medicinal Chemistry, 32(01): 61-73.)
[8] 张叶, 邢跃先, 王梓钰, 等. 2021. 玉米镰孢穗腐病菌接种方法的研究[J]. 东北农业科学, 46(03): 64-69.
(Zhang Y, Xing Y X, Wang Z Y, et al.2021. Study on inoculation methods of ear rot caused by Fusarium verticilloides and Fusarium graminearum in maize[J]. Journal of Northeast Agricultural Sciences, 46(03): 64-69.)
[9] 张子君, 孙亚男, 王涛, 等. 2021. 番茄灰叶斑病人工接种技术研究及抗病种质资源筛选[J]. 园艺与种苗, 41(11): 8-10.
(Zhang Z J, Sun Y N, Wang T, et al.2021. Artificial inoculation of grey leaf spot on tomato and screening of resistant sources of tomato[J]. Horticulture & Seed, 41(11): 8-10.)
[10] 朱静坚. 2018. 浙贝母种植模式调查与探讨[J]. 浙江农业科学, 59(03): 380-381+387.(Zhu J J. 2018. Investigation and discussion on planting pattern of Fritillaria thunbergii[J]. Journal of Zhejiang Agricultural Sciences, 59(03): 380-381+387.)
[11] Holzlöhner P, Hanack K.2017. Generation of murine monoclonal antibodies by hybridoma technology[J]. Journal of Visualized Experiments, 119: 54832. |
|
|
|
