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<sup>*</sup>

doi:10.13523/j.cb.2202053

中国生物工程杂志

2022, Vol. 42

Issue (7): 54-61 DOI: 10.13523/j.cb.2202053

技术与方法

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^*

汪琨,赵福运,徐云飞,袁小凤,赵伟春^**(

)

浙江中医药大学生命科学学院杭州 310053

Preparation of Monoclonal Antibody Against Fusarium solani and Development of Its Colloidal Gold Immunochromatographic Strip

Kun WANG,Fu-yun ZHAO,Yun-fei XU,Xiao-feng YUAN,Wei-chun ZHAO^**(

)

School of Life Sciences, Zhejiang Chinese Medical University, Hangzhou 310053, China

全文: PDF(2834 KB) HTML

摘要： 目的利用浙贝母(Fritillaria thunbergii)根腐病病原菌之一的茄病镰刀菌(Fusarium solani)为抗原制备分泌特异性单克隆抗体(单抗)的细胞株,开发基于单抗的胶体金免疫层析试纸条,为检测浙贝母根腐病提供技术支撑。方法以茄病镰刀菌全菌体蛋白质提取液为抗原,利用杂交瘤技术制备能分泌针对茄病镰刀菌特异性单抗的细胞株;间接ELISA和Western blot检测单抗的特异性、效价、灵敏度、结合蛋白等特性;采用柠檬酸三钠还原法制备胶体金颗粒并标记单抗,制备胶体金免疫层析试纸条。结果获得3株能分泌针对茄病镰刀菌的特异性单抗的细胞株,分别命名为FsA3、FsG6、FsD4。其中FsA3检测灵敏度为24.41 ng/mL,FsG6、FsD4检测灵敏度均为12.21 ng/mL。FsA3、FsG6、FsD4均针对茄病镰刀菌强烈反应,且不与细极链格孢(Alternaria tenuissima)、交链格孢(A. alternata)、灰葡萄孢(Botrytis cinerea)、木贼镰刀菌(F. equiseti)、变红镰刀菌(F. incarnatum)、尖孢镰刀菌(F. oxysporum)、茎点霉属真菌(Phoma sp.)、Phomopsis oblonga等抗原发生交叉反应。基于FsG6的胶体金免疫层析试纸条检测浙贝母无菌组培苗提取液时只出现质控线,加入100 ng茄病镰刀菌抗原或浙贝母根腐病病株样品时,均出现肉眼可见的质控线和检测线。结论制备的单抗和试纸条具有简单、快速、特异性和灵敏度高的优点,可用于检测浙贝母病株中分离到的茄病镰刀菌,这为浙贝母根腐病田间快速准确检测提供了技术支撑。

关键词： 浙贝母; 根腐病; 茄病镰刀菌; 单克隆抗体; 胶体金免疫层析试纸条

Abstract:

Objective: The cell lines secreting specific monoclonal antibodies (McAbs) were prepared by using Fusarium solani, one of the pathogenic fungi causing root rot of Fritillaria thunbergii, and the colloidal gold immunochromatographic test strip based on McAbs was developed to provide scientific basis for detecting root rot of F. thunbergii. Methods: Hybridoma technology was used to obtain cell lines that could secrete specific McAbs against F. solani using the whole protein extract of F. solani as the antigen. The specificity, titer, sensitivity and binding protein of McAbs were detected by indirect ELISA and Western blot. Colloidal gold particles were prepared by trisodium citrate reduction method and McAbs were labeled to prepare colloidal gold immunochromatographic strip. Results: Three cell lines secreting specific McAbs against F. solani were obtained, which were named as FsA3, FsG6 and FsD4. The detection sensitivity of FsA3 was 24.41 ng / mL, and that of both FsG6 and FsD4 was 12.21 ng / mL. FsA3, FsG6 and FsD4 had strong reactions to F. solani, and had no cross-reaction to Alternaria tenuissima, A. alternata, Botrytis cinerea, F. equiseti, F. incarnatum, F. oxysporum, Phoma sp., and Phomopsis oblonga. The colloidal gold immunochromatographic strip based on FsG6 showed only a quality control line when detecting the tissue culture seedlings of F. thunbergii. When 100 ng F. solani antigen or the samples of F. thunbergii infested with root rot disease were detected, there were visible quality control lines and test lines. Conclusion: The specificity and sensitivity of the McAbs and test strip are sufficient to detect F. solani isolated from diseased strains of F. thunbergii, which provides the technical support for the rapid detection of root rot of F. thunbergii in the field.

Key words: Fritillaria thunbergia Root rot Fusarium solani Monoclonal antibody Gold immunochromatographic strip

收稿日期: 2022-02-28 出版日期: 2022-08-03

ZTFLH:

Q816

基金资助: *浙江省基础公益研究计划(LGN21C140004);浙江省基础公益研究计划(LGN21H280002);浙江省大学生创新创业训练计划(S202110344082)

通讯作者: 赵伟春 E-mail: weichunzhao@zcmu.edu.cn

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引用本文:

汪琨,赵福运,徐云飞,袁小凤,赵伟春. 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, 2022, 42(7): 54-61.

Kun WANG,Fu-yun ZHAO,Yun-fei XU,Xiao-feng YUAN,Wei-chun ZHAO. Preparation of Monoclonal Antibody Against Fusarium solani and Development of Its Colloidal Gold Immunochromatographic Strip. China Biotechnology, 2022, 42(7): 54-61.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2202053 或 https://manu60.magtech.com.cn/biotech/CN/Y2022/V42/I7/54

图1 单抗效价检测

图2 单抗灵敏度检测

图3 单抗的特异性检测

图4 抗体的类型及亚类鉴定

图5 单抗结合蛋白的Western blot检测

图6 浙贝母对茄病镰刀菌单抗检测灵敏度的影响

图7 样品处理方法对单抗FsG6检测灵敏度的影响

图8 基于FsG6的胶体金免疫层析试纸条对茄病镰刀菌抗原的检测结果

图9 基于FsG6的胶体金免疫层析试纸条的田间试用

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