真菌病毒Beauveria bassiana chrysovirus 2 (BbCV2)

doi:10.16409/j.cnki.2095-039x.2023.01.049

中国生物防治学报 ›› 2024, Vol. 40 ›› Issue (3): 583-591.DOI: 10.16409/j.cnki.2095-039x.2023.01.049

• 研究论文 • 上一篇

真菌病毒Beauveria bassiana chrysovirus 2 (BbCV2)

路杨^1,2, 郭文博^1,2, 隋丽^1,2, 张庆贺³, 赵宇¹, 张正坤¹, 李启云^1,2,4

1. 吉林省农业科学院植物保护研究所/吉林省农业微生物重点实验室/农业农村部东北作物有害生物综合治理重点实验室, 长春 136100;
2. 吉林农业大学植物保护学院, 长春 130118;
3. 吉林省农业环境保护与农村能源管理总站, 长春 130021;
4. 吉林农业科技学院, 吉林 132101

收稿日期:2023-05-04 发布日期:2024-06-07
通讯作者: 张正坤,博士,研究员,E-mail:zhangzhengkun1980@126.com;李启云,博士,研究员,E-mail:qyli1225@126.com。
作者简介:路杨,博士,研究员,E-mail:jluluyang@126.com;郭文博,硕士研究生,E-mail:1004202279@qq.com。

Preparation and Application of Polyclonal Antibodies against the Coat Protein of Fungal Virus Beauveria Bassiana Chrysovirus 2 (BbCV2)

LU Yang^1,2, GUO Wenbo^1,2, SUI Li^1,2, ZHANG Qinghe³, ZHAO Yu¹, ZHANG Zhengkun¹, LI Qiyun^1,2,4

1. Institute of Plant Protection, Jilin Academy of Agricultural Sciences/Key Laboratory of Agricultural Microbiology, Jilin Province/Key Laboratory of Comprehensive Management of Crop Pests, Northeast China, Changchun 136100, China;
2. College of Plant Protection, Jilin Agricultural University, Changchun 130118, China;
3. Jilin Provincial Agricultural Environmental Protection and Rural Energy Management Station, Changchun 130021, China;
4. Jilin Agricultural Science and Technology College, Jilin 132101, China

Received:2023-05-04 Published:2024-06-07
Contact: 吉林省科技厅中青年科技创新创业卓越人才（团队）项目（创新类)(20230508011RC);国家自然科学基金（32271683）
Supported by:
10.16409/j.cnki.2095-039x.2023.01.049

摘要/Abstract

摘要： 球孢白僵菌Beauveria bassiana是一种在害虫生物防治中应用广泛的虫生真菌，真菌病毒能够影响球孢白僵菌的致病力和生物学性状，但真菌病毒相关蛋白的抗体制备和利用其进行病毒检测和定位鲜有报道。本研究原核表达了一株普遍流行并且造成寄主球孢白僵菌毒力下降的双链RNA（double stranded RNA，dsRNA）病毒Beauveria bassiana chrysovirus 2（BbCV2）的外壳蛋白（Coat protein，CP），并制备了多克隆抗体，进行了病毒在寄主球孢白僵菌胞内和胞外的检测，以及利用间接免疫荧光进行病毒在寄主内的定位。结果表明，表达的BbCV2-CP蛋白主要以包涵体的形式存在，相对分子质量约为85.7 kD；经间接ELISA方法测定，制备的BbCV2-CP兔源多抗效价达到1:8192000；Western blot结果显示，制备的BbCV2兔源多抗能与抗原蛋白进行特异性结合，且能检测出感染寄主球孢白僵菌的BbCV2；ELISA测定结果表明病毒BbCV2可在寄主真菌体内复制，并能够游离到寄主体外；经间接免疫荧光观察，发现BbCV2病毒定位于真菌细胞核中。本研究建立了球孢白僵菌真菌病毒的血清学检测体系，为球孢白僵菌-真菌病毒互作机制研究提供材料。

关键词: 球孢白僵菌, 真菌病毒BbCV2, 多克隆抗体, 免疫荧光, 亚细胞定位

Abstract: Beauveria bassiana is a widely used entomogenous fungus in pest biocontrol. Mycoviruses can affect the pathogenicity and biological characteristics of B. bassiana. However, there are few reports on the preparation of antibodies against fungal virus-related proteins and their use for virus detection and localization. In the present study, the coat protein (CP) of a double stranded RNA (dsRNA) virus, Beauveria bassiana chrysovirus 2 (BbCV2), which is epidemic and hypovirulent to host fungi B. bassiana, was expressed prokaryoticly. And polyclonal antibodies were prepared for the detection of the virus in and out of the host fungi body and location the virus in the host fungi spores using indirect immunofluorescence. The results showed that the expressed BbCV2-CP protein mainly existed in the form of inclusion body, with a relative molecular weight of approximately 85.7 kD. By indirect ELISA method, the prepared BbCV2-CP polyclonal antibody had a titer of 1: 8192000. The results of Western blot showed that the prepared BbCV2-CP polyclonal antibody specifically bound to the antigen protein and recognized BbCV2 infected B. bassiana strains; The ELISA assay results showed that the virus BbCV2 replicated in the host fungus and was able to dissociate outside the host. According to the indirect immunofluorescence observation, the BbCV2 virus localized in the fungal nucleus. This study has established a serological detection system for the mycovirus infecting B. bassiana, providing materials for the study of the interaction between B. bassiana and mycovirus.

Key words: Beauveria bassiana, mycovirus BbCV2, polyclonal antibody, immunofluorescence, subcellular localization

中图分类号:

S476.1

路杨, 郭文博, 隋丽, 张庆贺, 赵宇, 张正坤, 李启云. 真菌病毒Beauveria bassiana chrysovirus 2 (BbCV2)[J]. 中国生物防治学报, 2024, 40(3): 583-591.

LU Yang, GUO Wenbo, SUI Li, ZHANG Qinghe, ZHAO Yu, ZHANG Zhengkun, LI Qiyun. Preparation and Application of Polyclonal Antibodies against the Coat Protein of Fungal Virus Beauveria Bassiana Chrysovirus 2 (BbCV2)[J]. Chinese Journal of Biological Control, 2024, 40(3): 583-591.

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真菌病毒Beauveria bassiana chrysovirus 2 (BbCV2)

Preparation and Application of Polyclonal Antibodies against the Coat Protein of Fungal Virus Beauveria Bassiana Chrysovirus 2 (BbCV2)

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