球孢白僵菌真菌病毒BbPmV-4外壳蛋白多克隆抗体制备及应用  

Preparation and Application of Polyclonal Antibodies Against Beauveria bassiana Mycovirus BbPmV-4 Coat Protein

作　　者：郭文博 路杨[1,2] 隋丽 赵宇[1] 邹晓威[1] 张正坤[1,2] 李启云[1,2,3] GUO Wen-bo;LU Yang;SUI Li;ZHAO Yu;ZOU Xiao-wei;ZHANG Zheng-kun;LI Qi-yun(Institute of Plant Protection,Jilin Academy of Agricultural Science,Jilin Key Laboratory of Agricultural Microbiology,Key Laboratory of Integrated Pest Management on Crops in Northeast China,Ministry of Agriculture and Rural Affairs,Changchun 130033;College of Plant Protection,Jilin Agricultural University,Changchun 130118;Jilin Agricultural Science and Technology College,Jilin 132101)

机构地区：[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130033 [2]吉林农业大学植物保护学院,长春130118 [3]吉林农业科技学院,吉林132101

出　　处：《生物技术通报》2023年第10期58-67,共10页Biotechnology Bulletin

基　　金：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);国家自然科学基金项目(32271683)。

摘　　要：真菌病毒Beauveria bassiana polymycovirus 4(BbPmV-4)的感染能够提高寄主球孢白僵菌的致病力,然而其在寄主中的复制、传播和与寄主互作机理尚不清楚。本研究构建了真菌病毒BbPmV-4外壳蛋白(coat protein,CP)的原核表达载体pET-28a(+)∷BbPmV-4-CP,转化大肠杆菌Escherichia coli BL21(DE3),进行蛋白原核表达,免疫日本大耳兔制备了多克隆抗体,并利用间接ELISA,Western blot和免疫荧光等血清学方法进行病毒在寄主胞内定位和胞外复制的检测。结果表明,所表达BbPmV-4-CP为一种可溶性蛋白,相对分子质量约为28.56 kD;所制备的BbPmV-4-CP兔源多克隆抗体效价为1∶256000;Western blot检测验证了所制备抗体能够识别对应抗原蛋白及寄主球孢白僵菌中的病毒BbPmV-4;利用所获取的多抗BbPmV-4-CP对含病毒BbPmV-4的球孢白僵菌培养上清液及沉淀中的病毒含量进行间接ELISA检测,结果显示,培养上清液中存在病毒,表明真菌病毒BbPmV-4可在寄主球孢白僵菌体内复制并游离到寄主体外;免疫荧光检测结果显示该病毒定位于真菌细胞核上。本研究制备了高效价和特异性的真菌病毒多克隆抗体,建立了球孢白僵菌真菌病毒的血清学检测技术体系,为研究真菌病毒在寄主体内的复制及其与寄主真菌的互作机理研究提供实验材料,并且可以利用病毒感染及其互作基因调控寄主毒力,创制高毒力球孢白僵菌菌株。Beauveria bassiana polymycovirus 4(BbPmV-4)increases the virulence of host fungus Beauveria bassiana;however,the mechanism of its replication,transmission and interaction with host fungi is still unclear.In the present study,the prokaryotic expression vector of mycovirus BbPmV-4 coat protein(CP)was constructed,designated as pET-28a(+)∷BbPmV-4-CP,which was transformed into Escherichia coli BL21(DE3)for prokaryotic expression.The Japanese big ear rabbits were immunized to prepare polyclonal antibodies,which were used to detect the intracellular localization and extracellular replication of the virus by immunological methods including indirect ELISA,Western blot and immunofluorescence,respectively.The results showed that the expressed BbPmV-4-CP was a soluble protein with a relative molecular weight of approximately 28.56 kD,the titer of prepared rabbit polyclonal antibody against which was 1∶256 000.Western blot analysis confirmed that the prepared antibody recognized the corresponding antigen protein and the virus BbPmV-4 in the host strain of B.bassiana.The mycelia of nontoxic healthy strains and B.bassiana containing virus BbPmV-4 were collected for liquid culture,and the virus content in the supernatant and precipitation of the culture was detected by indirect ELISA using the prepared antibody.The results showed that there was virus in the supernatant,indicating that mycovirus BbPmV-4 replicated in the host fungal cells and dissociated outside.The indirect immunofluorescence detection results revealed that the virus was located on the fungal nucleus.In this study,highly effective and specific polyclonal antibodies against mycovirus coat protein are prepared,and a serological detection system for mycovirus is established,which may provide experimental materials for studying the replication of mycovirus in host fungi and the mechanism of its interaction with host fungi,and hypervirulent mycovirus infection and its interactive genes may be used to regulate host virulence and create high virulence strains of B.

关 键 词：球孢白僵菌 真菌病毒 外壳蛋白 多克隆抗体 定位 免疫荧光 血清学检测 

分 类 号：S476.12[农业科学—农业昆虫与害虫防治]