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

doi:10.13560/j.cnki.biotech.bull.1985.2023-0383

生物技术通报 ›› 2023, Vol. 39 ›› Issue (10): 58-67.doi: 10.13560/j.cnki.biotech.bull.1985.2023-0383

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

郭文博¹^,²(), 路杨¹^,², 隋丽¹, 赵宇¹, 邹晓威¹, 张正坤¹^,²(), 李启云¹^,²^,³()

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

收稿日期:2023-04-22 出版日期:2023-10-26 发布日期:2023-11-28
通讯作者: 张正坤，男，博士，研究员，研究方向：生防微生物研究与利用；E-mail: zhangzhengkun1980@126.com；
李启云，男，博士，研究员，研究方向：生防微生物研究与利用; E-mail: qyli1225@126.com
作者简介:郭文博，女，硕士研究生，研究方向：微生物农药研究与利用；E-mail: 1004202279@qq.com；
路杨为本文共同第一作者
基金资助:
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)

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

GUO Wen-bo¹^,²(), LU Yang¹^,², SUI Li¹, ZHAO Yu¹, ZOU Xiao-wei¹, ZHANG Zheng-kun¹^,²(), LI Qi-yun¹^,²^,³()

1. 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
2. College of Plant Protection, Jilin Agricultural University, Changchun 130118
3. Jilin Agricultural Science and Technology College, Jilin 132101

Received:2023-04-22 Published:2023-10-26 Online:2023-11-28

摘要/Abstract

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∶256 000；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。

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

Abstract:

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. bassiana.

Key words: Beauveria bassiana, mycovirus, coat protein, polyclonal antibody, localization, serological detection

郭文博, 路杨, 隋丽, 赵宇, 邹晓威, 张正坤, 李启云. 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[J]. 生物技术通报, 2023, 39(10): 58-67.

GUO Wen-bo, LU Yang, SUI Li, ZHAO Yu, ZOU Xiao-wei, ZHANG Zheng-kun, LI Qi-yun. Preparation and Application of Polyclonal Antibodies Against Beauveria bassiana Mycovirus BbPmV-4 Coat Protein[J]. Biotechnology Bulletin, 2023, 39(10): 58-67.

图/表 8

表1 引物信息

Table 1 Quote sequence information

引物名称 Name	注释 Annotation	序列 Sequence（5'-3'）	长度 Size/bp	退火温度 Annealing temperature/℃
PmV-4-FP	BbPmV-4-CP正向引物，含有Xho I酶切位点及保护碱基	CCGCTCGAGATGTCGCTCCACGATGTCATTTCCA	34	65
PmV-4-RP	BbPmV-4-CP反向引物，含有BamH I酶切位点及保护碱基	CGCGGATCCCTATTTGCCCGCGGCCTCGGTGGCG	34	65

图1 BbPmV-4基因片段的PCR扩增及重组质粒鉴定 A：BbPmV-4基因片段扩增的RT-PCR（M：DL2000；1：RT-PCR）；B：pET-28a-BbPmV-4-CP载体的双酶切鉴定（M：DL10000；1：pET-28a-BbPmV-4-CP载体Xho I和BamH I双酶切产物）

Fig. 1 PCR amplification of BbPmV-4 gene fragment and the identification of the recombinant plasmid A: RT-PCR of BbPmV-4 gene fragment amplification（M: DL2000; 1: RT-PCR）;B: Identification of pET-28a-BbPmV-4-CP vector by double digestion（M: DL10000;1: pET-28a-BbPmV-4-CP vector Xho I and BamH I double digestion products）

图2 目的蛋白BbPmV-4-CP表达鉴定重组蛋白的诱导表达SDS-PAGE鉴定；1：IPTG诱导表达前上清液；2：IPTG诱导表达后上清液；3：IPTG诱导表达前沉淀；4：IPTG诱导表达后沉淀

Fig. 2 Expression and identification of target protein BbPmV-4-CP Identification of induced expressed recombinant protein by SDS-PAGE. 1: Supernatant expressed before IPTG induction. 2: Supernatant expressed after IPTG induction. 3: Precipitation expressed before IPTG induction. 4: Precipitation expressed after IPTG induction

图3 重组蛋白BbPmV-4-CP的纯化与鉴定A：重组蛋白纯化的SDS-PAGE分析（M：蛋白分子质量标准；1：蛋白上样液；2：收集的洗涤液；3-6：10、20、250 mmol/L咪唑洗脱液）；B：重组蛋白BbPmV-4的Western blot检测（1：空白对照；2-3：BbPmV-4-CP）；C：重组蛋白BbPmV-4-CP超滤的SDS-PAGE分析（1：重组蛋白BbPmV-4-CP超滤液）

Fig. 3 Purification and identification of the recombinant protein BbPmV-4-CP A: Purified SDS-PAGE analysis of recombinant protein（M: Protein marker. 1: Protein loading solution. 2: Protein washing solution. 3-6: 10, 20, 250 mmol/L imidazole eluent）. B: Western blot assay of recombinant protein BbPmV-4（1: Bla-nk control. 2-3: BbPmV-4-CP）. C: Ultrafiltration SDS-PAGE of recombinant protein BbPmV-4-CP（Recombinant protein BbPmV-4-CP ultrafiltration solution）

图4 间接ELISA法检测效价

Fig. 4 Titer detected by indirect ELISA

图5 BbPmV-4-CP抗体特异性检测结果1：空白对照；2：阳性对照（BbPmV-4-CP蛋白）；3-5：阴性对照（无病毒菌株BbOFDH1-5）；6-8：携带病毒菌株BbOFJY

Fig. 5 Specificity detection of for the antibody of BbPmV-4-CP 1: Blank control. 2: Positive control（BbPmV-4-CP protein）. 3-5: Negative control（virus-free strain BbOFDH1-5）. 6-8: Virus-harboring strain BbOFJY

图6 间接ELISA检测BbPmV-4病毒含量结果 A：96孔板在菌液中对含BbPmV-4病毒的菌株BbOFJY的相对定量检测（1：空白对照；2：阳性对照BbPmV-4-CP；3：阴性对照-无病毒菌株BbOFDH1-5上清液；4：携带病毒的菌株BbOFJY上清液；5：阴性对照-无病毒菌株BbOFDH bar菌液沉淀；6：携带病毒的菌株BbOFJY菌液沉淀）；B：菌株BbOFJY菌液上清及沉淀中病毒含量检测

Fig. 6 Indirect ELISA detection results of BbPmV-4 virus contents A: Relative quantitative detection of BbPmV-4 virus containing strain BbOFJY in fungal liquid using 96 well plate（1: Blank control. 2: Positive control, BbPmV-4-CP. 3: Negative control, supernatant of virus-free strain BbOFDH1-5. 4: Supernatant of virus-harboring strain BbOFJY. 5: Negative control, precipitation of virus-free strain BbOFDH1-5. 6: Precipitation of virus-harboring strain BbOFJY）. B: Detection of virus content in the supernatant and precipitation of strain BbOFJY

图7 间接免疫荧光检测（×60） A、B、C：BbPmV-4-CP多克隆抗体鉴定感染BbPmV-4病毒的球孢白僵菌菌株BbOFJY；D、E：阴性对照-无病毒菌株BbOFDH1-5；比例尺为5 μm

Fig. 7 Indirect immunofluorescence assay（×60） A, B, C: Polyclonal antibodies BbPmV-4 against BbPmV-4-CP identified B. bassiana strain BbOFJY infected by virus BbPmV-4. D, E: Negative control, virus-free strain BbOFDH1-5. The scale bar is 5 μm

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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

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