生物技术通报 ›› 2023, Vol. 39 ›› Issue (10): 58-67.doi: 10.13560/j.cnki.biotech.bull.1985.2023-0383
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()
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;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; 基金资助:
GUO Wen-bo1,2(), LU Yang1,2, SUI Li1, ZHAO Yu1, ZOU Xiao-wei1, ZHANG Zheng-kun1,2(), LI Qi-yun1,2,3()
Received:
2023-04-22
Published:
2023-10-26
Online:
2023-11-28
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。
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.
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 |
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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