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

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

Abstract

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

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.

Figures/Tables 8

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

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）

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

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）

Fig. 4 Titer detected by indirect ELISA

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

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

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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