Germination-Arrest Bacillus subtilis Spores as An Oral Delivery Vehicle of Grass Carp Reovirus (GCRV) Vp7 Antigen Augment Protective Immunity in Grass Carp (Ctenopharyngodon idella)
Abstract
:1. Introduction
2. Materials and Methods
2.1. Strains and Growth Conditions
2.2. Construction of Plasmids and Recombinant B. Subtilis Strains
2.3. Spore Preparation and Germination Analysis
2.4. Preparation of the Recombinant Vp7 Protein and Rabbit Anti-Vp7 Antibody
2.5. Immunoblotting Analysis of Spore Coat Proteins
2.6. Immunofluorescence Analysis
2.7. Preparation of Experimental Fish and Spore-Coated Feed Pellets
2.8. Oral Administration, Sample Collection, and Challenge
2.9. Indirect Enzyme-Linked Immunosorbent Assay (ELISA) of Anti-Vp7 IgM Antibody
2.10. Total RNA Isolation, cDNA Synthesis and Quantitative Reverse Transcription PCR (qRT-PCR)
2.11. Statistical Analysis
3. Results
3.1. Recombinant B. Subtilis Strains
3.2. Spore Germination of Recombinant B. Subtilis Strains
3.3. Expression and Localization of Vp7 in the Recombinant Spores
3.4. Immune Responses of Grass Carp Following Oral Immunization
3.5. Protection of Grass Carp against GCRV after Oral Administration
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Plasmid | Characteristic Property or Genotype | Origen or Reference | |
---|---|---|---|
pJS700 | An integrative vector containing the integrative fragment amyE::erm-cotB | [25] | |
pJS1947 | A derivative of pJS700, containing the integrative fragment amyE::erm-cotB-vp7 | This study | |
pJS1956 | An integrative vector containing the integrative fragment cwlJ::erm-cotB | This study | |
pJS1985 | An integrative vector containing the integrative fragment sleB::npt-cotC | This study | |
pJS2000 | A derivative of pJS2016, containing the integrative fragment cwlJ::erm-cotB-vp7 | This study | |
pJS2020 | A derivative of pJS1976, containing the integrative fragment sleB::npt-cotC-vp7 | This study | |
pJS1621 | The vp7 gene in pET28a | This study | |
B. subtilis | Relevant Genotype | Expressed Fusion Antigen 1 on Spore Surface | Origen or Reference |
168 (trp-) | Wild type (WT) | - | BGSC 2 |
DR700 | amyE::erm-cotB | - | This study |
DR1947 | amyE:erm-cotB-vp7 | CotB-Vp7, 60.7 kDa | This study |
DR1956/1985 | cwlJ::erm-cotB; SleB::npt-cotC | - | This study |
DR2000/1985 | cwlJ::erm-cotB-vp7; sleB::npt-cotC | CotB-Vp7, 60.7 kDa | This study |
DR2000/2020 | cwlJ::erm-cotB-vp7; sleB::npt-cotC-vp7 | CotB-Vp7, 60.7 kDa; CotC-Vp7, 44.4 kDa | This study |
Spore | No. of Spores 2 | CFU | Germination Efficiency 3 |
---|---|---|---|
WT168 | 4.64 × 108 | 4.58 × 108 | 99% |
DR700 | 4.32 × 108 | 4.51 × 108 | 104% |
DR1956DR1985 | 4.85 × 108 | 2.14 × 104 | 0.0044% |
Group | Cumulative Mortality Rate (%) | RPS(%) | p Value 2 |
---|---|---|---|
Positive | 74.45 ± 3.14 | -- | -- |
DR1947 | 53.33 ± 2.72 | 28.09 ± 1.79 | 0.036/-- |
DR2000/1985 | 36.67 ± 1.57 | 50.66 ± 0.30 | 0.008/0.031 |
DR2000/2020 | 32.22 ± 3.14 | 56.73 ± 2.82 | 0.007/0.024 |
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Sun, R.; Zhang, M.; Chen, H.; Wei, Y.; Ning, D. Germination-Arrest Bacillus subtilis Spores as An Oral Delivery Vehicle of Grass Carp Reovirus (GCRV) Vp7 Antigen Augment Protective Immunity in Grass Carp (Ctenopharyngodon idella). Genes 2020, 11, 1351. https://doi.org/10.3390/genes11111351
Sun R, Zhang M, Chen H, Wei Y, Ning D. Germination-Arrest Bacillus subtilis Spores as An Oral Delivery Vehicle of Grass Carp Reovirus (GCRV) Vp7 Antigen Augment Protective Immunity in Grass Carp (Ctenopharyngodon idella). Genes. 2020; 11(11):1351. https://doi.org/10.3390/genes11111351
Chicago/Turabian StyleSun, Rui, Miao Zhang, Hui Chen, Yao Wei, and Degang Ning. 2020. "Germination-Arrest Bacillus subtilis Spores as An Oral Delivery Vehicle of Grass Carp Reovirus (GCRV) Vp7 Antigen Augment Protective Immunity in Grass Carp (Ctenopharyngodon idella)" Genes 11, no. 11: 1351. https://doi.org/10.3390/genes11111351