Immunogenicity of eGFP-Marked Recombinant Lactobacillus casei against Transmissible Gastroenteritis Virus and Porcine Epidemic Diarrhea Virus
Abstract
:1. Introduction
2. Materials and Methods
2.1. Virus, Plasmid, Bacterium, and Cell Lines
2.2. Construction of the eGFP-Marked Recombinant Lactobacillus Strain
2.3. Identification of Proteins Expressed by the Recombinant Strain
2.4. Immunizations
2.5. ELISA Assay
2.6. TGEV and PEDV Neutralization by Mouse Immune Sera
2.7. Antigen-Induced Proliferation of Lymphocytes In Vitro
2.8. Statistical Analysis
3. Results
3.1. Construction of eGFP-Marked Recombinant Lactobacillus Strain
3.2. Expression of Proteins of Interest by Recombinant Lactobacillus Strain
3.3. Immune Responses in Mice Induced by Recombinant Lactobacillus Strain
3.4. TGEV and PEDV Neutralization
3.5. Lymphocyte Proliferation
4. Discussion
5. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Plasmids | Relevant Characteristics | Description/Reference |
---|---|---|
pMD18-T-6D | As a template for PCR to amplify DNA fragment 6D | Liu et al. 2011 [31] |
peGFP-N1 | As a template for PCR to amplify DNA fragment eGFP | Wang et al. 2016 [32] |
pMD18-T-COE | 3169 bp; Ampr; core neutralizing epitope (COE) of PEDV strain HLJ-2012 | This study |
pMD19-Ts | 2692 bp; Ampr | Takara (DaLian, China) |
pMD19-Ts-6D | 3325 bp; Ampr; the amplified DNA fragment 6D was inserted into pMD19-Ts | This study |
pMD19-Ts-eGFP | 3469 bp; Ampr; the amplified DNA fragment eGFP was inserted into pMD19-Ts | This study |
pMD18-T-6D-COE | 3766 bp; Ampr; 6D was inserted into pMD18-T-COE | This study |
pPG-T7g10 | 4965 bp; Cmr; HCE promoter of Geobacillus toebii D-AAT; poly-γ-glutamate synthase A (PgsA) anchor; constitutive expression plasmid | Song et al., 2014 [33] |
pPG-T7g10-6D-COE | 6034 bp; Cmr; 6D-COE was inserted into pPG-T7g10 | This study |
pPG-T7g10-eGFP-6D-COE | 6793 bp; Cmr; eGFP was inserted into pPG-T7g10-6D-COE | This study |
pPGF-T7g10-eGFP-6D-COE | 6596 bp; Cmr gene of pPG-T7g10-eGFP-6D-COE was deleted | This study |
Gene | ID | Primer Sequences | Length(bp) | Accession No./Reference |
---|---|---|---|---|
6D | Fs1 | 5′-GAGCTCGCAGGTACCAGATCTTGTT-3′ | 612 | Liu et al., 2011 [31] |
Rs1 | 5′-ACGCGTGAGTCTAGAGGATCCGCCAC-3′ | |||
COE | Fs2 | 5′-ACGCGTGGTGGAGGAGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGGTTACTTTGCCATCATTT-3′ | 420 | JX512907 |
Rs2 | 5′-GGGCCCAACGTCCGTGACACCTTC-3′ | |||
eGFP | Fe | 5′-CGAGCTCATGGTGAGCAAGGGCGA-3′ | 720 | U55762.1 |
Re | 5′-GGGGTACCCGATCCGCCACCGCCAGAGCCACCTCCGCCTGAACCGCCTCCACCCTTGTACAGCTCGTCCATGC-3′ |
Group | No. of Mice | Dose/Treatment | No. of Treatments |
---|---|---|---|
Phosphate-buffered saline (PBS) | 15 | 200 μL of PBS | 3 |
rLpPG-T7g10 | 15 | 200 μL of 1010 CFU mL−1 | 3 |
rLpPG-T7g10-6D-COE | 15 | 200 μL of 1010 CFU mL−1 | 3 |
rLpPGF-T7g10-eGFP-6D-COE | 15 | 200 μL of 1010 CFU mL−1 | 3 |
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Yu, M.; Wang, L.; Ma, S.; Wang, X.; Wang, Y.; Xiao, Y.; Jiang, Y.; Qiao, X.; Tang, L.; Xu, Y.; et al. Immunogenicity of eGFP-Marked Recombinant Lactobacillus casei against Transmissible Gastroenteritis Virus and Porcine Epidemic Diarrhea Virus. Viruses 2017, 9, 274. https://doi.org/10.3390/v9100274
Yu M, Wang L, Ma S, Wang X, Wang Y, Xiao Y, Jiang Y, Qiao X, Tang L, Xu Y, et al. Immunogenicity of eGFP-Marked Recombinant Lactobacillus casei against Transmissible Gastroenteritis Virus and Porcine Epidemic Diarrhea Virus. Viruses. 2017; 9(10):274. https://doi.org/10.3390/v9100274
Chicago/Turabian StyleYu, Meiling, Li Wang, Sunting Ma, Xiaona Wang, Yusai Wang, Ya Xiao, Yanping Jiang, Xinyuan Qiao, Lijie Tang, Yigang Xu, and et al. 2017. "Immunogenicity of eGFP-Marked Recombinant Lactobacillus casei against Transmissible Gastroenteritis Virus and Porcine Epidemic Diarrhea Virus" Viruses 9, no. 10: 274. https://doi.org/10.3390/v9100274