A Linear Surface Epitope in a Proline-Rich Region of ORF3 Product of Genotype 1 Hepatitis E Virus
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cells and Transfection
2.2. Plasmids
2.3. Expression and Purification of VP13
2.4. Immunization and Monoclonal Antibody Preparation
2.5. Enzyme-Linked Immunosorbent Assay (ELISA)
2.6. Western Blotting
2.7. Immunofluorescence Assay (IFA)
2.8. Statistical Analysis
3. Results
3.1. A VP13 Mab only Reacts with VP13 of Genotype 1 HEV
3.2. The Linear Epitope Spans Residues aa66-75 of VP13 Protein
3.3. The Residue Methionine 70 Is Essential for the Interaction of the Linear Epitope with the Mab
3.4. IFA Detection of the Linear Epitope in HEV-Infected Cells Using the Mab
3.5. The Peptide aa66–75 Is Genotype-Specific in Reacting with HEV-Positive Human Serum Samples
4. Discussion
5. Conclusions
Acknowledgments
Author Contributions
Conflicts of Interest
References
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Primer a | Sequences (5′ to 3′) b | Used for |
---|---|---|
VP13-F1 | GCTCGAGGTTCGCGACCATGCGCCCTC | Cloning of VP13-D5 |
VP13-R1 | CGAATTCTTACATCGGGGGCGAAGGGGTTG | Cloning of VP13-D5 |
VP13-R2 | CGAATTCTTACGGCCGCAGCGGTGACATCG | Cloning of VP13-D6 |
VP13-R3 | CGAATTCTTACACGAGGTCCAGCCCCGGCC | Cloning of VP13-D7 |
VP13-R4 | CGAATTCTTAGGGCGGGTTGGCGAACACG | Cloning of VP13-D8 |
VP13-R5 | CGAATTCTTACGGAGCCGAGTGGTCGGGCG | Cloning of VP13-D9 |
VP13-F3 | GCTCGAGGTATGTCACCGCTGCGGCCGG | Cloning of VP13-D10 |
VP13-R6 | CGAATTCTTAGCGGCGCGGCCCCAGC | Cloning of VP13-D10 |
VP13-F2 | GCTCGAGGTATGCCACCGCTGCGGCCGG | Cloning of VP13-D10 mutant |
H3F30 | AATTCCCTTCGCCCCCGATGTCACCGCTGCGGCCGC | Cloning aa66–75 |
H3R27 | TCGAGCGGCCGCAGCGGTGACATCGGGGGCGAAGGG | Cloning aa66–75 |
H3F31 | AATTCCCTTCGCCCCCGATCTCACCGCTGCGGCCGC | Cloning aa66–75 with mutation M70I |
H3R28 | TCGAGCGGCCGCAGCGGTGAGATCGGGGGCGAAGGG | Cloning aa66–75 with mutation M70I |
T2H3F3 | AATTCCCTTTGCCCCAGATGTTGCCGCTGCGTCCGC | Cloning aa66–75 of type 2 VP13 with mutation T70M |
T2H3R3 | TCGAGCGGACGCAGCGGCAACATCTGGGGCAAAGGG | Cloning aa66–75 of type 2 VP13 with mutation T70M |
KH3F5 | AATTCCCTTCGCCGCCGATGTCGTTTCACAATCCGC | Cloning aa66–75 of type 3 VP13 with mutation I70M |
KH3R6 | TCGAGCGGATTGTGAAACGACATCGGCGGCGAAGGG | Cloning aa66–75 of type 3 VP13 with mutation I70M |
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Yang, Y.; Lin, S.; Nan, Y.; Ma, Z.; Yang, L.; Zhang, Y. A Linear Surface Epitope in a Proline-Rich Region of ORF3 Product of Genotype 1 Hepatitis E Virus. Viruses 2016, 8, 227. https://doi.org/10.3390/v8080227
Yang Y, Lin S, Nan Y, Ma Z, Yang L, Zhang Y. A Linear Surface Epitope in a Proline-Rich Region of ORF3 Product of Genotype 1 Hepatitis E Virus. Viruses. 2016; 8(8):227. https://doi.org/10.3390/v8080227
Chicago/Turabian StyleYang, Yonglin, Shaoli Lin, Yuchen Nan, Zexu Ma, Liping Yang, and Yanjin Zhang. 2016. "A Linear Surface Epitope in a Proline-Rich Region of ORF3 Product of Genotype 1 Hepatitis E Virus" Viruses 8, no. 8: 227. https://doi.org/10.3390/v8080227