Recombinant Fusion Protein Joining E Protein Domain III of Tick-Borne Encephalitis Virus and HSP70 of Yersinia pseudotuberculosis as an Antigen for the TI-Complexes
Abstract
:1. Introduction
2. Materials and Methods
2.1. Construction of Chimeric Plasmids
2.2. Expression of the Recombinant Plasmids
2.3. Recombinant Protein Purification
2.4. Fluorescence Spectroscopy
2.5. Calorimetry
2.6. Preparation of TI-Complexes
2.7. Animals and Immunization
2.8. ELISA and Statistical Analyses
2.9. Computational Analysis of the Intrinsic Disorder Predisposition of the Chimeric HSP70/EIII Protein
3. Results and Discussion
4. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Primer | Sequence |
---|---|
dnaK-X-Nco-dir | 5′-ATATCCATGGCGATGGGTAAAATTATTGGTATCGAC-3′ |
dnaK-X-Sac-rev | 5′-TATAGAGCTCGCTTTTTTGTCTTTTACTTCTTCGAATTC-3′ |
E3-X-Sac-L-dir | 5′-TATAGAGCTCGGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGGTCTTACATACACAATGTGCGACAAGACGAAATTCAC-3′ |
E3-X-Sal-stop-rev | 5′-TATA GTCGAC TTA GTTAAAGGCACCGCCAAGAACTGT-3′ |
Hsp70 | HSP70/EIII | HSP70/EIII + MGDG U. lactuca | |
---|---|---|---|
Tm1, °C | 54.6 ± 0.1 | 54.2 ± 0.1 | 55.4 ± 0.1 |
ΔH1, kcal/mol | 92.1 ± 0.7 | 99.5 ± 1.7 | 96.9 ± 1.4 |
Tm2, °C | - | 58.4 ± 0.3 | 63.2 ± 0.1 |
ΔH2, kcal/mol mol−1 | - | 79.9 ± 1.5 | 103.0 ± 1.9 |
Tm3, °C | 66.4 ± 0.2 | 67.1 ± 0.1 | 67.7 ± 0.1 |
ΔH3, kcal/mol | 81.5 ± 1.5 | 116.0 ± 1.5 | 170.0 ± 1.9 |
Tm4, °C | 68.5 ± 0.4 | 70.1 ± 0.6 | 71.3 ± 0.5 |
ΔH4, kcal/mol | 129.0 ± 1.3 | 124.0 ± 1.9 | 126.0 ± 2.6 |
Protein | Spectral Forms (%) | |||
---|---|---|---|---|
S | I | II | III | |
HSP70 | 13.6 ± 0.3 | 58.5 ± 0.5 | 12.2 ± 0.3 | 15.7 ± 0.2 |
HSP70/EIII | 19.5 ± 0.5 | 45.7 ± 0.4 | 34.8 ± 0.5 | 0 |
HSP70/EIII + MGDG U. lactuca | 19.3 ± 0.5 | 42.8 ± 0.5 | 0 | 37.9 ± 0.4 |
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Golotin, V.; Sanina, N.; Davydova, L.; Chopenko, N.; Mazeika, A.; Roig, M.; Shnyrov, V.; Uversky, V.N.; Kostetsky, E. Recombinant Fusion Protein Joining E Protein Domain III of Tick-Borne Encephalitis Virus and HSP70 of Yersinia pseudotuberculosis as an Antigen for the TI-Complexes. Biomolecules 2018, 8, 82. https://doi.org/10.3390/biom8030082
Golotin V, Sanina N, Davydova L, Chopenko N, Mazeika A, Roig M, Shnyrov V, Uversky VN, Kostetsky E. Recombinant Fusion Protein Joining E Protein Domain III of Tick-Borne Encephalitis Virus and HSP70 of Yersinia pseudotuberculosis as an Antigen for the TI-Complexes. Biomolecules. 2018; 8(3):82. https://doi.org/10.3390/biom8030082
Chicago/Turabian StyleGolotin, Vasily, Nina Sanina, Ludmila Davydova, Natalia Chopenko, Andrey Mazeika, Manuel Roig, Valery Shnyrov, Vladimir N. Uversky, and Eduard Kostetsky. 2018. "Recombinant Fusion Protein Joining E Protein Domain III of Tick-Borne Encephalitis Virus and HSP70 of Yersinia pseudotuberculosis as an Antigen for the TI-Complexes" Biomolecules 8, no. 3: 82. https://doi.org/10.3390/biom8030082