Development of a Novel Q-body Using an In Vivo Site-Specific Unnatural Amino Acid Incorporation System
Abstract
:1. Introduction
2. Materials and Methods
2.1. Materials
2.2. Homology Modeling
2.3. Construction of Mutant Antibody and Nef Genes
2.4. Antibody Expression and Purification
2.5. Antigen Expression and Purification
2.6. Fluorescence Measurements
2.7. Western Blotting
2.8. ELISA
3. Results
3.1. Homology Modeling of anti-Nef Antibody
3.2. Construction of the Nef Q-body
3.3. Antigen-dependent Changes in Fluorescence Intensity of the Labeled Antibodies
3.4. Involvement of Internal Trp Residues in the Quenching of Cy3 at Position L67L
3.5. Antigen-dependent Changes in Fluorescence Intensity of Labeled Antibodies
3.6. Analysis of the anti-Nef Antibody Epitope
3.7. Decrease in Response Resulting from Mutation of the Antigen (Nef)
4. Discussion
5. Conclusions
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
References
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Primer Name | Nucleotide Sequence (5′-3′) |
---|---|
303_T66LAzY_F | ctacgccgtttcctagttagattctggtgt |
303_T66LAzY_R | acaccagaatctaactaggaaacggcgtag |
303_L67LAzY_F | cgccgtttccacttaggattctggtgtccc |
303_L67LAzY_R | gggacaccagaatcctaagtggaaacggcg |
303_S69LAzY_F | ttccactttagattagggtgtcccaaaaag |
303_S69LAzY_R | ctttttgggacaccctaatctaaagtggaa |
303_D101LAzY_F | tgaagattttgcatagtattactgtctcca |
303_D101LAzY_R | tggagacagtaatactatgcaaaatcttca |
303_S18LAzY_F | tccatcctccttataggcctctctgggaga |
303_S18LAzY_R | tctcccagagaggcctataaggaggatgga |
303_W41HF_F | cgactatatgcacttcgtgaagcagaggcc |
303_W41HF_R | ggcctctgcttcacgaagtgcatatagtcg |
303_W52HF_F | acagggcctggagttcattggatggattga |
303_W52HF_R | tcaatccatccaatgaactccaggccctgt |
303_W55HF_F | ggagtggattggattcattgatcctgagaa |
303_W55HF_R | ttctcaggatcaatgaatccaatccactcc |
303_W118H4F_F | tacaagggatgtcttcggcgcagggaccac |
303_W118HF_R | gtggtccctgcgccgaagacatcccttgta |
303_W41LF_F | tggttacttaagcttccttcagcagaaacc |
303_W41LF_R | ggtttctgctgaaggaagcttaagtaacca |
Nef_del2to11_F | agtagtccatgggatggcctgctgtaagg |
Nef_R | aagtgtagcggtcacgctgcgcgtaaccac |
Nef_W13A_F | tagtgtgattggagcacctgctgtaaggga |
Nef_W13A_R | tcccttacagcaggtgctccaatcacacta |
Gene | Template | Primers |
---|---|---|
T66LAzY | wt anti-Nef antibody | 303_T66LAzY_F, 303_T66LAzY_R |
L67LAzY | wt anti-Nef antibody | 303_L67LAzY_F, 303_L67LAzY_R |
S69LAzY | wt anti-Nef antibody | 303_S69LAzY_F, 303_S69LAzY_R |
D101LAzY | wt anti-Nef antibody | 303_D101LAzY_F, 303_D101LAzY_R |
S18LAzY | wt anti-Nef antibody | 303_S18LAzY_F, 303_S18LAzY_R |
L67LAzY_W41HF | L67LAzY | 303_W41HF_F, 303_W41HF_R |
L67LAzY_W52HF | L67LAzY | 303_W52HF_F, 303_W52HF_R |
L67LAzY_W55HF | L67LAzY | 303_W55HF_F, 303_W55HF_R |
L67LAzY_W118HF | L67LAzY | 303_W118HF_F, 303_W118H4F_R |
L67LAzY_W41LF | L67LAzY | 303_W41LF_F, 303_W41LF_R |
NefΔ2–10 | Nef wt | Nef_del2to11_F, Nef_R |
Nef W13A | Nef wt | Nef_W13A_F, Nef_W13A_R |
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Kurumida, Y.; Hayashi, N. Development of a Novel Q-body Using an In Vivo Site-Specific Unnatural Amino Acid Incorporation System. Sensors 2018, 18, 2519. https://doi.org/10.3390/s18082519
Kurumida Y, Hayashi N. Development of a Novel Q-body Using an In Vivo Site-Specific Unnatural Amino Acid Incorporation System. Sensors. 2018; 18(8):2519. https://doi.org/10.3390/s18082519
Chicago/Turabian StyleKurumida, Yoichi, and Nobuhiro Hayashi. 2018. "Development of a Novel Q-body Using an In Vivo Site-Specific Unnatural Amino Acid Incorporation System" Sensors 18, no. 8: 2519. https://doi.org/10.3390/s18082519
APA StyleKurumida, Y., & Hayashi, N. (2018). Development of a Novel Q-body Using an In Vivo Site-Specific Unnatural Amino Acid Incorporation System. Sensors, 18(8), 2519. https://doi.org/10.3390/s18082519