Construction of scFv Antibodies against the Outer Loops of the Microsporidium Nosema bombycis ATP/ADP-Transporters and Selection of the Fragment Efficiently Inhibiting Parasite Growth
Abstract
:1. Introduction
2. Results
2.1. Constructing of scFv Immune Library, Its Panning and Selection of NbAAC1-Specific Abs
2.2. Originality of Selected scFv Sequences
2.3. Heterologous Expression of Selected scFv Fragments in Sf9 Cells
2.4. Expression of NbAAC1-Specific scFv Abs Differently Affected the Parasite Intracellular Growth
2.5. The Effectiveness of scFv5 to Suppress Intracellular Growth of N. bombycis Was Confirmed by Additional Infections and qPCR Assays
2.6. Western Blot Analysis of Infected Transformants with Abs against N. bombycis β-Tubulin Confirmed the Inhibitory Activity of scFv5
2.7. Identification of Complementarity Determining Regions (CDRs) of the scFv5 Unique VH Domain
3. Discussion
4. Materials and Methods
4.1. Bacterial Overexpression of Chimeric Proteins and Development of Immune scFv Library
4.2. Antigen Preparation and Immunization of Mice
4.3. Construction of the scFv Immune Library
4.4. Library Panning
4.5. Bacterial Expression and Analysis of Selected scFv Fragments
4.6. Heterologous Expression of scFv Abs in Sf9 Cells and Their Infection with N. bombycis Spores
4.7. qPCR Analysis of N. bombycis Growth in Infected Cultures
4.8. Analysis of N. bombycis β-Tubulin Accumulation in Infected Transformants
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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VH | 9 * | 8 | 7 | 6 | 5 | 4 | 3 | 2 | ||
---|---|---|---|---|---|---|---|---|---|---|
1 | 86.2 | 89.7 | 89.7 | 87.6 | 45.6 | 86.2 | 48.3 | 84.5 | ||
2 | 76.3 | 83.6 | 83.1 | 85 | 54.4 | 82.8 | 50.4 | 36.1 | 2 | |
3 | 50 | 49.1 | 50.8 | 52.2 | 84.2 | 49.1 | 88.6 | 37 | 3 | |
4 | 81.9 | 93.1 | 90.5 | 85 | 46.5 | 36 | 36.9 | 100 | 5 | |
5 | 47.4 | 46.5 | 48.2 | 48.7 | 37.8 | 94.8 | 87.7 | 37 | 6 | |
6 | 85.8 | 86.7 | 86.7 | 82.8 | 36.9 | 83.5 | 83.3 | 38 | 7 | |
7 | 87.3 | 92.2 | 81 | 94.8 | 36.9 | 93 | 86.8 | 38 | 8 | |
8 | 85.3 | 35.3 | 34.5 | 36.2 | 42.3 | 35.7 | 36 | 40.7 | 9 | |
8 | 7 | 6 | 5 | 3 | 2 | 1 | VL |
scFv | Cq Values * and Number of Transcripts in 1 μL of cDNA Samples (in Brackets) | ΔCq ** and Number of NbPTP2 Transcripts per 103 SfCOXI Ones (in Brackets) | N Samples and Variants with ΔCq ≥ 7 (in Brackets) | |
---|---|---|---|---|
SfCOXI | NbPTP2 | |||
1 | 13.8 ± 0.2 (7.1 × 106) | 20.9 ± 0.6 (20.7 × 103) | 7.1 ± 0.6 a (2.9) | 9 (6) |
2 | 14.2 ± 0.3 (5.4 × 106) | 21.6 ± 0.5 (12.3 × 103) | 7.4 ± 0.6 a (2.3) | 9 (4) |
3 | 15.3 ± 0.5 (2.7 × 106) | 22.2 ± 0.4 (7.9 × 103) | 6.9 ± 0.2 a (3) | 6 (3) |
4 | 14.7 ± 0.2 (3.9 × 106) | 19.9 ± 0.7 (43.3 × 103) | 5.2 ± 0.6 b (11) | 5 (1) |
5 | 13.5 ± 0.3 (8.6 × 106) | 22 ± 0.3 (9.2 × 103) | 8.5 ± 0.5 a (1.1) | 8 (8) |
6 | 14.3 ± 0.2 (5.1 × 106) | 20.1 ± 0.4 (37. 4 × 103) | 5.8 ± 0.4 b (7.3) | 9 (2) |
7 | 14 ± 0.3 (6.2 × 106) | 21.8 ± 0.3 (10.6 × 103) | 7.8 ± 0.5 a (1.7) | 4 (4) |
8 | 14.2 ± 0.5 (5.4 × 106) | 20.2 ± 0.4 (34.7 × 103) | 6 ± 0.6 b (6.4) | 8 (1) |
9 | 14.7 ± 0.3 (3.9 × 106) | 22.7 ± 0.5 (5.4 × 103) | 8 ± 0.3 a (1.4) | 6 (6) |
scFv | Spores per Cell | Cq Values * and Number of Transcripts in 1 μL of cDNA Samples (in Brackets) | ΔCq ** and Number of NbPTP2 Transcripts per 106 SfCOXI Ones (in Brackets) | |
---|---|---|---|---|
SfCOXI | NbPTP2 | |||
1 | 10 | 15.5 ± 0.4 (2.3 × 106) | 23.2 ± 0.2 (3.8 × 103) | 7.7 ± 0.5 a (1.7 × 103) |
5 | 10 | 14.4 ± 0.2 (5.1 × 106) | 25.1 ± 0.3 (1.2 × 103) | 10.7 ± 0.4 b (2.4 × 102) |
7 | 10 | 14.8 ± 0.2 (3.7 × 106) | 23.7 ± 0.2 (2.6 × 103) | 8.9 ± 0.2 a (7 × 102) |
9 | 10 | 13.8 ± 0.3 (7.1 × 106) | 22.1 ± 0.3 (8.5 × 103) | 8.3 ± 0.9 a (1.2 × 103) |
1 | 3 | 14.7 ± 0.1 (3.9 × 106) | 22.4 ± 0.1 (6.8 × 103) | 7.7 ± 0.2 a (1.7 × 103) |
5 | 3 | 15.5 ± 0.3 (2.3 × 106) | 26.1 ± 0.2 (4.4 × 102) | 10.6 ± 0.2 b (1.9 × 102) |
7 | 3 | 15.3 ± 0.6 (2.7 × 106) | 24.1 ± 0.9 (1.9 × 103) | 8.8 ± 0.4 a (7 × 102) |
9 | 3 | 14.9 ± 0.2 (3.4 × 106) | 23.9 ± 0.2 (2.2 × 103) | 9 ± 0.3 a (6.5 × 102) |
scFv | Cq * | ΔCq ** | |
---|---|---|---|
SfCOXI-2 | NbPTP2-2 | ||
2 | 19.4 ± 0.1 | 21.7 ± 0.1 | 2.3 ± 0.1 a |
3 | 19.7 ± 0.8 | 22.1 ± 0.4 | 2.4 ± 0.5 a |
5 | 20.6 ± 0.4 | 26.4 ± 1 | 5.8 ± 0.6 b |
7 | 18.1 ± 0.3 | 20.4 ± 0.4 | 2.3 ± 0.4 a |
8 | 19.2 ± 0.1 | 19.6 ± 0.1 | 0.4 ± 0.1 a |
eGFP | 18.5 ± 0.2 | 19.1 ± 0.2 | 0.6 ± 0.2 a |
Primer | Sequence (5′-3′) |
---|---|
NbAAC3 rev a | TGCAAGCTTACGGTGCAGCACGACGAATGTTGTC |
pOPE101seq for | AAGAGGAGAAATTAACCATGA |
pOPE101seq rev | TCATTAGCACAGGCCTCTAGA |
pOPEpIB for b | ACGGAGCTCGGTACCTGCTGCTGGCAGCTCAGCCGGCCATG |
pOPEpIB rev c | GCTGAATTCTCGAGTTAATGATGATGGTGATGATGGGATAG |
GFP for d | CATGAATTCATGGTGAGCAAGGGCGAGGAGCTG |
GFP rev e | TCACTCGAGTTACTTGTACAGCTCGTCCATGCCGA |
pIB seq for | CGCAACGATCTGGTAAACAC |
pIB seq rev | GACAATACAAACTAAGATTTAGTCAG |
SfCOXI for | TTTGAGCAGGAATAGTAGGT |
SfCOXI rev | TAAAGATGGGGGTAAAAGT |
SfCOXI-2 for | TACCGCATTTTTATTATTATTATC |
SfCOXI-2 rev | GTTTCCTTTTTACCTCTTTCTTGA |
NbPTP2 for | TGGCATCAGTAGCTCCTCCTCAAG |
NbPTP2 rev | ACGGCCCTAGCTGCTGTTTCAA |
NbPTP2-2 for | CAATAATCCAGCCGAGTGTCAA |
NbPTP2-2 rev | AGTGGGGTACCTTCAGCAGTTT |
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Dolgikh, V.V.; Senderskiy, I.V.; Timofeev, S.A.; Zhuravlyov, V.S.; Dolgikh, A.V.; Seliverstova, E.V.; Ismatullaeva, D.A.; Mirzakhodjaev, B.A. Construction of scFv Antibodies against the Outer Loops of the Microsporidium Nosema bombycis ATP/ADP-Transporters and Selection of the Fragment Efficiently Inhibiting Parasite Growth. Int. J. Mol. Sci. 2022, 23, 15307. https://doi.org/10.3390/ijms232315307
Dolgikh VV, Senderskiy IV, Timofeev SA, Zhuravlyov VS, Dolgikh AV, Seliverstova EV, Ismatullaeva DA, Mirzakhodjaev BA. Construction of scFv Antibodies against the Outer Loops of the Microsporidium Nosema bombycis ATP/ADP-Transporters and Selection of the Fragment Efficiently Inhibiting Parasite Growth. International Journal of Molecular Sciences. 2022; 23(23):15307. https://doi.org/10.3390/ijms232315307
Chicago/Turabian StyleDolgikh, Viacheslav V., Igor V. Senderskiy, Sergej A. Timofeev, Vladimir S. Zhuravlyov, Alexandra V. Dolgikh, Elena V. Seliverstova, Diloram A. Ismatullaeva, and Bakhtiyar A. Mirzakhodjaev. 2022. "Construction of scFv Antibodies against the Outer Loops of the Microsporidium Nosema bombycis ATP/ADP-Transporters and Selection of the Fragment Efficiently Inhibiting Parasite Growth" International Journal of Molecular Sciences 23, no. 23: 15307. https://doi.org/10.3390/ijms232315307