Generation of a Highly Reactive Chicken-Derived Single-Chain Variable Fragment against Fusarium verticillioides by Phage Display
Abstract
:1. Introduction
2. Results and Discussion
2.1. Antigen Preparation and Immunization
2.2. Selection of Phage Clones Reactive to Antigens by Phage Display
2.3. Identification of Phage Clones by Phage ELISA
2.4. Antigen Binding of Soluble Antibodies
2.5. Three-Dimension Structures of scFv Antibodies
2.6. Characterization of FvCA4 Antibody
2.7. Localization Labeling of FvCA4 Antibody Binding
2.8. Immunological Detection of Mycelium and Contaminated Samples
3. Experimental Section
3.1. Antigen Preparation and Immunization
3.2. Library Construction
3.3. Panning
3.4. Phage ELISA
3.5. Sequencing
3.6. Expression and Purification
3.7. ELISA Assays
3.8. Immunoblot Analyses
3.9. Immunofluorescence Microscopy
3.10. Sample Preparation for Immunological Detection
4. Conclusions
Acknowledgments
References
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Round | Input (pfu) | Output (pfu) | Ratio (%) |
---|---|---|---|
1 | 2.6 × 1012 | 1.5 × 104 | 5.8 × 10−9 |
2 | 4.5 × 1012 | 6.0 × 104 | 1.3 × 10−8 |
3 | 5.4 × 1012 | 1.2 × 106 | 2.2 × 10−7 |
Species | Host | Reactivity of FvCA4 |
---|---|---|
F. verticillioides | maize | +++ |
F. verticillioides | rice | +++ |
F. proliferatum | maize | +++ |
F. proliferatum | potato | +++ |
F. proliferatum | wheat | +++ |
F. oxysporum f.sp. matthiolae | stock | +++ |
F. oxysporum f.sp. zingiberi | turmeric | +++ |
F. oxysporum f.sp. niveum | watermelon | +++ |
F. poae | wheat | +++ |
F. culmorum | wheat | +++ |
F. asiaticum | wheat | ++ |
F. solani | mung bean | +++ |
F. tricinctum | wheat | +++ |
A. flavus | peanut | − |
S. sclerotiorum | rapeseed | − |
R. cerealis | wheat | − |
M. fructicola | peach | − |
V. dahliae | cotton | − |
Name | Nucleotide Sequence |
---|---|
VH-cDNA | 5’-CGGTGGGGGACATCTGAGTGGG-3’ |
VL-cDNA | 5’-AGGGGTGGAGGACCTGCACCTC-3’ |
CPDVHF | 5’-ATCTAGGCATCCCTTGGCCCAGCCGGCCATGGCTGCCGTGACGTTGGACGAGTCC-3’ |
CHIC-Gly | 5’-GCCAGAGCCACCTCCGCCTGAACCGCCTCCACCTGAGGAGACGATGACCTCGGTCCC-3’ |
CHIC-Ser | 5’-GGCGGAGGTGGCTCTGGCGGTGGCGGATCGGCGCTGACTCAGCCGTCCTCGGTG-3’ |
CPDVLB | 5’-TGACCTGCGAGGATGCGCGGCCGCGTCGACGGGCTGGCCTAGGACGGTCAG-3’ |
pHENpel | 5’-GCAGCCGCTGGATTGTTATTACTCGC-3’ |
pHENmyc | 5’-ATTCAGATCCTCTTCTGAGATGAG-3’ |
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Hu, Z.-Q.; Liu, J.-L.; Li, H.-P.; Xing, S.; Xue, S.; Zhang, J.-B.; Wang, J.-H.; Nölke, G.; Liao, Y.-C. Generation of a Highly Reactive Chicken-Derived Single-Chain Variable Fragment against Fusarium verticillioides by Phage Display. Int. J. Mol. Sci. 2012, 13, 7038-7056. https://doi.org/10.3390/ijms13067038
Hu Z-Q, Liu J-L, Li H-P, Xing S, Xue S, Zhang J-B, Wang J-H, Nölke G, Liao Y-C. Generation of a Highly Reactive Chicken-Derived Single-Chain Variable Fragment against Fusarium verticillioides by Phage Display. International Journal of Molecular Sciences. 2012; 13(6):7038-7056. https://doi.org/10.3390/ijms13067038
Chicago/Turabian StyleHu, Zu-Quan, Jin-Long Liu, He-Ping Li, Shu Xing, Sheng Xue, Jing-Bo Zhang, Jian-Hua Wang, Greta Nölke, and Yu-Cai Liao. 2012. "Generation of a Highly Reactive Chicken-Derived Single-Chain Variable Fragment against Fusarium verticillioides by Phage Display" International Journal of Molecular Sciences 13, no. 6: 7038-7056. https://doi.org/10.3390/ijms13067038