Strategies for Vaccine Design Using Phage Display-Derived Peptides
Development of peptide vaccines through the phage display technology is a powerful strategy that relies on short peptides expressed in the phage capsid surface to induce highly targeted immune responses. Phage display-derived immunogenic peptides can be u
- PDF / 208,959 Bytes
- 13 Pages / 504.567 x 720 pts Page_size
- 41 Downloads / 174 Views
troduction The phage display technology (PD), a selection strategy of polypeptides displayed in the surface of filamentous bacteriophage against targets, was introduced in 1985 by George Smith. PD is based on DNA recombination, resulting in expression of foreign peptide variants on the outer surface of phage clones [1]. Using an in vitro selection process based on binding affinity, or biopanning, the ligands are eluted from targets and enriched under many cycles of selection. The exposed peptides in the selected phage clones are characterized by DNA sequencing and then identified [2]. Phage-displayed peptide libraries became one of the most powerful technologies for selecting peptide ligands for specific target molecules [3]. The random peptides fused to the bacteriophage capsid selected against a specific target are considered mimetic in relation to their native epitopes and can be used as antigenic and immunogenic molecules. Immunogenic carriers are able to generate antibodies against recombinant peptides expressed in the N-terminal region of the phage surface, which may crossreact with the native antigen target, suggesting that expressed mimotopes can be used as candidate vaccinal subunits. Differently from working with entire proteins or whole organisms in conventional vaccines with multiple epitopes and unnecessary antigenic load, peptide vaccines are an attractive strategy that relies on short peptides to induce highly targeted immune responses. The single immunogenic region of the phage-fused peptide is able to eliminate cross-reactions and avoid allergic responses, which are usually observed in complex antigens that share common epitopes with other proteins. Besides, it may facilitate production as a recombinant or chemically synthesized antigen subunit for vaccinal purposes without the large-scale Sunil Thomas (ed.), Vaccine Design: Methods and Protocols, Volume 2: Vaccines for Veterinary Diseases, Methods in Molecular Biology, vol. 1404, DOI 10.1007/978-1-4939-3389-1_28, © Springer Science+Business Media New York 2016
423
424
Luiz R. Goulart and Paula de S. Santos
urification strategy needed for whole proteins [4–6]. The present p chapter presents protocols for identification of highly reactive peptides against antibodies based on phage display and procedures to demonstrate their potential as immunogens in vaccine formulations (Fig. 1).
2 Materials 2.1 Peptide Selection Components
1. Coating buffer: 0.1 M NaHCO3, pH 8.6. 2. Polystyrene microplates (Nunc Maxisorp). 3. PhD-12 or PhD-C7C phage library (New England Biolabs, Beverly, MA, USA). Store at −20 °C. 4. Tris-buffered saline (TBS): 50 mM Tris–HCl, pH 7.5, 150 mM NaCl. Sterilize by autoclaving 20 min at 15 psi. 5. Blocking solution (TBS/BSA 3 %): add 3 g of BSA (bovine serum albumin) to 100 mL with TBS. 6. Washing solution (TBS-T 0.1 %): add 1 mL of Tween 20 to 1 L with TBS. 7. Elution buffer: 0.2 M glycine in water. Adjust pH to 2.2 with HCl. Add 1 mg/mL of BSA. Sterilize by filtration and store at 4 °C. 8. Neutralization buffer: 1 M Tris b
Data Loading...
