Generation and validation of canine single chain variable fragment phage display libraries

Area of Science:

• Immunology
• Molecular Biology
• Veterinary Medicine

Background:

• Single-chain variable fragments (scFvs) are the smallest antibody fragments retaining antigen specificity.
• scFvs hold significant potential for therapeutic antigen targeting in vivo.
• Developing canine-specific antibody fragments is crucial for veterinary medicine.

Purpose of the Study:

• To design and validate degenerate primers for amplifying canine immunoglobulin heavy (VH) and light (VL) chain variable regions.
• To construct scFv fragments by combining VH and VL amplicons using splicing by overlap extension PCR.
• To demonstrate the feasibility of generating canine scFv phage display libraries and isolating antigen-specific scFvs.

Main Methods:

• Degenerate primers were used to amplify canine VH and VL genes from lymphocyte cDNA.
• Splicing by overlap extension PCR was employed to join VH and VL amplicons with a flexible linker, forming scFv constructs.
• scFv constructs were expressed on the surface of M13 bacteriophage for library generation and panning.

Main Results:

• Successful amplification and combination of canine VH and VL genes into scFv constructs.
• Expression of functional scFvs on M13 bacteriophage, creating scFv phage display libraries.
• Isolation of canine parvovirus (CPV)-specific scFvs from a library generated from immunized dog splenocytes.

Conclusions:

• The developed approach is feasible for generating diverse canine scFv libraries.
• This method paves the way for isolating canine antigen-specific scFvs.
• Isolated scFvs could be tested as targeting agents for canine infectious, inflammatory, and neoplastic diseases.