An efficient method for variable region assembly in the construction of scFv phage display libraries using independent strand amplification.
Phage display library technology is a common method to produce human antibodies. In this technique, the immunoglobulin variable regions are displayed in a bacteriophage in a way that each filamentous virus displays the product of a single antibody gene on its surface. From the collection of different phages, it is possible to isolate the virus that recognizes specific targets. The most common form in which to display antibody variable regions in the phage is the single chain variable fragment format (scFv), which requires assembly of the heavy and light immunoglobulin variable regions in a single gene. In this work, we describe a simple and efficient method for the assembly of immunoglobulin heavy and light chain variable regions in a scFv format. This procedure involves a two-step reaction: (1) DNA amplification to produce the single strand form of the heavy or light chain gene required for the fusion; and (2) mixture of both single strand products followed by an assembly reaction to construct a complete scFv gene. Using this method, we produced 6-fold more scFv encoding DNA than the commonly used splicing by overlap extension PCR (SOE-PCR) approach. The scFv gene produced by this method also proved to be efficient in generating a diverse scFv phage display library. From this scFv library, we obtained phages that bound several non-related antigens, including recombinant proteins and rotavirus particles.
Centro de InmunoBioTecnología (IBT), Programa Disciplinario de Inmunología, Instituto de Ciencias Biomédicas (ICBM), Facultad de Medicina, Universidad de Chile, Santiago, Chile.
Pablo
P
Sotelo
Noberto
N
Collazo
Roberto
R
Zuñiga
Matías
M
Gutiérrez-González
Diego
D
Catalán
Carolina Hager
CH
Ribeiro
Juan Carlos
JC
Aguillón
María Carmen
MC
Molina
D018076:DNA, Complementary / Q000235:genetics*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000235:genetics; D019151:Peptide Library; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D057127:Single-Chain Antibodies / Q000235:genetics* / Q000276:immunology
D018076:DNA, Complementary; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D019151:Peptide Library; D057127:Single-Chain Antibodies