Efficient amplification of light and heavy chain variable regions and construction of a non-immune phage scFv library.
Non-immune phage scFv library is one of the most attractive resources for therapeutics, diagnostics and basic research. As a matter of fact, quality of the library is limited by inefficient PCR cloning of antibody genes using degenerated primers. PCR using this type of primers is difficult to optimize conditions for efficient amplification, and therefore causes loss of antibody diversities. To overcome this problem, we described a novel two-step amplification of V(kappa) and V(H) genes with newly designed primer sets. Initially, we amplified V(kappa) and V(H) genes from their signal sequences to the joining region to keep antibody diversity as large as possible. Thereafter, highly degenerated primers were used to amplify the V(kappa) and V(H) genes from the framework region 1 to the joining region. The V(kappa) and V(H) genes from the second PCR then were linked by PCR overlapping extension to generate the scFv library. Fifteen clones from the library were randomly picked and sequenced, and the diversity of full-length scFvs was confirmed. Expression capability of clones in the library was 80% after confirmation using colony hybridization. The results demonstrated the efficiency of this strategy and the primer sets for construction of the scFv library.
Koohapitagtam
Maneerat
M
Center for Agricultural Biotechnology, Kasetsart University, Nakhon Pathom, 73140, Thailand.
Rungpragayphan
Suang
S
Hongprayoon
Ratchanee
R
Kositratana
Wichai
W
Sirinarumitr
Theerapol
T
D000818:Animals; D003001:Cloning, Molecular; D017931:DNA Primers / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D014644:Genetic Variation; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007147:Immunoglobulin Light Chains / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D051379:Mice; D019151:Peptide Library; D016133:Polymerase Chain Reaction / Q000379:methods*; D014170:Transformation, Genetic
D017931:DNA Primers; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D019151:Peptide Library