Construction of a human Ig combinatorial library from genomic V segments and synthetic CDR3 fragments.
A naive combinatorial Ig library was constructed from semi-synthetic V genes consisting of human genomic V segments and synthetic CDR3 fragments. VH and V kappa segments were amplified from human genomic DNA by polymerase chain reaction using V subgroup-specific primers. The amplified VH and V kappa segments were combined with synthetic oligonucleotides containing a J region and CDR3 with amino acid sequence variations, resulting in complete V genes. These V genes were cloned into a phagemid expression vector in a single-chain form fused to the carboxyl-terminus of the M13 minor coat protein III. Phagemid particles displaying the single chain hybrid proteins on their surface were screened with Con A as Ag. Several clones showing specific binding to Con A were obtained after four rounds of selection and were further analyzed for their binding properties and DNA sequences. This method provides a novel way to create a naive combinatorial library without using mRNA from B lymphocytes as template. The method should be useful to isolate human antibodies that react with self-Ag.
Akamatsu
Y
Y
Protein Design Labs, Inc., Mountain View, CA 94043.
Cole
M S
MS
Tso
J Y
JY
Tsurushita
N
N
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003208:Concanavalin A / Q000276:immunology; D015723:Gene Library; D005803:Genes, Immunoglobulin; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction
D007135:Immunoglobulin Variable Region; D003208:Concanavalin A