Patent ID: 8178320

Claim:
A method for preparing a single chain antibody library having a repertory of at least 10 11 comprising: step (a) amplifying nucleic acid sequences comprising the CDR1 and CDR2 regions and the CDR3 region of the VH and VL region in an immunoglobulin gene by PCR using a cDNA library as a template to generate gene fragments; step (b) shuffling said gene fragments between a first gene fragment and a second gene fragment, wherein the first gene fragment comprises the CDR1 and CDR2 regions and the second gene fragment comprises the CDR3 region in each of the VH and VL regions to produce a VH library and a VL library; step (c) inserting said VH library into a first non-expression vector comprising an f1 ori and a first drug-resistance gene, and said VL library into a second non-expression vector comprising an f1 ori and a second drug-resistance gene, wherein one of said first and second non-expression vectors is a promoter-free vector comprising a third drug-resistance gene with a Shine-Dalgarno (SD) sequence and the other non-expression vector is a Shine-Dalgarno (SD) sequence-free vector and wherein the third drug resistance gene is different from the first and second drug resistance genes; step (d) shuffling gene fragments between the VH region in the VH library and the VL region in the VL library by introducing both the VH library-incorporated non-expression vector and VL library-incorporated non-expression vector into a host; step (dd) recombining the two non-expression vectors to create recombined expression vectors comprising an antibody gene whereby co-expression of the antibody gene and the third drug resistance gene on a single mRNA is enabled, said expression vectors constituting a single-chain antibody library having a repertory of at least 10 11 ; step (e) displaying a single chain antibody comprising VH and VL regions obtained by shuffling on the surface of a phage; and selecting said hosts through use of the third drug resistance gene, wherein the selected hosts comprise the recombined expression vectors.