期刊
NATURE BIOTECHNOLOGY
卷 18, 期 1, 页码 75-80出版社
NATURE AMERICA INC
DOI: 10.1038/71958
关键词
antibody engineering; phage display; recombination; Cre recombinase; filamentous phage; single-chain Fv (scFv)
The creation of large phage antibody libraries has become an important goal in selecting antibodies against any antigen. Here we describe a method for making libraries so large that the complete diversity cannot be accessed using traditional phage technology. This involves the creation of a primary phage scFv library in a phagemid vector containing two nonhomologous lox sites. Contrary to the current dogma, we found that infecting Ore recombinase-expressing bacteria by such a primary library at a high multiplicity of infection results in the entry of many different phagemid into the cell. Exchange of VH and VL genes between such phagemids creates many new VH/VL combinations, all of which are functional. On the basis of the observed recombination, the library is calculated to have a diversity of 3 x 10(11). A library created using this method was validated by the selection of high affinity antibodies against a large number of different protein antigens.
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