A new combination of mutated loxPs in a vector for construction of phage antibody libraries.
In the construction of large antibody libraries by in vivo recombination, two non-homogeneous loxP sites are required for the exchange of V genes between phagemids to create many new VH-VL combinations. The mutated loxP511 was designed not to recombine with the wild-type loxP (loxPwt) in early studies and a combination of the two has been used to construct antibody libraries. But recent reports have shown that recombination occurs between loxPwt and loxP511. This suggests that the combinational use of loxP511 and loxPwt might lead to the loss of the V gene diversity of antibody libraries. Therefore, it is necessary to find a new combination of loxPs to avoid the excision recombination in the antibody library. In this study, we found that the excision recombination between loxP511 and loxP2272, another mutated loxP sequence, was undetectable within one phagemid, while the excision recombination between loxP511 and loxPwt occurred at a frequency of 40%, higher than that reported previously. Furthermore, the in vivo recombination of different phagemids with loxP511 and loxP2272 showed that the V gene exchange was efficiently mediated to produce new VH-VL combinations. It was concluded that the loxP511 and loxP2272 combination was more favorable for reducing the excision recombination and constructing large phage antibody libraries with high diversity.
Shanghai Medical College, Fudan University, Shanghai 200032, China.
Yu
Y
Gan
Xin-Tai
XT
Zhao
D000906:Antibodies; D001483:Base Sequence; D004251:DNA Transposable Elements / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics*; D005822:Genetic Vectors; D007135:Immunoglobulin Variable Region / Q000235:genetics; D019426:Integrases / Q000235:genetics*; D009154:Mutation; D019151:Peptide Library; D011995:Recombination, Genetic; D014764:Viral Proteins / Q000235:genetics*
D000906:Antibodies; D004251:DNA Transposable Elements; D004279:DNA, Viral; D007135:Immunoglobulin Variable Region; D019151:Peptide Library; D014764:Viral Proteins; C045073:Cre recombinase; D019426:Integrases