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Short Communication
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Acta
Biochim Biophys Sin 2005,37:495-500 doi:10.1111/j.1745-7270.2005.00068.x A New Combination of Mutated
loxPs in a Vector for Construction of Phage Antibody Libraries Yu GAN1,2 and
Xin-Tai ZHAO2* 1 Shanghai Medical College, Fudan
University, Shanghai 200032, China; 2 Laboratory for Cellular and Molecular
Immunology, Cancer Institute, Shanghai Jiaotong University, Shanghai 200032,
China Abstract 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. Key words mutated loxP; excision
recombination; phage antibody library |
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Copyright©2005 Acta Biochimica et
Biophysica Sinica |