Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.603815
Title: Using the loxP/Cre site-specific recombination system in phage display technology
Author: Hartley, O.
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 1997
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Abstract:
A technique for highly efficient combinatorial transformation of heavy and light chain repertoires (combinatorial infection and in vivo recombination) was used to create a phage antibody library of previously unattainable diversity (6.5 x 1010). From this library, antibody fragments of high affinity (up to 3 nM) were isolated directly, providing experimental proof of the value of large repertoires. Subsequently a peptide display library was designed by analogy, where repertoires of ten residue 'exons' were employed in place of antibody genes. Combinatorial infection and in vivo recombination was used in the creation of a highly diverse (>1011) combinatorial peptide library. From this library peptide ligands with exquisite specificity for several proteins were isolated. The useful diversity of large combinatorial phage antibody repertoires is limited by the number of phage which can be accommodated in the panning technique. Extra diversity can be sampled by re-pairing the heavy chain gene of a single selected clone with the original input light chain gene repertoire, or vice versa. The variants can then be reselected to isolate antibodies with improved affinities. A model '3-lox' phage system was developed in which heavy and light chain genes could be shuffled efficiently and independently of each other. This system could now be used to generate large phage-antibody libraries, and should permit rapid shuffling and re-selection of either chain in selected antibodies. A further use for the diversity provided by combinatorial infection and in vivo recombination was then envisaged. A phage display vector was designed into which every element of an antibody repertoire could be shuffled with every element of a library encoding antigens.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.603815  DOI: Not available
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