|
PROJECT DESCRIPTION:
Library of
phage-displayed single chain variable fragment (ScFv) of antibody
has been use for deriving tailor-made antigen-specific monoclonal
antibody (mAb) in the last decade. Furthermore, affinity enhancement
of ScFv can be achieved by in vitro mutation. However, in addition
to the shortage of retrieving a smaller repertoire pool, the use of
mRNA as antibody template is also hampered by the inability of
retrieving immunoglobulin genes (Ig) mRNA of poor immunogenic
targets, e.g. haptens, and Ig genes that masked by class selection,
clonal deletion, negative selection, self-tolerance and
non-productive exon joining. Here a novel platform technology for
antibody engineering was developed. Briefly, a set of degenerated
primers, which covers most of Ig genes, was used to recover the
variable regions of immunoglobulin heavy and £e/£f-light chains (VH
& VL-£e/£f) from sorted CD+19 lymphocytic genomic DNA with a
semi-nested PCR method. Moreover, a supplementary PCR step was used
to recover defective Ig genes resulting from non-productive exon
joining and to introduce diversity into the CDR3 region of
immunoglobulin by mimicking somatic recombination. Feasibility of
applying current method for preparation of antigen-specific antibody
was confirmed by having constructed a small scFv phage-displayed
library (5.16 x 105 recombinants) from CD19+ cells that derived from
a Balb/C mouse which was immunized with phenyl-oxazolone (phOx) -
chicken serum albumin (CSA) conjugate. After 5 rounds of panning
against phOx conjugated to bovine serum albumin (BSA), potential
candidate clones (9.7 x 105 recombinants) were identified. Clones
(288) were randomly picked and their reactivity against phOx were
determined by phageELISA. Forty-four highly reactive clones, of
which reactivity towards phOx were 1.5-fold higher than the mean
value of the sample set, were isolated and further analyzed. After
sequence determination, phylogenic analysis suggested that the
derived Ig genes were grouped into different classes and significant
sequence variations were found within the CDR3 region of Ig genes in
each class. Furthermore, with the use of phOx-BSA conjugate as free
ligand, competitive phageELISA indicated significant differences in
affinity among different clones. In conclusion, our approach offers
a fast and simple way to retrieve the variable region of Ig genes
and simultaneously introducing sequence diversity in the CDR3 region
of antigen recognition domain. |
