Biology Reference
In-Depth Information
subtype H9N2. Four rounds of panning were carried out, each with a slight increase in
stringency to isolate high-affinity peptide ligands.
Table 1 shows the heptapeptide sequences obtained from four rounds of panning
the peptide library against AIV subtype H9N2. Seventeen out of 35 phages analyzed
from the fourth round represented the sequence NDFRSKT and other major sequences
found in the fi nal round of panning were LPYAAKH and ILGDKVG. A new sequence
carrying the peptide QHSTKWF emerged during the fourth round of panning repre-
sented 10% of the total phages sequenced.
Table 1.
Heptapeptides binding to AIV subtype H9N2 and streptavidin selected from the phage
display random peptide library.
Rounds of panning
Heptapeptide sequences
Frequency of sequences (%)
4<h round
NDFRSKT
47
QHSTKWF
10.5
LPYAAKH
5
ILGDKVG
5
Unrelated sequences
23
Panning of Streptavidin
3rd round Streptavidin
HPQFLSL
55
GLYNHPQ
27
Unrelated sequences
18
Biopanning of the phage library against streptavidin (the positive control) gave
a consensus sequences containing HPQ motif which totally represented 82% of the
total phages screened from the third round of panning and these results are in good
agreement to the reported fi ndings [20-22]. No recognizable consensus sequence was
observed with BSA, which served as a negative control. The peptide NDFRSKT was
named as P1 (C-P1--cyclic form; L-P1--Linear form; FP-P1--fusion phage display-
ing this peptide).
Estimation of Binding Abilities of Selected Phage Clones
Recombinant phages selected from the fourth round of panning were further analyzed
for their binding specificity by phage-ELISA which was carried out with all the four
recombinant phages in varying phage concentrations against two different virus con-
centrations (5 μg and 10 μg/100 μl). The results (Figure 1) showed that all the phages
selected from the biopanning were able to bind the virus efficiently and the higher
the concentration of the recombinant phages, the higher the signal irrespective of the
concentration of the virus.
