including egg allergy. SIT involves the administration of allergen
starting at very small amounts and increasing to a higher maintenance
dosing, to the patients to promote tolerance by the immune
system (Akdis and Akdis, 2011). Egg allergic patients react to each
allergen at different intensities; therefore, it will be important to
administer the allergens separately in SIT to better manage adverse
reactions. However, isolating egg white allergens without contamination
from the remaining allergens is extremely difficult, time
consuming and expensive. Production of recombinant variants of
allergens is an effective alternative to natural allergens, since the
allergen is produced independently of the others. In this study, we
aimed to produce recombinant egg white allergens and compared
their IgE reactivity within a defined Australian patient group. The
recombinant versions of Gal d 1, 2 and 3 that we produced showed
IgE reactivity when tested against allergic patients’ sera.
When PCR amplifying cDNA sequences for cloning purposes, it
is vitalto use methods that generate minimal errors. Gal d 1, 2 and 4
were successfully amplified using the standard PCR methods, however
for Gal d 3 a long-range PCR was used to amplify its longer
length. The PCR products shown in Fig. 1 did not appear on the
gel exactly at the right molecular weight. This can be attributed to
the conditions of gel run including the percentage of agarose in the
gel. The amount of DNA loaded onto the gel may also have affected
the predicted size. The subsequent cloning in pTrcHisA vector and
sequencing (results not shown) confirmed that the PCR amplifi-
cation of cDNA was successful. Gal d 2 and Gal d 3 showed 99%
similarity to the published sequences on IUIS/NCBI, this may be
attributed to PCR or sequencing error due to their larger number of
nucleotides compared to Gal d 1 and Gal d 4 which showed 100%
similarity to the published sequences. The Express I
q E. coli strain
was chosenfor expressionofthe allergens because thisBL21derivative
is tolerant to toxic proteins. Gal d 1 is a trypsin inhibitor and
Gal d 4 is an antibacterial agent, therefore this strain of E. coli was
ideal for protein expression.