Background: Sensitivity to the fungus Alternaria alternata is a common caus
e of asthma, Epidemiologic studies from a variety of locations worldwide in
dicate that A alternata sensitivity is closely linked with the development
of asthma. Furthermore, A alternata sensitivity has been associated with se
vere and potentially fatal attacks of asthma,
Objective: The diagnosis of A alternata sensitivity is hampered by the lack
of standardized and well-characterized allergenic extracts. Molecular clon
ing of allergens offers the possibility of providing large quantities of pu
rified, well-characterized allergens not only for diagnostic purposes but a
lso for studying the pathogenesis of A alternata sensitivity, We used molec
ular cloning to identify, purify, and produce a majors alternata allergen i
n quantity.
Methods: We prepared messenger (m)RNA from A alternata to produce a complem
entary (c)DNA library The library was screened for A alternata allergens by
using sera from A alternata-sensitive individuals. A recombinant allergen
was isolated, the cDNA sequence was determined, and the protein was express
ed in Pichia pastoris,
Results: A unique A alternata allergen, rAlt a 2, was identified. A 2.2-kb
cDNA sequence was obtained that has homology with a common transposable reg
ion and mouse RNA-dependent eukaryote initiation factor-2 a-kinase but no h
omology to any known allergen, No N-glycosylation sites were found in the c
DNA sequence. The recombinant allergen was recognized by IgE antibodies in
the sera of 16 of 26 (61%) individuals allergic to A alternata, which defin
es Alt a 2 as a major allergen.
Conclusions: We have molecularly cloned a unique major A alternata allergen
, rAlt a 2. Identification and expression of the recombinant allergen shoul
d enhance the development of standardized A alternata allergenic extracts a
nd provide stable reagents for investigating the pathogenesis of A alternat
a sensitivity.