An 18 kDa protein isolated from saliva of the cat flea, Ctenocephalides felis, elicits
a positive intradermal skin test (IDST) in 100 and 80% of experimental and clinical
flea allergic dogs, respectively. Using solid-phase enzyme-linked immuno assay (ELISA),
this protein detected IgE in 100 and 80% of experimental and clinical flea allergic
dogs, respectively. A cDNA (pFSI) encoding a full-length Cte f 1 protein was isolated
from a C. felis salivary gland cDNA library, using a combination of PCR and hybridization
screening. This cDNA is 658 bp in length, and contains an open reading frame of 528
bp. The open reading frame encodes a protein of 176 amino acids, consisting of an
18 amino acid signal sequence and a 158 amino acid mature protein. The calculated
molecular weight and pI of the mature protein are 18106 Da and 9.3, respectively.
The protein, named Cte f 1, is the first novel major allergen described for canine
flea allergy. Recombinant Cte f 1 (rCte f 1) was expressed in Escherichia coli, Pichia
pastoris and baculovirus infected Trichoplusia ni cells. Approximately, 90% of the
rCte f 1 expressed in E. coli accumulated in insoluble inclusion bodies, which could
be refolded to a soluble mixture of disulfide isomers with partial IgE binding activity.
Small quantities of an apparently correctly refolded form of rCte f 1, which had IgE
binding activity equal to the native antigen, was isolated from the soluble fraction
of E. coli cells. However, P. pastoris and baculovirus infected insect cells expressed
and secreted a fully processed, correctly refolded and fully active form of rCte f
1. Mass spectrometry analysis of the active forms of rCte f 1confirmed that eight
intact disulfide bonds were present, matching the number observed in the native allergen.
The relative ability of rCte f 1 to bind IgE in the serum of flea allergic animals,
produced in these three expression systems, matched that of the native allergen. Competition
ELISA demonstrated that approximately 90% of the specific IgE binding to native Cte
f 1 could be blocked by the different forms of rCte f 1.