Abstract:

For acquiring more information about crab allergens, a 21 kD protein was purified from Scylla paramamosain by
ammonium sulfate fractionation, anion exchange chromatography and gel filtration column chromatography. The protein
was identified as a novel allergen having IgE binding activity with serum from shellfish-allergic patients. According to the
results of Western blotting and matrix assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF/
TOF-MS) studies, the protein was confirmed as a sarcoplasmic calcium binding protein (SCP). The method of switching
mechanism at RNA termini-rapid amplification of cDNA ends (SMART-RACE) was applied to clone the cDNA of SCP.
A full length cDNA with 986 bp was obtained, which included an open reading frame (ORF) coding for 193 amino acid
residues with a predicted molecular weight of 21.94 kD and theoretical isoelectric point of 4.44. The protein had a high
homology with SCP from other shellfishes, but the homology with insects was low. Its tertiary structure was constructed
using Phyre 2.0 server and the results showed that the SCP had five helix-loop-helix motifs, namely EF-hand domains, two
of which formed two Ca2+ binding sites. Furthermore, bioinformatics analysis predicted that this SCP contained four linear
and three conformational epitopes.

Key words: Scylla paramamosain, novel allergen, sarcoplasmic calcium binding protein, purification, mass spectrometry;cloning, bioinformatics analysis