Abstract
Tropomyosin (TM) from Chinese mitten crab ( Eriocheir sinensis) was purified to homogeneity and TM genes were amplified from three species of crab (Chinese mitten crab, mud crab and swimming crab), respectively. Sequence analysis showed that all three cloned DNA fragments had open reading frames of 855 bp, predicted to encode proteins with 284 amino acid residues. Sequence alignment revealed that the three tropomyosins share high homology to tropomyosins from other crustaceans. Chinese mitten crab TM gene was further recombined with the vector of pGEX-4T-3 and expressed in Escherichia coli JM109. The expressed protein revealed a band of about 62 kDa on SDS–PAGE, suggesting the successful expression of glutathione S-transferase–tropomyosin (GST–TM) fusion protein. Immunoblotting analysis using sera from subjects with crustacean allergy confirmed that the expressed fusion protein reacted positively with these sera, indicating tropomyosin is a major allergen of Chinese mitten crab.
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