A new anti-lipopolysaccharide factor (EsALF-3) from Eriocheir sinensis with antimicrobial activity

The cDNA of a new Eriocheir sinensis ALF (designated as EsALF-3) was obtained based on EST analysis. The full-length cDNA was of 956 bp, consisting of an open reading frame (ORF) of 369 bp encoding a polypeptide of 123 amino acids. In the deduced amino acid sequence of EsALF-3, there were two highly conserved cysteine residues to define the LPS binding site, and eight positively charged residues existed between the two cysteine residues. The phylogenetic analysis revealed that EsALF-3 was clustered with the ALFs from some crustaceans, forming a separate sister branch to the group of EsALF-1 and EsALF-2 from E.sinensis. The mRNA transcript of EsALF-3 was detected in all the examined tissues of crabs, including haemocytes, hepatopancreas, gill, muscle, heart and gonad. The recombinant protein of EsALF-3 represented antimicrobial activity against Listonella anguillarum, Escherichia coli and Bacillus subtilis with minimal inhibitory concentration (MIC) values of 33.75 μg mL ^-1, 270 μg mL ^-1 and 135 μg mL ^-1, respectively. These results together indicated that there were multiplicate ALF isoforms coexist in E. sinensis, and the diversity of the immune effector molecules in innate immune system, such as ALF, helps Chinese mitten crab to deal with various pathogens in the aquatic environment.