Molecular cloning, expression pattern and antimicrobial activity of a new isoform of anti-lipopolysaccharide factor from the swimming crab Portunus trituberculatus.

A new isoform of anti-lipopolysaccharide factor (PtALF5) was cloned from eyestalk cDNA library of swimming crab Portunus trituberculatus. The full-length cDNA of PtALF5 was 1045 bp encoding 120 amino acids. PtALF5 shared lower amino acid similarity with other ALFs, yet it contained the conserved LPS-binding domain and was clearly member of the ALF family. The genomic fragment of PtALF5 contained two exons separated by one intron. Several tandem repeats were found in intron. The mRNA transcript of PtALF5 was predominantly expressed in the hemocytes but barely detectable in muscle. After challenge with Vibrio alginolyticus, a main pathogen causing high mortality in P. trituberculatus, the PtALF5 transcript in hemocytes showed a clear time-dependent response expression pattern with obvious decrease at 6 h and significant increase at 24 h. The recombinant PtALF5 protein revealed antimicrobial activity against Gram-negative bacteria V. alginolyticus and Pseudomonas aeruginosa, but did not inhibit the growth of the tested Gram-positive bacteria and fungus. These results together suggest that PtALF5 is a potent antibacterial protein against Gram-negative bacteria infection, and might function as a promising therapeutic agent in disease control of crab aquaculture.