Molecular Cloning, Recombinant Expression, and Characterization of Lysozyme from Chinese Shrimp Fenneropenaeus chinensis

Lysozyme catalyzes the hydrolysis of bacterial cell walls and acts as an innate immunity molecule against the invasion of bacterial pathogens. We cloned the cDNA of lysozyme from Fenneropenaeus chinensis and named it as Fc-lysozyme (FcLyz in short). The full length of the gene is of 709 bp, and the open reading frame (477 bp) encodes 158 amino acids. The predicted protein has a signal peptide (-1∼-18 residue) and the molecular weight of the mature protein (residue 1-140) is of 16.2 kD. A Lyz 1 domain (residue 1-130) in the FcLyz was identified by SMART analysis. The results of semi-quantity RT-PCR showed that FcLyz was constitutively expressed in tested tissues in a low level in normal shrimp, and up-regulated in hemocytes, heart, hepatopancreas and gill of bacterial challenged shrimp. The DNA fragment of mature Fc-Lys was subcloned to pET-30a (+) expression vector, and the recombinant plasmid was transformed into Escherichia coli BL21 (DE3) and then induced by isopropylthio-β-D-galactoside (IPTG). The antibacterial activity of the purified recombinant FcLys was analyzed and minimal inhibitory concentration (MIC) was assayed. The recombinant protein showed high antibacterial activity against some Gram-positive bacteria and the MIC reached 3.43 μM, while it showed relatively low activity against Gram-negative bacteria. All together, the Fc-Lys was regulated by pathogen infection and had antibacterial activity. This suggested that the FcLyz may be one of the important effector molecules against pathogens in innate immunity of the shrimp.