Identification and functional characterization of a g-type lysozyme gene of Labeo rohita, an Indian major carp species.

ABSTRACT

Lysozyme, an important secretory innate immune component, possesses antimicrobial activity against broad spectrum of bacteria and viruses. In the present study, complete CDs (558 bps) of g-type lysozyme of rohu (Labeo rohita) was amplified and translated for a putative protein of 185 amino acids. The domain architecture and tertiary structure was also predicted for the protein. Its expression profile was studied in three infection models (bacteria Aeromonas hydrophila, poly IC, a dsRNA viral analogue and an ectoparasite Argulus siamensis) in liver and kidney tissues of rohu. An up-regulation of 630-fold and 420-fold of the gene was observed at 48 h in liver and anterior kidney tissues respectively, after A. hydrophila infection. Significant increase in transcript level was noticed in both liver (0.8-fold) and kidney (480-fold) after 1 h and 12 h of poly IC induction, respectively. Similarly, expression of lysozyme g transcripts was increased 6000-fold after 7 d of A. siamensis infection in liver tissue. The recombinant protein of g-type lysozyme of rohu (rLr-lysG) of 20.19 kDa was produced in Escherichia coli system and the lysozyme activity of rLr-lysG was found to be most active at pH 6.0 and temperature 35 °C. The potential lytic activity was found to be against A. hydrophila (UL = 0.53) followed by for E. tarda (UL = 0.45) whereas the lytic activity was the least against S. aureus (UL = 0.35) and M. lysodeikticus (UL = 0.34), at pH 6.0 and temperature 35 °C. The normal serum level of protein was estimated using indirect ELISA and was found to be very low (0.12-0.15 µg/ml). These results suggested that g-type lysozyme of rohu might be a potent immunostimulant against microbial infections, with a major role in innate immunity.