Molecular cloning, recombinant expression and characterization of lysozyme from Chinese shrimp Fenneropenaeus chinensis.
- Author:
Xingjiang BU
1
;
Xinjun DU
;
Wenjie ZHOU
;
Xiaofan ZHAO
;
Jinxing WANG
Author Information
1. School of Life Sciences, Shandong University, Jinan 250100, China.
- Publication Type:Journal Article
- MeSH:
Amino Acid Sequence;
Animals;
Anti-Bacterial Agents;
biosynthesis;
Base Sequence;
Cloning, Molecular;
DNA, Complementary;
genetics;
Escherichia coli;
genetics;
metabolism;
Molecular Sequence Data;
Muramidase;
biosynthesis;
genetics;
Penaeidae;
enzymology;
genetics;
Recombinant Proteins;
biosynthesis;
genetics;
pharmacology
- From:
Chinese Journal of Biotechnology
2008;24(5):723-732
- CountryChina
- Language:Chinese
-
Abstract:
Lysozyme hydrolyses bacterial cell walls and acts as a nonspecific innate immunity molecule against the invasion of bacterial pathogens. We cloned the cDNA of lysozyme from Fenneropenaeus chinensis and named Fc-lysozyme (FcLyz in short). The full length of the gene was of 709 bp, and the open reading frame (477 bp) encoded 158 amino acids. The predicted protein had a signal peptide (-1--18 residue) and molecular weight of the mature protein (residue 1-140) was of 16.2 kD. A Lyz 1 domain (residue 1-130) in the lysozyme was found by SMART analysis. The results of semiquantity 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, the recombinant plasmid was transformed into Escherichia coli BL21 (DE3) and then induced by isopropylthio-beta-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 MIC reached 3.43 micromol/L, and 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 molecules against pathogens in innate immunity of the shrimp.
