Cloning, Expression And Function Analysis Of The G-type Lysozyme And Glutathione Peroxidase 2 From Lampetra Japonica

The lysozyme is a key protein of the innate immune system to fight bacterial pathogens infections. Our work has been focused on the molecular characterization, expression analysis on the g-type lysozyme from lamprey(Lampetra japonica). Moreover, the antibacterial activity was studied using a recombinant protein produced in Escherichia coli. The cloned lamprey cDNA of g-type lysozyme was 701 bp in length with an open reading frame(ORF) of 555 bp. Real-time PCR results showed that the lamprey g-type lysozyme transcript was detected in multiple tissues with the highest expression in intestine. Furthermore, upon in vivo stimulation of lampreys with lipopolysaccharide(LPS), the lysozyme was significantly up regulated in buccal gland and head kidney. The bacteriolytic activity of recombinant lysozyme was optimal at pH 7.5 and at a temperature of 35℃ using Micrococcus lysodeikticus as substrate. In addition, from scanning electron microscopy(SEM) analysis, M. lysodeikticus cells were damaged by the lysozyme. Collectively these data suggest that the identified g-type lysozyme should be involved in the innate immune responses of lamprey against pathogen infection.Glutathione peroxidase 2(GPx2) is an important antioxidant enzyme in vivo that enzymatically reduces hydrogen peroxide to limit reactive oxygen species(ROS) harmful effects in living organisms. In this study, the full-length GPx2 gene and the genomic DNA sequences were cloned from the blood cDNA library of Lampetra japonica(L. japonica). The sequence analysis showed that the full-length cDNA of 868 bp includes an open reading frame(ORF) of 564 bp plus 53 bp of the 5?-untranslated region(5?UTR) and 251 bp of the 3?-untranslated region(3?UTR). Genomic DNA sequences included a two exon-one intron structure. Phylogenetic analysis revealed that GPx2(LjGPx2) is closely related to other GPx2. The real-time PCR results demonstrated that the GPx2 gene was widely expressed in adult lamprey in which higher levels in buccal glands, heart, ovum, gonad. Furthermore, upon in vivo stimulation of lampreys with lipopolysaccharide(LPS), LjGPx2 mRNA was upregulated in gonad. The results obtained in the present study will help us to understand the roles of GPx2 in immune responses in jawless vertebrates.