Molecular Cloning,expression Pattern,antibody Production And Immunological Studies Of The Lyn-like In Lamprey,lampetra Japonica

The tyrosine kinase Lyn,a member of Src family kinase(SFKs),plays a very important role in cell growth,differentiation,migration and adhesion.In this study,a homolog of Lyn was identified in the lamprey,Lamptra japonica for the first time and was named Lyn-like,which was cloned and got a complete open reading frame(ORF).The cDNA fragment of lamprey Lyn-like contains a 1515-bp ORF and encodes a protein that contains 504 amino acids.Multiple sequence alignment analysis revealed that it has four conserved domains including a Src homology(SH)4 domain,a SH3 domain,a SH2 domain,a protein kinases catalytic(PKC)domain and a variable Unique domain(UD).The identities among Lyn-like and Lyn molecules in unique domain are relatively higher than that among Lyn and Hck molecules,although Lyn-like possesses high sequence similarity with Lyn and Hck molecules of mammals.The sequences were searched from fishes to mammals in the Genebank database and the phylogenetic analysis the results showed that Lyn and Hck molecules are grouped into two obvious distinct phylogenetic clusters,and Lyn-like is grouped as a single branch in Lyn cluster.These results indicated that Lyn-like is a homolog of Lyn rather than Hck.The real-time quantitative PCR assay showed the Lyn-like m RNA in lamprey immune related tissues distributed widely.The levels of Lyn-like mRNA were significantly up-regulated in the lymphocytes and supraneural myeloid bodies,and similar expression patterns of Lyn-like protein were confirmed by western blot analysis after stimulation with mixed antigens.The immunofluorescence assay demonstrated that Lyn-like was expressed in variable lymphocyte receptor(VLR)B~+lymphocyte sub group as a cytoplasmic protein.Our findings indicated that the Lyn-like identified in lampreys might play a significant role in the VLRB~+-mediated adaptive immune response.