Cloning, Expression And Immunological Research Of Fyn-like Cording CDNA From Lampetra Japonica

Fyn is a member of the non-receptor protein tyrosine kinase avian sarcoma virus transforming gene(Src) family, it plays a very important role in cell growth, survival, apoptosis, tumor formation, the adhesion between cells and immune response. Lamprey is one of the most primitive jawless vertebrates; it becomes a highly valuable research model because it connects the vertebrates and invertebrates in evolution. It not only has the innate immune system, but also has the adaptive immune system at the same time. In the researches nowadays, the study of the immune systems of lampreys has always been the hot spot of the current studies.In the current study, a Fyn-like is discovered from a cDNA library of supraneural myeloid body in the lamprey(Lampetra japonica) for the first time. The open reading frame(ORF) were cloned and get a complete ORF.The cDNA of lamprey Fyn-like contained a 1424-bp 3’-UTR and a 1611-bp open reading frame(ORF) which encodes a 537 amino acids protein. Multi-alignment analysis indicated it has high sequence homology with Fyn, Src and Yes from other vertebrates. It also has three conserved domains inclouding a Src homology(SH) 3 domain, a SH2 domain and a protein kinases catalytic(PKc) domain. The expression levels of Fyn-like on the transcriptional and the post-translational levels were detected in five organs(gill, supraneural myeloid bodies, lymphocyte-like cells, kidneys and hearts) of lampreys by real time quantitative PCR and western blot experiments, respectively. Real-time PCR assay has revealed that the level of Fyn-like was obviously upregulated in the gill and leukocyte after LPS stimulation and the Western blot experiment get the same results.These results show that non-receptor protein tyrosine kinase Fyn-like is likely to be involved in immune response. It was found that Fyn-like proteins are mainly distributed in the cytoplasm by immunofluorescence experiment. It may also participate in multiple signaling pathways in the body, and adjust the cell biology function. The relative expression quantities of Fyn-like protein were also tested every 8 hours after boost immunization. It was found that the expression quantity of Fyn-like protein was highest at the 16 h, and then reduced gradually. at last, our present findings also provide the necessary information for researching the evolutionary relationship between the members of Src family.