Molecular Cloning, Expression Pattern,bioinformatics Analysis And Immunological Studies Of The Syk In Lamprey, Lampetra Japonica

Syk(Spleen tyrosine kinase), a member of Syk family of cytoplasmic non-receptor tyrosine kinases, is a key component of B cell receptor signaling and regulates multiple physiological functions of B lymphocytes in vertebrates. In this study, a piece of EST sequence that is surely Syk sequence which homologous sequence alignment and sea lampreys database comparison has been found in the blood cDNA library of Lampetra japonica after searching database. Through 3’RACE and open reading frame experiments result, and successfully get the full length of cDNA of Syk in Lampetra japonica.Through comparing with the sequence of Syk amino acid in species of different species, the phylogenetic tree has been constructed that analysis the relationship of species.Furthermore, we also analyzed the structure prediction, conserved motif by MEME and antigen epitope.Through performing Real Time PCR assay, Syk is analyzed for the control group and the mixed bacteria stimulate lamprey relative expression.The result show when the unstimulated Syk widely expressed in these tissues. When bacteria stimulate by mixing in all tissues above Syk has upregulated this amount, especially obivious in gill, leukocyte cells, kidney and heart.The expression of the supraneural body have also been raised. Furthermore detected by Western Blot, Syk in the gills, supraneural body and leukocyte cells of myeloid bodies expressed when the unstimulated. After mixed bacteria stimulate the expression of Syk in supraneural body and leukocyte cells was significantly upregulated above were statistically significant. It is suggested that Syk participate lamprey immunoregulation plays a crucial role.In the further step, we built prokaryotic expression vectors and successfully expressed in E.coli BL21. Immunoprecipitation was conducted to precipitate the two proteins that might interact with Syk of the mass spectrography. By analyzing the cellular localization of leukocyte cells before and after immunization, the results show that Syk before and after immunization were present in the cytoplasm.The above data, at the molecular level will help to explore the origin and evolution of the adaptive immune system Syk protein and study some of the new features of vertebrates provide clues.