Cloning, Expression And Bioinformatics Analysis Of Lck-like Gene From Lampetra Japonica

Lymphocyte-specific protein tyrosine kinase (Lck) is an important kind of SRC family protein tyrosine kinase (PTK) that is widely expressed in organisms. It is the primary signal downstream activated by TCR and involved in phosphorylation of protein molecules during the process of signal transduction. The research shows that Lck plays a very important role in mediating TCR signal transduction and the development, differentiation, proliferation and activation of T cells. Lampetra japonica, as the interim species of invertebrates and vertebrates, is regarded as the new model organism to study the origin and evolution of vertebrates. The current study on Lck mainly focuses on mammals while it is still unknown whether Lck can be found in Lampetra japonica, the most ancient jawless vertebrate. In this paper, with the help of molecular biology and bioinformatics measures, a piece of sequence that is homologous with Lck has been found in the lab-built blood cDNA library of Lampetra japonica, then we proceeded with gene colone, expression and bioinformatics analysis, which might help us to make further study on the oringin and evolution of Lck protein in vertebrates and the signal transduction pathway of Lck-mediated Lampetra japonica at the molecular level.In this study, a piece of EST sequence that is highly homologous with SRC family kinase has been found in the lab-built blood cDNA library of Lampetra japonica after searching more than3300pieces of EST sequences. After making homology analysis, we found this piece of EST sequence highly homologous with the ORF3’ter minal sequence of SRC-like gene in Lampetra reissneri. According to the structure features of particular gene, we designed the primer, spliced following3’RACE,5’RACE and the sequencing result, and successfully get the full length of cDNA of Lck-like in Lampetra japonica. Through comparing with the primary sequence of Lck amino acid in species of different stages from lower to higher level, the phylogenetic tree has been constructed based on neighbouring and other methods. Furthermore, we also analyzed the secondary structure, predicted the tertiary structure, the hydrophilicity, the disulfide bond, the antigenic epitope and the distribution of conserved motif.Through performing Real Time PCR assay, we designed specifified primer and set GAPDH as internal control gene. We analyzed the relative expression level of Lck-like in heart, supraneural body, kidney, intestine, liver, gill, leukocyte of normal saline-stimulated and LPS-stimulated Lampetra japonica, and the results indicated that Lck-like was widely expressed in the above tissues. After being further stimulated by LPS, the expression level of Lck-like in the above tissues was upregulated, especially obivious in heart with statistical significance. This shows that Lck-like plays a very important role in signal transduction.In the further step, we built prokaryotic expression vectors pGEX-4T-l-Lck-like and successfully expressed in E.coli BL21.In conclusion, the discovery of Lck-like in Lampetra japonica, the existing and most ancient jawless vertebrate, provides us the clue to better understand the origin and evolution of adaptive immune system in ancient vertebrates. The specific approach to be involved in signal transduction, however, is still unknown.