Cloning, Expression And Immunological Research Of Tec Cording CDNA From Lampetra Japonica

Tyrosine-protein kinase Tec(Tec), a kind of non-receptor tyrosine kinase, is primarily found to be expressed in T cells, B cells, hematopoietic cells and liver cells as a cytoplasmic protein. Tec is proved involve in G-protein-coupled receptors and interleukin-mediated signaling pathways, and regulate many biological processes. It is also an important regulatory molecule of T cell receptor signaling. In the current study, we successfully cloned a cDNA fragment of Tec homolog from the blood cDNA library of Lampetra japonica. The cDNA fragment of Tec of L. japonica(Lja-Tec) contained a 1923-bp open reading frame, a 69-bp 5’-untranslated region(UTR) and a 1037-bp 3’-UTR. Lja-Tec contains a pleckstrin homology domain, a Src homology(SH) 3 domain, a SH2 domain and a tyrosine kinase domain by its sequence functional domains prediction. Moreover, bioinformatics methods were used for analyzing Tec sequence. A phylogenic tree was reconstructed with 24 Tecs selected from representative species of jawed and jawless vertebrates from lamprey to mammals, and the molecular evolution profile of conserved motif of those Tecs was analyzed by MEME software. We found that Lja-Tec was clustered as the out-group of the vertebrate’s evolutional branch, and this is consisted with the evolutionary process.Lja-Tec was detected to be widely distributed in immune related tissues such as supraneural myeloid bodies, gills, lymphocyte-like cells, kidneys and hearts of lamprey by Real-time quantitative PCR. We found that the transcription levels in lymphocyte-like cells and gills were significantly up-regulated after injection with mixed pathogens. At the same time, the expression of Lja-Tec was analyzed in above tissues before and after stimulation by Western blot. We found that the expression of Lja-Tec was significantly up-regulated in lymphocyte-like cells after stimulation. These results indicated that Tec may be involved in the immune response and play an important role in the lamprey adaptive immunity.We constructed the expression vector Pet32a(+)-Tec, and transformed the vector into E.coli BL21 for expression by induction with 0.1mmol/L IPTG, 30?C. Tec recombinant protein is mainly expressed in the deposit as a insoluble protein. We obtained the purified Tec recombinant protein by the histidine affinity chromatography with a purification method suitable for inclusion body. The Western blot analysis was performed with an anti-Lja-Tec antibody to determine the expression pattern of Tec recombinant protein and Tec natural protein. The results showed that Tec protein in lymphocyte-like cells have obvious change before and after stimulation of mixed pathogens in lamprey. Then, the interacting proteins to the Lja-Tec were detected by immunoprecipitation, and the results still need further verification. The Immunofluorescence result showed that Tec is a cytoplasmic protein localized in lymphocyte-like cells.In summary, the above research provided important clues for the further study of Lja-Tec function and immune mechanism of the Agnatha.