Cloning,Expression,Antibody Production And Immunological Related Research On SLP-76 Homolog Of Lampetra Japonica

SLP-76 molecule as an adaptor protein exists in many of hematopoietic cell lineages;its protein sequence structure contains three characteristic domains,such as N-terminal sterile alpha motif(SAM)domain and C-terminal Src homology 2(SH2)domain and a proline-rich region(PRR)in between the other two domains.SLP-76 is critical for downstream signaling of T cell receptor(TCR)and its conjugate protein.Lamprey is a representative species of jawless vertebrates in Cyclostomata.It has attracted extensive attention of immunologists because of its adaptive immune defense system mediated by variable lymphocyte receptors(VLRs).In order to explore the existence and function of SLP-76 molecule in lamprey,we carried out the following works.In this study,the ORF region of lamprey’s SLP76(Lja-SLP76)was amplified using total RNA from the peripheral blood lymphocytes of Lampetra japonica.The coding region of Lja-SLP76 was 1590 bp,encoding a protein containing 529 amino acids.The protein sequence and structure of Lja-SLP76 were predicted and analyzed,and it was found that Lja-SLP76 had three conserved characteristic domains like vertebrate SLP-76,and had high sequence consistency.These results indicate that the SLP-76 family molecule is found in the lampreys,a kind of jawless vertebrate.The quantitative real-time PCR method was used to detect the differential expression of Lja-SLP76 m RNA in the immune related tissues of lamprey after 12 hours of mixed bacteria stimulation.It was found that the transcription level of Lja-SLP76 m RNA was significantly up-regulated in peripheral blood lymphocytes and gills after stimulation,indicating that Lja-SLP76 participates in the immune response of lamprey.Based on the antigenic epitope prediction,the antigenic region for the preparation of Lja-SLP76 antibody was designed.The truncated Lja-SLP76 antigen segment was expressed by prokaryotic expression system and purified by affinity chromatography to obtain the truncated Lja-SLP76 recombinant protein.The truncated Lja-SLP76 recombinant protein was used as antigen to immunize healthy New Zealand rabbits to prepare polyclonal antibodies.After five times of enhanced immunization,rabbit anti-Lja-SLP76 polyclonal antibodies with a titer of 1:128000 were finally obtained.The expression of Lja-SLP76 in the immune related tissues of lamprey was detected by Western blotting after stimulated by LPS,PHA and mixed bacteria for 24 hours.The results showed that the expression of Lja-SLP76 in peripheral blood lymphocytes of lamprey was significantly down regulated,but the expression of Lja-SLP76 in gills and supraneural myeloid bodies was significantly up-regulated after LPS stimulation.The interaction between Lja-SLP76 protein and membrane proteins such as myonectin,myosin,actin,cytokeratin,oxidized low density lipoprotein and glycosyltransferase protein was determined by immunoprecipitation technique,which indicated that Lja-SLP76 may be closely related to the membrane localization of signal transduction complex.Our results lay a foundation for further study on the molecular mechanism of Lja-SLP76 involved in VLR signal transduction.