Molecular Cloning,Expression Pattern,Antibody Production And Immunological Studies Of The Bam32 Molecule In Lamprey,Lampetra Japonica

Bam32 is also called DAPP1(dual adaptor for phosphotyrosine and 3-phosphoinositides)or PHISH(3? phosphoinositide-interacting SH2 domain containing protein).As a lymphocyte adaptor protein,Bam32 plays important roles in B cell receptor signalling and antibody affinity maturation in germinal centres.So far,most of the studies about Bam32 are restricted in vertebrate,but little is known about the existence and function of Bam32 in jawless vertebrate.Lamprey is a representative of jawless vertebrate,we chose it as research subject to identify and verify the existence and function of Bam32 so as to provide some clues for deeply understanding the immune response mechanism of adaptive immune system of jawless vertebrate.In the present study,a piece of c DNA sequence which is homologous to vertebrate Bam32 was identified in cDNA library of Lampetra japonica after searching the sequencing database.The full length cDNA sequence of Bam32 of Lampetra japonica was successfully cloned through rapid-amplification of 3' cDNA ends technique and its open reading frame(ORF)was amplified by pyrobest DNA polymerase.Bam32 of Lamptra japonica(Lja-Bam32)contains a 747-bp ORF and encodes a protein that contains 249 amino acids.Multiple sequence alignment analysis revealed that it has two conserved domains including a SH2 domain(Src homology 2 domain),a PH domain(Pleckstrin Homology),and a Tyrosine phosphorylation site.The phylogenetic tree has been re-constructed by using neighbor-jointing methods after comparing the sequences of Bam32 in species of different Classes.The grouping results of these molecules are accordance to the evolutional relationship of these animals.Furthermore,we have successfully built Lja-Bam32 prokaryotic expression vectors and expressed in E.coli BL21 strain.The recombinant protein of Lja-Bam32 is obtained and purified by the inclusion body protein purification methods.The rabbit anti-Lja-Bam32 polyclonal antibody was generated by immuning the New Zealand rabbit whith the purified recombinant proteins,the antibody titer and specificity were checked by ELISA method and western blot,respectively.The mRNA level of Lja-Bam32 is detected by Q-PCR method and compared between animal group with or without stimulated by mixed antigens.The results showed that,compared to none immune stimulation group,the lja-bam32 mRNA levels expressed in lymphocyte-like cells,gill and supraneural myeloid body were increases significantly in quantity stimulation by mixed bacteria.Western blot assay showed that the relative expression levels of Lja-Bam32 protein increased significantly in lymphocytes and supraneural myeloid bodies after mixed bacteria stimulation.In conclusion,Lja-Bam32 may paticipate in the immune response process of the lymphocyte-like B cells of the jawless vertebrate.