Prokaryotic Expression,Antibodies Preparation And Immunological Studies Of L-BLNK-like From Lampetra Japonica

B cell linker,as a intracellular adaptor protein,is widely expressed in B lymphocytes and macrophages.In mammals,BLNK mainly participates in adaptive immunity.In adaptive immunity,BLNK plays an important role in the BCR signaling pathways and is essential for B lymphocyte proliferation,differentiation and apoptosis.The origin of the adaptive immunity can be dated back to the oldest vertebrates Lampetra japonica.Lampetra japonica is agreed to be the oldest jawless vertebrate by now,the fossils traced back to the ordovician.Lampetra japonica has both innate immunity and adaptive immunity,therefore lamprey is an important model for study the origin of adaptive immunity.The early experiment had certified that L-BLNK-like is present in Lampetra japonica.While what function is L-BLNK-like in the Lampetra japonica? Whether L-BLNK-like participates in the signaling induced by VLR in Lampetra japonica? In this paper,we report the molecular cloning,prokaryotic expression,antibodies preparation and characterization of L-BLNK-like which was found among the extensive EST sequences from the cDNA library of Lampetra japonica blood.Our researches may contribute to provide important clues for the research of the signaling induced by VLR in Lampetra japonica.In this paper,a conservative domain of Lampetra japonica L-BLNK-like was linked with the vector pET-28 a to constitute recombinant plasmid.The fusion protein was induced by IPTG and purified by affinity chromatography.Mass spectrometry results showed that the L-BLNK-like is the right one.Rabbit anti-L-BLNK-like polyclonal antibodies were developed following rabbit immuned by Lampetra japonica rL-BLNK-like.After ELISA,the specificity of the antibodies against the L-BLNK-like protein was confirmed by western blot.The antibody could not only detect the rL-BLNK-like,but also be detected in Lampetra japonica.Western blot analysis showed broad distribution of L-BLNK-like in various tissues,and after Lampetra japonica was stimulated with LPS,the level of L-BLNK-like was up-regulated in leukocytes by FACS analysis.As expect,the level of VLRB was also up-regulated in leukocytes from LPS-stimulated Lampetra japonica.Scanning micrographs and western blot analysis demonstrated that VLRB and L-BLNK-like present in the same leukocytes.The results indicated that L-BLNK-like may play an important role in adaptive immunity and may participate in the signaling induced by VLR in Lampetra japonica.These may provide some clues for further studies about adaptive immunity in Lampetra japonica.