Gene Cloning, Expression And Biological Activity Of A B Cell Linker (BLNK) Homologue From Lampetra Japonica

B cell linker, as a key protein, is highly conserved in evolution. BLNK are present in B cell and macrophages and possesss various biological functions, including signal transmission and mediation of cell proliferation, differentiation and apoptosis. Agnathans, represented by lamprey, are agreed to be the oldest vertebrates possessing the adaptive immune defenses by now. The study of jawless vertebrate provides a theoretical basis for the origin of adaptive immune defense.So far researches have focused mainly on BLNK in mammalian; little is known about the existence in lamprey. Here, we report the molecular cloning and characterization of a homologue from L. japonica by analyzing the expressed sequence tags (ESTs) of the blood cDNA library. Our researches may contribute to understand the relationship of BLNK and vertebrates.In this paper, a single EST homologous to BLNK was found among the extensive EST sequences from the cDNA library of L. japonica blood. The EST of BLNK is 594 bp and containing SH2 domain. We have successfully cloned BLNK gene using RACE method. Then we study on primary structure alignment, advanced structure prediction, reconstructing a phylogenetic tree and conserved motifs distribution of this sequence. There is no signal peptide in Lj- BLNK. Lj- BLNK shows high sequence similarities with BLNK in other species. As seen from the sequence comparison and phylogenetic tree, BLNK genes are divided into three clusters, mammals, birds, reptiles in a cluster, bony fishes cluster in a cluster, amphibians and lamprey in a cluster. It is consistent with biological objective laws of natural evolution.To determine the expression of Lj- BLNK gene in various tissues, we performed real time quantitative PCR assay with GAPDH as an internal control. Our result demonstrated that Lj- BLNK gene was ubiquitously expressed in a variety of tissues including hearts, livers, kidneys, gills, intestines and white blood cells. Obviously, the level of Lj- BLNK gene expression in blood cells and hearts remained at a high level in LPS and PHA stimulated group, indicating that Lj- BLNK can act as a very effective to protect lamprey from damage and therefore ensure its own survival.Then the Lj- BLNK gene cloned into pET32a was expressed as a histidine tag fusion protein in Escherichia coli BL21 (DE3) with the treatment of IPTG. And we prepared polyclonal antibody against rLj- BLNK. These above results demonstrate that B cell linker may exist in lampreys, the precise signal transmission mechanism of lamprey remained to be elucidated.