[Cloning and expression of VLRB of Lampetra japonica and generation of the corresponding monoclonal antibodies].

Abstract

The agnathans (lampreys and hagfishes) are representatives of the jawless vertebrates. The receptor molecules of adaptive immune system in lampreys are different from the antigen receptors in mammal vertebrates. The unique receptor molecules of lampreys are known as variable lymphocyte receptors (VLR). There are three types of VLRs in lampreys, VLRA, VLRB, and VLRC. Multimeric antigen-specific VLRB antibodies are secreted by VLRB+ lymphocytes and constitute the major components of the humoral arm of the lamprey adaptive immune system. Oligomeric VLRB antibodies are composed of four or five disulfide-linked dimeric subunits, which are similar to IgM antibodies in structure and function. In this study, the conservative c-terminal of Lampetra japonica VLRB was cloned and expressed in BL21 E. coli. The recombinant VLRB protein was purified by Ni2+ affinity chromatography column. After Balb/c mice immunity, cell fusion, the positive clones were screened by indirect enzyme-linked immunosorbent assay (ELISA). Finally, the hybridoma cells that produced specific anti-VLRB monoclonal antibodies were obtained. In order to get a large number of antibodies against VLRB, the hybridoma cells were injected into the abdominal cavity of Balb/c mice and the antibodies were purified by protein G sepharose. The results of ELISA indicated that the valence of anti-VLRB antibodies was 1:40000. Western blotting assay showed that the antibodies were able to detect both recombinant VLRB and secreted VLRB in lamprey sera. Flow cytometry analysis also revealed the existence of VLRB on the surface of lymphocytes. In summary, the anti-VLRB monoclonal antibodies provided a major tool for studying lamprey adaptive immune system.