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

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.

[Research progress on the MACPF/CDC family of pore-forming toxins].

Pore-forming toxins (PFTs) are commonly associated with bacterial and other prokaryote pathogenesis. In eukaryotes, PFTs play important roles in immunity, embryonic development and neural-cell migration. Two families of globular protein PFTs: the cholesterol-dependent cytolysins (CDCs) and the membrane attack complex/perforin superfamily (MACPF), arrest researchers' attention due to their unique molecular configuration and the function. CDCs are produced by Gram-positive bacteria and disrupt the membrane of host cells during infection. In eukaryotes, MACPF proteins have both lytic and non-lytic roles. With perfringolysin O (PFO) for example, the structure and molecular mechanism of CDCs are relatively well characterized. Recently crystallographic together with biochemical studies revealed that although MACPF and CDCs were extremely divergent at the amino acid sequence level, their common fold suggests that lytic MACPF proteins use a CDC-like mechanism of membrane disruption. This paper focuses on the structure, molecular mechanism of pore-formation and the research hotspot of the two families of PFTs, providing valuable clues for the future research in this field.

[Progress of adaptive immunity system of agnathan vertebrates].

Extant jawless vertebrates, represented by lampreys and hagfishes, have innate immune receptors with variable domains structurally resembling T/B-cell receptors. However, they lack cardinal elements of adaptive immunity shared by all jawed vertebrates: T/B-cell receptors and major histocompatibility complex molecules. Thus, it is widely believed that adaptive immunity is unique to jawed vertebrates. Recently, this belief was overturned by the discovery of agnathan antigen receptors, known as variable lymphocyte receptors. These receptors generate diversity in their antigen-binding sites through assembling highly diverse leucine-rich repeat modules. The crystal structures of hagfish variable lymphocyte receptor monomers indicate that they adopt a horseshoe-shaped structure and likely bind antigens through the hypervariable concave surface. Secreted variable lymphocyte receptors form pentamers or tetramers of dimers bind antigens with high specificity and avidity. This indicates that jawed and jawless vertebrates have developed antigen receptors independently. This paper provides valuable clues for studying the origin and evolution of adaptive immunity system.

[Preparation of monoclonal antibodies against multiple antigens].

OBJECTIVE: To prepare monoclonal antibodies (mAbs) against multiple antigens by single cell fusion. METHODS: BALB/c mice were immunized with the multiple antigens, namely alpha fetoprotein (AFP), carcinoembryonic antigen (CEA), HBsAgiHBcAg and HBeAg, and hybridomas were employed using PEG as the fusing agent. The hybridoma cells were respectively screened with AFP, CEA, HBsAg, HBcAg and HBeAg by enzyme-linked immunosorbent assay and limited dilution. The mAbs were purified by protein G affinity chromatography, its subtype was identified, the affinity constants (K(a)) were determined and the specificity was analyzed by Western blotting. RESULTS: Twenty hybridoma cell lines were obtained by single cell fusion, including 5 cell lines against AFP, 6 against CEA, 3 against HBsAg, 4 against HBcAg, and 2 against HBeAg. The subtypes of some hybridoma cell lines positive for the mAbs were identified as the immunoglobulin G1 (IgG1), with K(a) ranging from 1x10(9) M(-1) to x10(11) M(-1). Western blot analysis showed that all the mAbs strongly and specifically bound to their respective antigens. CONCLUSION: The mAbs against multiple antigens have been obtained by single cell-fusion, which increases the production of mAbs and reduces the time of preparation.