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Abstract: B-cell activating factor (BAFF), a member of the TNF family, is critical to the survival, proliferation, maturation, and differentiation of B-cells. In the present study, a CpBAFF was amplified from the white-spotted catshark (Chiloscyllium plagiosum) using RT-PCR and RACE (rapid amplification of cDNA end) techniques. To our knowledge, this is the first report of any BAFF gene being cloned from a cartilaginous fish. The open reading frame (ORF) of CpBAFF cDNA consists of 819 bases encoding a protein of 272 amino acids. This protein was found to contain a predicted transmembrane domain, a putative furin protease cleavage site, and a typical TNF homology domain corresponding to other identified BAFF homologues. Sequence alignment showed that CpBAFF shares 37–57% identity with BAFF amino acid sequences reported in other vertebrates. Three-dimensional structure modeling analysis revealed a soluble mature portion of CpBAFF (CpsBAFF) with a long D–E loop specific to the BAFF gene, which has not been found in other reported TNF proteins. Phylogenetic reconstruction showed that CpBAFF is most closely related to other fish BAFFs and clusters with BAFF genes from higher vertebrates (reptiles, birds, and mammals). Real-time quantitative RT-PCR demonstrated that CpBAFF mRNA expression was high in the spleen but moderate in the kidney and branchia. Recombinant CpsBAFF fused to NusA-His6-tag was efficiently expressed in Escherichia coli BL21 (DE3), and a molecular weight of approximately 83kDa was determined using SDS-PAGE and Western blotting. In vitro MTT assay indicated that the purified pET43.1a (+)-CpsBAFF protein can co-stimulate the proliferation of mammalian B-cells with anti-IgM in a dose-dependent manner. The present findings not only present novel information that may be relevant to shark immunity but also provide some new insights into the origins and evolution of immunity in all vertebrates. [Copyright &y& Elsevier]

Abstract: B-cell activating factor (BAFF), belonging to the TNF family, is a critical cytokine for B-cell survival, proliferation, maturation and differentiation. In the present study we cloned the cDNA of zebrafish (Danio rerio) BAFF (designated zBAFF) by reverse transcription-PCR (RT-PCR). The open reading frame (ORF) of zBAFF consists of 807 bases encoding a protein of 268 amino acids. The deduced amino acid sequence of its cDNA possessed the TNF family signature, a transmembrane domain, and three cysteine residues, which are the typical characteristics of TNF gene in mammals and birds. Phylogenetic analysis exhibits the highest identity score 67.6, 61.4 and 66.9% with the rainbow trout, tetraodon and salmon counterparts, respectively. The identity to avian and mammalian BAFFs ranges from 49.7 to 53.8%. Recombinant soluble zBAFF (zsBAFF) was fused with a small ubiquitin-related modifier gene (SUMO) to enhance the soluble expression level in Escherichia coli BL21 (DE3). The resulting fused protein SUMO-zsBAFF was highly expressed in BL21 (DE3) with a molecular weight of 38kDa. The fusing protein was purified using metal chellate affinity chromatography (Ni-NTA) and cleaved by a SUMO-specific protease, then confirmed by SDS-PAGE and Western blotting analysis. In vitro, the MTT assay indicated that the purified zsBAFF as well as SUMO-zsBAFF proteins were able to promote spleen lymphocyte survival in a dose-dependent manner also to co-stimulate the proliferation of mammalian B-cells with anti-IgM. Thus, the fusion protein represents a readily obtainable source of biologically active zsBAFF that may prove useful in further studies on zebrafish BAFF and its receptors. [Copyright &y& Elsevier]