Molecular cloning, functional characterization and phylogenetic analysis of B-cell activating factor in zebrafish (Danio rerio).

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 38 kDa. 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.

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