Molecular structure, expression and bioactivity characterization of TNF13B (BAFF) gene in mefugu, Takifugu obscurus

Abstract

B-cell activating factor of the TNF family (BAFF) induces B-cell survival, proliferation, immunoglobulin secretion and plays a role in enhancing immune responses. In the present study, a BAFF homolog has been identified in mefugu Takifugu obscurus, and its biological activities have been characterized. The mefugu BAFF (fBAFF) cDNA is 789 bp in length and translates into a 262-aa protein. The fBAFF genomic sequence consists of six exons and five introns, is approximately 1.8 kb in size. Amino acid sequence comparison indicated that fBAFF possessed the TNF signatures, a transmembrane domain, a putative furin protease cleavage site and three cysteine residues, which were the typical characteristics of TNF gene in mammals and birds. The predicted three-dimensional (3D) structure of the fsBAFF monomer analyzed by comparative protein modeling revealed that it was very similar to its human counterpart. Real-time quantitative PCR (qPCR) analysis revealed that fBAFF was predominantly expressed in mefugu lymphoid tissue spleen. The SUMO-fsBAFF and GFP/fsBAFF efficiently expressed in Escherichia coli Rosetta (DE3) were confirmed by SDS-PAGE and Western blotting analysis. After purification, the GFP/fsBAFF fusion protein obtained similar fluorescence spectrum to those of GFP. Laser scanning confocal microscopy analysis showed GFP/fsBAFF could bind to its receptors. In vitro, the MTT assays and flow cytometric analysis indicated that SUMO-fsBAFF could promote the proliferation of mefugu splenocytes or mouse splenic B cells together with/without anti-mouse IgM. These findings indicate that SUMO-fsBAFF plays an important role in proliferation of mefugu B cells and has functional cross-reactivity among mefugu and other mammalians. Therefore, BAFF may be a potential immunologic factor for enhancing immunological efficacy in fish.