Molecular cloning, expression and functional analysis of TNF13b (BAFF) in Japanese sea perch, Lateolabrax japonicus.

Abstract Members of the tumor necrosis factor (TNF) family play key roles in the regulation of inflammation, immune responses and tissue homeostasis. Here we describe the identification of the Japanese sea perch ( Lateolabrax japonicus ) homologue of mammalian B cell activating factor of the TNF family (BAFF/BLyS) (designated LjBAFF). The cDNA contains an open reading frame (ORF) of 783 nucleotides that are translated into a predicted 260 amino acid protein. Like most known BAFFs, Japanese sea perch BAFF contains three cysteine residues (Cys 123 , Cys 218 and Cys 232 ) which are conserved in the aligned BAFF sequences and a furin protease cleavage site (R–K–K–R). Real-time quantitative PCR (qPCR) analysis revealed that LjBAFF could be detected in various tissues and predominantly expressed in lymphoid tissue spleen. The soluble BAFF (LjsBAFF) had been cloned into a pET28a vector to express the recombinant protein. The His-LjsBAFF was efficiently expressed in Escherichia coli BL21 (DE3) and its expression was confirmed by SDS-PAGE and Western blotting analysis. After purification, MTT assays and flow cytometric analysis revealed that LjsBAFF could promote the survival/proliferation of splenic B cells in vitro . Furthermore, bacterially expressed LjsBAFF induced the selective expansion of B cells in the spleen when administered to young mice. Our results suggest that like its mammalian counterparts, LjsBAFF plays an important role in the survival and/or proliferation of B cells.