Molecular cloning, expression, bioinformatics analysis and bioactivity characterization of TNF13B (BAFF) gene in bat (Vespertilio superans Thomas)

Abstract

B cell activating factor (BAFF) belonging to the tumor necrosis factor (TNF) family is critical to B cell survival, proliferation, maturation, and immunoglobulin secretion and to T cell activation. In the present study, the full-length cDNA of BAFF (designated bBAFF) from the bat (Vespertilio superans Thomas) was cloned using RT-PCR and rapid amplification of cDNA ends (RACE) techniques. The full-length cDNA of bBAFF consists of 986 bases including an 873bp open reading frame encoding 290 amino acids. Sequence comparison indicated that the amino acid of bBAFF possessed the TNF signature, a transmembrane domain, a putative furin protease cleavage site and three cysteine residues. The predicted three-dimensional (3D) structure of the bsBAFF monomer, analyzed by comparative protein modeling, revealed that it was very similar to its counterparts. Real-time quantitative PCR (qPCR) analysis indicated that bBAFF mRNA was predominantly expressed in bat lymphoid tissue spleen. The SUMO (Small Ubiquitin-like Modifier)-bsBAFF was efficiently expressed in Escherichia coliBL21 (DE3) and confirmed by SDS-PAGE and Western blotting analysis. Laser scanning confocal microscopy analysis showed that bsBAFF could bind to its receptors on B cells. In vitro, the MTT assays indicated that SUMO-bsBAFF was not only able to promote survival/proliferation of bat lymphocytes but also able to stimulate survival/proliferation of mouse B cells. These findings indicate that bsBAFF plays an important role in the survival/proliferation of B cells and has functional cross-reactivity among mammalians. The present findings may provide valuable information for research into the immune system of the bat.