Molecular cloning, bioinformatics analysis and functional characterization of B-cell activating factor in goat (Capra hircus)

Abstract

B-cell activating factor of the TNF family (BAFF) induces B cell survival, proliferation, immunoglobulin secretion and has a role in enhancing immune responses. In the present study, we amplified the cDNA of goat (Capra hircus) BAFF (designated gBAFF) from spleen by reverse transcription-PCR (RT-PCR). The open reading frame (ORF) of gBAFF covers 843 bp encoding 280 amino acids, with a 152-aa mature peptide. Sequence comparison indicated that the amino acid of gBAFF 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 soluble part of gBAFF (gsBAFF) analyzed by "comparative protein modelling" revealed that it was very similar to its counterparts. Real-time quantitative PCR (qPCR) analysis indicated that gBAFF mRNA was predominantly expressed in goat lymphoid tissue spleen. Recombinant gsBAFF was fused with a small ubiquitin-related modifier (SUMO) gene to enhance the soluble expression level in Escherichia coli BL21 (DE3). The resulting fused protein SUMO-gsBAFF was efficiently expressed and purified using metal chelate affinity chromatography (Ni-NTA), then confirmed by SDS-PAGE and Western blotting analysis. In vitro, the MTT assays and flow cytometric analysis indicated that SUMO-gsBAFF as well as gsBAFF could promote the survival/proliferation of goat splenic B cells or mouse splenic B cells. Therefore, BAFF may be a potential immunologic factor for enhancing immunological efficacy in goat.