Molecular structure, expression, cell and tissue distribution, immune evolution and cell proliferation of the gene encoding bovine ( Bos taurus) TNFSF13 (APRIL)

Abstract

A novel bovine cDNA has been isolated by EST assembly and subsequently confirmed by using RT-PCR and designated bovine A Proliferation-Inducing Ligand belonging to TNF family (bAPRIL). The open reading frame (ORF) of this cDNA covers 753 bp, encoding 250 amino acids. The functional soluble part of bAPRIL (bsAPRIL) shows 97% and 92% identity with its pig and human counterparts, respectively, at the level of the primary protein structure. The bAPRIL genomic sequence consists of six exons and five introns, is approximately 1.8 kb in size, and maps to bovine chromosome 19q. Real-time quantitative PCR (qPCR) analysis revealed that bAPRIL is predominantly expressed in bovine lymphoid tissues spleen. The predicted three-dimensional (3D) structure of the bsAPRIL monomer analyzed by “comparative protein modelling” revealed that it is very similar to its mouse counterpart. The bsAPRIL and EGFP/bsAPRIL were efficiently expression in Escherichia coli BL21 (DE3) and its expression was confirmed by SDS-PAGE and Western blotting analysis. After purification, the EGFP/bsAPRIL fusion protein obtained similar fluorescence spectrum to those of EGFP. Laser scanning confocal microscopy analysis showed EGFP/bsAPRIL could bind to its receptor. In vitro, bsAPRIL could promote the proliferation of bovine or mouse splenic B cells together with/without SAC or anti-mouse IgM. Furthermore, compared to mouse soluble APRIL, the bovine soluble APRIL has the similar proliferation to mouse B cell. Those findings indicated that bsAPRIL plays an important role in proliferation of bovine B cells and has functional cross-reactivity among cow and other mammalians. Therefore, APRIL may be a potential immunologic factor for enhancing immunological efficacy in animals.