Search

• Cat (Felis catus) BAFF and its receptor BAFF-R were cloned. • csBAFF, EGFP/csBAFF, and cBAFF-R were expressed in E. coli and purified using a Ni2+ column. • csBAFF cellular localization was assessed. • csBAFF promoted B-cell survival in vitro. • Ligand–receptor interaction was assessed. B-cell survival depends on signals induced by binding of B-cell activating factor (BAFF) to its receptor (BAFF-R). In this study, the full-length cDNAs of cat BAFF (cBAFF) and BAFF-R (cBAFF-R) were amplified from the spleen by reverse transcription PCR. The open reading frame of cBAFF cDNA encodes a protein of 285 amino acids containing a predicted transmembrane domain and a furin protease cleavage site, similar to mammalian, avian, and reptile BAFFs. The cBAFF-R gene encodes a 189 amino acid protein. Real-time quantitative PCR analyses revealed that the two genes are predominantly expressed in the spleen. csBAFF, EGFP/csBAFF, and cBAFF-R were efficiently expressed in Escherichia coli BL21 (DE3), as confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting analyses. After purification, the EGFP/csBAFF fusion protein showed a fluorescence spectrum similar to that of EGFP. Confocal laser scanning microscopy showed that EGFP/csBAFF bound to its receptor. In vitro , csBAFF promoted the survival of cat and mouse splenic B cells with/without a priming agent (Staphylococcus aureus Cowan 1, SAC) or anti-mouse IgM. Furthermore, it stimulated the survival of mouse B cells, similar to msBAFF. Recombinant cBAFF-R blocked the function of sBAFF in vitro. These findings indicate that csBAFF plays an important role in the survival of cat B cells and has functional cross reactivity between cats and other mammals, and suggest a role for the BAFF–BAFF-R system in regulating B-cell survival. Therefore, BAFF and BAFF-R show promise for enhancing the immune systems of animals. [ABSTRACT FROM AUTHOR]

Abstract: Full-length cDNA encoding TWEAK and Fn14 from bovine was isolated. We used bioinformatics to analyze the gene structure, function, evolutionary relationships, and predicted three-dimensional structure of proteins and binding sites. Real-time quantitative PCR (Q-PCR) analysis revealed that both TWEAK and Fn14 are constitutively expressed in various tissues in bovine. Bovine TWEAK and Fn14 interaction analysis by yeast two-hybrid. Our results suggest that the TWEAK–Fn14 pathway is evolutionarily highly conserved. It will be helpful for investigation on the biological role of the TWEAK/Fn14 system in this important animal model. Furthermore, it provides insight into the molecular evolution of the emerging TWEAK and Fn14 families. [Copyright &y& Elsevier]

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 753bp, 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 1.8kb 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. [Copyright &y& Elsevier]