Molecular structure, expression analysis and functional characterization of APRIL (TNFSF 13) in goat (Capra hircus)
2011
Luo, Cai-Hong | Ai, Hong-Xin | Zhou, Xu-Ming | Min, Cui | Liang, Zhen-Ning | Zhang, Jia-Xin | Song, Ren | Liu, Mei-Yan | Zhang, Shuang-Quan
A proliferation-inducing ligand (APRIL) is an important member of the tumor necrosis factor (TNF) superfamily. In the present study, a novel cDNA was isolated from the spleen of goat by RT-PCR and designated as goat APRIL (gAPRIL). The open reading frame (ORF) of this cDNA covered 753bp, encoding a protein of 250 amino acids. Sequence comparison showed that gAPRIL contains a predicted transmembrane domain, a putative furin protease cleavage site, and two cysteine residues, which are the typical characteristics of TNF gene in mammals. The predicted three dimensional (3D) structure of soluble part of the gAPRIL (gsAPRIL) monomer analyzed by comparative protein modeling revealed that it is very similar to its counterparts. Real-time PCR analysis revealed that gAPRIL was constitutively expressed in various tissues. Recombinant gsAPRIL fused with NusA tag was efficiently produced in Escherichia coli BL21 (DE3) and then analyzed by the SDS-PAGE as well as western blot. Laser scanning confocal microscopy analysis showed gsAPRIL could bind to its receptors. In vitro, the MTT and flow cytometric methods revealed that purified gsAPRIL protein was not only able to promote survival/proliferation of goat splenocytes, but also able to stimulate survival/proliferation of mouse B cells. These results indicated that gAPRIL plays an important role in survival/proliferation of goat splenocytes and provided a basis for investigating its potential to be used as an immunoadjuvant for enhancing vaccine efficacy and as an immunotherapeutic in goats.
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