Molecular structure, phylogenetic analysis, tissue distribution, and function characterization of interferon-γ-inducible lysosomal thiol reductase (GILT) gene in sheep (Ovis aries).

Interferon-γ-inducible-lysosomal thiol reductase (GILT) plays a key role in the processing and presentation of MHC class II-restricted antigen (Ag) by catalyzing disulfide bond reduction. In this study, a sheep cDNA, sGILT, encoding GILT protein was isolated from the spleen cDNA library of Ovis aries. It codes for a deduced protein of 244 amino acids with a putative molecular weight of 27.6 kDa, which has all the typical structural features of GILT protein including an active-site CXXC motif, a GILT signature sequence CQHGX(2)ECX(2)NX(4)C, and 5 conserved cysteines. Sequence comparison indicated the amino acid sequence of sGILT showed high identity to cow GILT (93.03%). Phylogenetic analysis showed that sGILT and cow GILT shared the greatest homology. The result of real-time PCR suggested that sGILT mRNA was expressed in a tissue-specific manner and obviously up-regulated in splenocytes and PBMCs after induction with lipopolysaccharide (LPS). Recombinant sGILT fused with His(6) tag was efficiently expressed in Escherichia coli BL21 (DE3) and purified by Ni-NTA affinity chromatography. Its expression was confirmed by SDS-PAGE and Western blotting analysis. Thiol reductase activity was assessed using antibody as the substrate. These results suggest that sGILT has the activity of disulfide bond reduction and indicate that sheep also express a protein that has been found to maintain first line of innate immune defense at basal level.

Molecular structure, expression analysis and functional characterization of APRIL (TNFSF 13) in goat (Capra hircus).

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.

Expression and purification of moricin CM4 and human ß-defensins 4 in Escherichia coli using a new technology.

Different strategies have been developed to produce small antimicrobial peptides using recombinant techniques. Here we report a new technology of biosynthesis of moricin CM4 and human ß-defensins 4 (HßD4) in the Escherichia coli. The CM4 and HßD4 gene were cloned into a vector containing the tags elastin-like peptide (ELP) and intein to construct the expression vector pET-EI-CM4 and pET-EI-HßD4. All the peptides, expressed as soluble fusions, were isolated from the protein debris by the method called inverse transition cycling (ITC) rather than traditional immobilized metal affinity chromatography (IMAC) and separated from the fusion leader by self-cleavage. Fully reduced peptides that were purified exhibited expected antimicrobial activity. The approach described here is a low-cost, convenient and potential way for generating small antimicrobial peptide.

Molecular characterization of the canine cytokine TWEAK (CD255) and its receptor, Fn14 (CD266).

TWEAK is a member of the tumor necrosis factor superfamily. The interaction of TWEAK with its receptor Fn14 regulates multiple cellular responses, including stimulation of proliferation, migration, apoptosis, angiogenesis, and induction of proinflammatory cytokines. This paper reports for the first time the molecular cloning of dog TWEAK and Fn14. The open reading frame (ORF) of dog TWEAK is 750bp, its genomic DNA consists of seven exons and six introns and is approximately 10kb in size by computer-assisted analysis. Sequence similarity at the amino acid level between dog TWEAK and human or mouse was 95 and 92%, respectively. The ORF of dog Fn14 contains 390bp. Sequence similarity at the amino acid level between dog Fn14 and human, or mouse, or frog was 95, 93 and 64%, respectively. Real-time quantitative PCR analysis revealed that both TWEAK and Fn14 are constitutively expressed in various tissues in dog. Furthermore, we verified dTWEAK interacted with dFn14 by yeast two-hybrid assay. 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.

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

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.