RESUMEN
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.
Asunto(s)
Cabras/genética , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/genética , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/metabolismo , Animales , Linfocitos B/citología , Linfocitos B/inmunología , Linfocitos B/metabolismo , Proliferación Celular , Clonación Molecular , Biología Computacional , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Escherichia coli/genética , Escherichia coli/metabolismo , Citometría de Flujo , Perfilación de la Expresión Génica , Cabras/metabolismo , Masculino , Ratones , Microscopía Confocal , Filogenia , Reacción en Cadena de la Polimerasa , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Bazo/citología , Bazo/inmunología , Bazo/metabolismo , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/inmunología , Miembro 13 de la Superfamilia de Ligandos de Factores de Necrosis Tumoral/aislamiento & purificaciónRESUMEN
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.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/aislamiento & purificación , Biotecnología/métodos , Escherichia coli/metabolismo , Expresión Génica , Proteínas de Insectos/aislamiento & purificación , beta-Defensinas/aislamiento & purificación , Péptidos Catiónicos Antimicrobianos/biosíntesis , Péptidos Catiónicos Antimicrobianos/genética , Péptidos Catiónicos Antimicrobianos/farmacología , Clonación Molecular , Escherichia coli/genética , Vectores Genéticos , Humanos , Proteínas de Insectos/biosíntesis , Proteínas de Insectos/genética , Proteínas de Insectos/farmacología , beta-Defensinas/biosíntesis , beta-Defensinas/genética , beta-Defensinas/farmacologíaRESUMEN
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.