RESUMEN
It has been shown that the Hepatitis C virus nonstructural NS3 protein possesses at least two enzymatic domains: a serine-protease domain and an adenosine triphosphatase (ATPase)/helicase domain. In this report, a truncated fragment of NS3 (26 kDa), representing main epitopes from the (ATPase)/helicase domain, has been expressed in Escherichia coli. The recombinant protein was purified by Ion Metal Affinity Chromatography (IMAC) with more than 90% purity. The recognition of B-cell linear epitopes in the NS3 protein was evaluated by immunoblot. The recombinant NS3 protein was reduced and carboxymethylated, and the recognition of either conformational and/or linear B-cell determinants was evaluated by ELISA. The inclusion of the recombinant NS3 protein in a third-generation diagnostic system UltraMicroELISA (UMELISA) allowed an increase in the sensitivity, due to the detection of a new variety of false-negative sera in blood donor test samples.
Asunto(s)
Adenosina Trifosfatasas/química , ADN Helicasas/química , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genética , Cromatografía de Afinidad , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Ensayo de Inmunoadsorción Enzimática , Epítopos , Escherichia coli/metabolismo , Fermentación , Immunoblotting , Modelos Genéticos , Plásmidos/metabolismo , Estructura Terciaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , TemperaturaRESUMEN
Little is known about the mechanism of hepatitis C virion assembly. So the capacity of the entire Hepatitis C virus core protein (HCcAg) produced in Pichia pastoris to form particles either in its native soluble state or after detergent treatment of HCcAg associated to cell debris were studied. Size exclusion chromatography suggested that HCcAg assembled into high molecular weight structures. HCcAg was also specifically recognized by a serum from a chronic HCV carrier patient. This antigen migrated with buoyant density values similar to those obtained for native nucleocapsid particles from infected patients when analyzed using sucrose density gradient centrifugation. The analysis by electron microscopy of purified HCcAg showed aggregates resembling virus-like particles (VLPs) with an average diameter of 30 nm. These results indicated that the HCcAg obtained from P. pastoris assembled into VLPs resembling HCV nucleocapsid particles in a mature stage. Such HCcAg aggregates characterized here could be a valuable tool to elucidate the mechanisms of HCV nucleocapsid assembly.
Asunto(s)
Hepacivirus/química , Pichia/virología , Proteínas del Núcleo Viral/química , Virión/química , Immunoblotting , Peso Molecular , Renaturación de Proteína , Proteínas del Núcleo Viral/metabolismoRESUMEN
The reconstitution of recombinant bacterial outer membrane proteins (OMPs) into their native conformations after purification has been the major problem in their use as effective vaccines. Liposomes have been shown to be an attractive approach, providing a native-like environment for these antigens. The meningococcal recombinant Opc (rOpc) protein, produced as inclusion bodies in Escherichia coli, was incorporated into phospholipid vesicles consisting of dipalmitoyl phosphatidylcholine and cholesterol. The incorporation of rOpc into the lipid bilayer was demonstrated, and the reconstitution of some native epitopes was tested using a set of monoclonal antibodies. Subcutaneous immunization of Balb/c mice with rOpc-containing vesicles resulted in the generation of a high level of specific antibodies. The elicited antibodies reacted with the native meningococcal protein and showed opsonic activity.
Asunto(s)
Proteínas de la Membrana Bacteriana Externa/química , Neisseria meningitidis/química , Proteínas Recombinantes/química , 1,2-Dipalmitoilfosfatidilcolina/química , Animales , Anticuerpos Monoclonales/metabolismo , Proteínas de la Membrana Bacteriana Externa/metabolismo , Western Blotting , Colesterol/química , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/metabolismo , Immunoblotting , Membrana Dobles de Lípidos/química , Liposomas/química , Ratones , Ratones Endogámicos BALB C , Proteínas Recombinantes/metabolismoRESUMEN
The immune response against hepatitis B surface and core antigens was evaluated by either coinoculation or independent intramuscular administration of pAEC compact DNA immunization vectors carrying their genes. The pAEC vectors bear just the essential elements for mammalian expression and bacterial amplification. Balb/c mice were immunized with 100 microg of each construct, either alone or in combination. In spite of lacking known immunostimulatory sequences (e.g., AACGTT), significant cellular (proliferative) and humoral immune responses were raised against both antigens. Coadministration of both plasmids maintained the immune response against the two antigens, without interference between them. Modulation of the antigen expression and further immune response, by using the Kozak's translation initiation sequence, was also analyzed. No differences due to its presence or absence were observed.
Asunto(s)
Antígenos del Núcleo de la Hepatitis B/genética , Antígenos del Núcleo de la Hepatitis B/inmunología , Antígenos de Superficie de la Hepatitis B/genética , Antígenos de Superficie de la Hepatitis B/inmunología , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/inmunología , Vacunas de ADN/genética , Vacunas de ADN/farmacología , Animales , Secuencia de Bases , Células COS , Femenino , Vectores Genéticos , Anticuerpos contra la Hepatitis B/biosíntesis , Inmunización , Activación de Linfocitos , Ratones , Ratones Endogámicos BALB C , Plásmidos/genética , Linfocitos T/inmunología , Transfección , Vacunas de ADN/inmunologíaRESUMEN
The nasal mucosa may provide a simple, non-invasive route to deliver DNA encoding genes that stimulate a specific immune response. Based on this, a new approach using pCMVbeta-gal plasmid DNA complexed to the Opc meningococcal outer membrane protein was assayed for. Optimal conditions of interaction were established between recombinant Opc protein and pCMVbeta-gal plasmid DNA. Complexes were fully characterized by electrophoresis analysis, DNAse resistance assay and transmission electron microscopy. DNA-protein complexes were also evaluated in in vitro transfection experiments. After the characterisation of complexes, Balb/c mice were intranasal (i.n.) and intramuscularly (i.m.) immunized. The humoral immune response against beta-galactosidase was measured by ELISA. The proliferative response in the spleen lymph nodes was also measured. Complexes administered by i.n. route induced both systemic and mucosal antibody responses. This behavior was not observed with the naked DNA. Finally, a lymphoproliferative response specific to beta-galactosidase induced by DNA-protein complexes was also detected.
Asunto(s)
Antígenos Bacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Plásmidos/inmunología , Vacunas de ADN/administración & dosificación , Vacunas de ADN/inmunología , Administración Intranasal , Animales , Antígenos Bacterianos/administración & dosificación , Antígenos Bacterianos/genética , Proteínas de la Membrana Bacteriana Externa/administración & dosificación , Proteínas de la Membrana Bacteriana Externa/genética , Células COS , Línea Celular , Esquemas de Inmunización , Ratones , Ratones Endogámicos BALB C , Plásmidos/administración & dosificación , Plásmidos/metabolismo , Plásmidos/ultraestructura , TransfecciónRESUMEN
P64k is a minor outer membrane protein from Neisseria meningitidis. This protein has been produced at high levels in Escherichia coli. We generated a group of monoclonal antibodies (mAbs) against recombinant P64k, which recognise four non-overlapping epitopes, as shown using competition assays with biotinylated mAbs. The P64k sequences involved in mAbs binding were mapped with synthetic overlapping peptides derived from the P64k protein, and located in the previously determined three-dimensional structure of the protein. These antibodies were also characterised by whole-cell ELISA and bactericidal tests against N. meningitidis. Only two of the recognised epitopes were exposed on the bacterial surface, and none of the mAbs showed bactericidal activity. The relationship between these results and the structural data on the epitopes bound by the mAbs is discussed.
Asunto(s)
Anticuerpos Antibacterianos/análisis , Anticuerpos Monoclonales/análisis , Antígenos Bacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/inmunología , Neisseria meningitidis/inmunología , Secuencia de Aminoácidos , Antígenos Bacterianos/biosíntesis , Antígenos Bacterianos/genética , Proteínas de la Membrana Bacteriana Externa/biosíntesis , Proteínas de la Membrana Bacteriana Externa/genética , Clonación Molecular , Dihidrolipoamida Deshidrogenasa/genética , Dihidrolipoamida Deshidrogenasa/inmunología , Mapeo Epitopo , Epítopos/química , Epítopos/inmunología , Datos de Secuencia Molecular , Estructura Secundaria de ProteínaRESUMEN
A common meningococcal antigen designated P64k has been identified, cloned and expressed in Escherichia coli. The recombinant antigen is highly immunogenic in several animal species and its immunogenicity in healthy human volunteers is under investigation. Recently, P64k has been used as an immunological carrier for weak immunogens. To characterize the B-cell epitopes on P64k, recognized by immune sera obtained from mice, rabbits and monkeys, multiple overlapping peptides were synthesized and screened for antibody binding. Peptides covering the complete sequence of the P64k protein, 59 in all, of 20 amino acids each (overlapped by 10 residues), were synthesized. A number of continuous epitopes were detected with all sera, when immune and pre-immune bleeds were compared. For mouse and monkey sera, a few major antigenic peptides were identified, while the recognition of the rabbit serum was much more heterogeneous. Despite variation in the exact location of continuous epitopes defined by different anti-P64k sera, we found an immunogenic core region within the molecule, composed of amino acids Asp(524)-Gly(533). Consistently, in this protein segment there was an amino acid stretch located in a beta-hairpin loop, which is exposed to the solvent in the previously determined three-dimensional structure of the protein. This region is protruding and accessible to a sphere with a radius of 9 A.
Asunto(s)
Linfocitos B/inmunología , Mapeo Epitopo/métodos , Neisseria meningitidis/inmunología , Fosfoproteínas/inmunología , Secuencia de Aminoácidos , Animales , Chlorocebus aethiops/inmunología , Femenino , Glicoproteínas de Membrana , Ratones , Ratones Endogámicos BALB C , Proteínas de Microfilamentos , Datos de Secuencia Molecular , Fragmentos de Péptidos/inmunología , Fosfoproteínas/química , Conformación Proteica , ConejosRESUMEN
The HIV-1 gp120 gene with natural signal sequence expressed in eukaryotic expression systems showed extremely low levels of synthesis and secretion. Several expression systems have been used to improve the secretion levels of gp 120. In mammalian cells, the efficient expression of gp120 fused to t-PA signal peptide has been previously reported. Here, the effects of t-PA and EPO signal peptides were compared as secretion sequences for expression of gp120 in COS-7 cells. The EPO's signal peptide is used for the first time as leader sequence for secretion of foreign proteins. Our results indicated that higher amounts of secreted gp 120 were obtained when vectors containing EPO signal peptide were used.
Asunto(s)
Eritropoyetina/genética , Proteína gp120 de Envoltorio del VIH/genética , VIH-1/genética , Señales de Clasificación de Proteína/genética , Secuencia de Aminoácidos , Animales , Células COS , Eritropoyetina/biosíntesis , Expresión Génica , Vectores Genéticos , Proteína gp120 de Envoltorio del VIH/biosíntesis , VIH-1/metabolismo , Humanos , Datos de Secuencia Molecular , Señales de Clasificación de Proteína/biosíntesis , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Recombinantes de Fusión/genética , Activador de Tejido Plasminógeno/biosíntesis , Activador de Tejido Plasminógeno/genética , TransfecciónRESUMEN
P64k protein from Neisseria meningitidis is well recognised in sera from individuals convalescent from meningococcal disease or vaccinated with the Cuban antimeningococcal vaccine VA-MENGOC-BC. The presence of the protein in more than 80 meningococcal strains has also been verified. It is immunogenic in animal models and the antibodies elicited show bactericidal activity against meningococci. To further investigate at the molecular level whether lpdA, the gene coding for P64k protein, is conserved among different N. meningitidis strains, a total of 20 strains isolated from different geographic areas were differentiated on the basis of restriction fragment length polymorphism (RFLP) patterns after polymerase chain reaction (PCR) amplification of the lpdA gene and restriction endonuclease digestion with HpaII. Although a total of five different PCR-RFLP patterns were present, nucleotide sequence determination showed that identity levels were as high as 93-99% among the N. meningitidis strains analysed.
Asunto(s)
Antígenos Bacterianos/genética , Proteínas de la Membrana Bacteriana Externa/genética , Genes Bacterianos , Neisseria meningitidis/genética , Secuencia de Aminoácidos , Vacunas Bacterianas/genética , Vacunas Bacterianas/inmunología , Secuencia Conservada , Vacunas Meningococicas , Datos de Secuencia Molecular , Neisseria meningitidis/clasificación , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Especificidad de la EspecieRESUMEN
By making use of recombinant DNA technology it is possible to characterize meningococcal outer membrane proteins (OMPs) capable of stimulating a host immune response. The lpdA gene, which codes for an OMP (P64k) from Neisseria meningitidis, was cloned in Escherichia coli. The recombinant protein was recognized by sera from patients convalescing from meningococcal disease. The monoclonal antibodies obtained against the recombinant protein recognized the natural protein on a Western blot, and monoclonal antibody 114 was assayed in ELISA with a panel of 85 N. meningitidis strains. The protein was recognized in 81 strains (95.3%); the strains that were not recognized were neither epidemic nor isolated from systemic disease. The complete amino acid sequence of P64k was obtained by automatic sequencing and MS.
Asunto(s)
Antígenos Bacterianos/genética , Proteínas de la Membrana Bacteriana Externa/genética , Secuencia de Aminoácidos , Antígenos Bacterianos/biosíntesis , Proteínas de la Membrana Bacteriana Externa/biosíntesis , Escherichia coli , Expresión Génica , Datos de Secuencia Molecular , Neisseria meningitidis , Proteínas Recombinantes/biosíntesis , Análisis de Secuencia de ADNRESUMEN
The meningococcal Opc protein has been expressed as inclusion bodies in Escherichia coli. After cell disruption and successive washing of the insoluble fraction, insoluble proteins were solubilized in presence of the chaotropic agent guanidium hydrochloride. The extract was applied to a Reverse Phase High Performance Liquid Chromatography (RP-HPLC)-C4 column, for further purification. The obtained recombinant Opc protein was refolded in vitro, by the addition of several compounds to the resuspended solution. Over time, the progress of renaturation was tested by immunoblot with the human monoclonal antibody LuNm03 against the meningococcal Opc protein. LuNm03 recognizes a conformational epitope on the native meningococcal Opc protein. Having established the optimal conditions of renaturation. Balb/c mice were immunized to study the humoral immune response. The human at immune response elicited in mice was measured by ELISA and immunoblot, while the functional activity of these antibodies was assayed in a bactericidal test. According to our results, it was possible to obtain a recombinant Opc protein folded in vitro, with a conformation suitable enough to generate functional antibodies in mice, capable of killing meningococci in the presence of human complement.
Asunto(s)
Anticuerpos Antibacterianos/inmunología , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/inmunología , Vacunas Bacterianas/inmunología , Pliegue de Proteína , Vacunas Sintéticas/inmunología , Animales , Proteínas de la Membrana Bacteriana Externa/aislamiento & purificación , Vacunas Bacterianas/química , Vacunas Bacterianas/aislamiento & purificación , Escherichia coli , Fermentación , Humanos , Inmunización , Ratones , Ratones Endogámicos BALB C , Desnaturalización Proteica , Vacunas Sintéticas/química , Vacunas Sintéticas/aislamiento & purificaciónRESUMEN
A methodology is presented for efficiently gaining structural information from electrophoresed proteins after on-gel detection by imidazole-sodium dodecyl sulfate-zinc reverse staining. As a consequence of reverse staining, (a) protein bands arise transparent against a deep white-stained background, limits of detection being in the femtomole range; (b) there is no loss of image when the gel is kept in distilled water (even during years); and (c) protein bands result immobilized, i.e., they do not diffuse upon gel storage. To recover reverse-stained proteins or fragments thereof from the gel, the immobilization of bands must first be abrogated by chelating the zinc ions from stain (protein mobilization). We had originally described mobilization at low pH by using citric acid. Here, we improve this procedure regarding the protein electrotransfer. We demonstrate that mobilization is efficiently done at neutral to alkaline pH by short-term (5 to 10 min) incubation of the gel in a buffer containing glycine or dithiothreitol prior to transfer. Moreover, mobilization was most simply performed by just adding the zinc chelating agent to the transfer buffer. Reverse staining and the new mobilization procedure made electrotransferring single protein bands from gel onto small-sized (13 x 5 mm2) PVDF membrane pieces in mini sandwich-like assemblies practical. Equipment is described for the protein electroblotting in such minisandwiches. Microsequence analysis of the electroblotted proteins showed initial yields in the range of those achieved when the transfer was done from unstained control gels. Protein bands kept in the reverse-stained gel for prolonged time periods (even for as long as 2 years) could be similarly analyzed.(ABSTRACT TRUNCATED AT 250 WORDS)
Asunto(s)
Proteínas/análisis , Electroforesis en Gel de Poliacrilamida , Focalización Isoeléctrica , Coloración y EtiquetadoRESUMEN
A protein constituent of the outer membrane from Neisseria meningitidis (hereafter called P64K) has been crystallized using the hanging drop technique. Crystals are tetragonal with unit cell dimensions a = b = 136.84 A and c = 78.44 A, compatible with a single monomer of 64 kDa in the asymmetric unit. When exposed to high intensity synchrotron radiation, these crystals diffract X-rays to at least 2.9 A resolution, indicating that a high resolution structure analysis is feasible.