RESUMEN
Recombinant protein rNS3 imitating helicase region (1356-1459 amino acid residues) of hepatitis C virus (HCV) was expressed in E. coli cells and used for BALB/c mice immunization. Seven hybrydoma clones producing monoclonal antibodies (MAbs) to rHS3 were obtained. All MAbs reacted in ELISA with NS3 protein from Murex anti-HCV Version III and in immunoblotting from RIBA 3. These MAbs detect 5 individual epitopes, 4 of which were conformational and 1 discontinuous. All MAbs could compete for rNS3 binding with serum antibodies from patients with chronic hepatitis C, which suggests that these MAbs can recognize the natural HCV NS3 protein.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos contra la Hepatitis C/inmunología , Proteínas no Estructurales Virales/inmunología , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Unión Competitiva , Epítopos/química , Epítopos/aislamiento & purificación , Escherichia coli/genética , Vectores Genéticos , Anticuerpos contra la Hepatitis C/aislamiento & purificación , Hepatitis C Crónica/sangre , Hepatitis C Crónica/diagnóstico , Humanos , Ratones , Ratones Endogámicos BALB C , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunología , Proteínas no Estructurales Virales/biosíntesis , Proteínas no Estructurales Virales/genéticaRESUMEN
A method has been developed for preparing and purifying recombinant polypeptide--hepatitis C virus core protein (HCcoreAg) expressed in E. coli from pFC105-302 plasmid coding for 150 N-terminal amino acids of HCcoreAg in the hybrid from the C-terminal with beta-galactosidase. HCcoreAg was purified by ion-exchange chromatography on P-11 phosphocellulose. The bulk of protein carrying HCcoreAg antigenic determinants was found in two polypeptides: with molecular weights 26 and 136 kD. Antigenic and immunogenic properties of the resultant polypeptide were studied. The 26 kD protein can be used in enzyme immunoassay and immunoblotting as antigen for detecting antibodies to the HCV core protein. The results of immunization of rabbits indicate a high immunogenic activity of the protein. High diagnostic value of HCcoreAg preparation has been demonstrated, for the rapid variant of indirect solid-phase enzyme immunoassay among other tests.
Asunto(s)
Epítopos/genética , Proteínas del Núcleo Viral/genética , Animales , Cromatografía por Intercambio Iónico , Epítopos/química , Epítopos/aislamiento & purificación , Hepatitis C/diagnóstico , Peso Molecular , Plásmidos , Conejos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Sensibilidad y Especificidad , Proteínas del Núcleo Viral/química , Proteínas del Núcleo Viral/aislamiento & purificaciónRESUMEN
Highly affine murine monoclonal antibodies (MAB) to recombinant nucleocapsid (core) protein of hepatitis C virus (rHCcAg) expressed in E. coli were obtained. The MABs were analyzed by solid-phase enzyme immunoassay (EIA), immunodot, immunoblotting, and competitive immunochemical analysis. For estimating the epitope specificity of MAB, several immunoreactive fragments of different length were cloned from the HCcAg region overlapping 160 N-terminal amino acid (a. a.) residues. Use of these fragments and the competitive EIA demonstrated that MAB recognize 4 non-overlapping epitopes, 2 of which are localized in the 1-80 a. a. and 2 other in the 80-150 a. a. regions. A protocol of EIA for detecting HCcAg using MABs to two nonoverlapping HCcAg epitopes has been designed. The sensitivity of double-site sandwich is 1 ng/ml for the recombinant protein.
Asunto(s)
Hepacivirus/inmunología , Proteínas del Núcleo Viral/inmunología , Animales , Anticuerpos Monoclonales/biosíntesis , Especificidad de Anticuerpos , Femenino , Hepacivirus/aislamiento & purificación , Immunoblotting , Técnicas para Inmunoenzimas , Ratones , Ratones Endogámicos BALB C , Proteínas Recombinantes/análisis , Proteínas Recombinantes/inmunología , Proteínas del Núcleo Viral/análisisRESUMEN
For investigation of proteins possessing assigned immunological properties, plasmids pPS31-42, pPS1-5, pPS2-17, and pPS1P-30 were constructed encoding the hepatitis B core protein (HBcAg) with N-terminally inserted immunodominant epitopes of preS regions (amino acids 31-36 or 94-105 of preS1, or 133-143 of preS2). Analysis of the hybrid proteins with the use of ELISA and immunoelectron microscopy showed that the insertions did not prevent specific aggregation of the protein molecules, the inserted sequences being exposed on the surface of the particles obtained, and both HBcAg and the corresponding preS determinants were antigenically active.
Asunto(s)
Antígenos del Núcleo de la Hepatitis B/genética , Epítopos Inmunodominantes/genética , Proteínas Virales/genética , Secuencia de Aminoácidos , Secuencia de Bases , Ensayo de Inmunoadsorción Enzimática , Virus de la Hepatitis B/inmunología , Microscopía Inmunoelectrónica , Datos de Secuencia Molecular , Plásmidos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/ultraestructuraRESUMEN
A set of plasmids was constructed determining synthesis of hybrid proteins with insertions of antigenic determinants of preS1 and preS2 regions of HBV in the middle part of HBcAg. The polypeptides containing the 31-36 or monomeric 94-105 preS1 epitopes were water-soluble and formed particles similar to the 27-nm ones of native HBcAg, possessing antigenic properties of both HBcAg and the inserted epitope, while the hybrids containing 133-143 of preS2 or a trimeric form of the 94-105 preS1 epitope became membrane-bound. Another hybrid encoded by plasmid pPS2M31 contained two regions of HBcAg modification: insertion of amino acids 133-143 a.a. (preS2 region) in the N-terminal part and 31-36 (preS1) in the middle part of the carrier. The immunogenicity of the epitope inserted into the middle part of the HBcAg molecule was an order of magnitude higher than that of the same epitope in the N-part of the protein; the fact might be important for constructing artificial immunogens.
Asunto(s)
Antígenos del Núcleo de la Hepatitis B/genética , Epítopos Inmunodominantes/genética , Secuencia de Aminoácidos , Virus de la Hepatitis B/inmunología , Microscopía Electrónica , Datos de Secuencia Molecular , Plásmidos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/ultraestructuraRESUMEN
Based on theoretical analysis of secondary structure and hydrophilicity data, the region (residues 73-89) of the HBV core-antigen presumably containing the antigenic determinant has been revealed. The epitope mapping of this region with the use of synthetic peptides, obtained by the pin technology and solid phase method, was carried out. Peptides were synthesized in two variants with different amino acids residues in the position 80 (Ala, Ile). Free peptides and their conjugates with bovine serum albumin were tested for antigenicity in ELISA. The results indicate that the fragment 78-83 is the shortest region of the core-antigen which reacts with antibodies from hepatitis B patients sera.
Asunto(s)
Antígenos del Núcleo de la Hepatitis B/química , Epítopos Inmunodominantes/análisis , Péptidos/química , Secuencia de Aminoácidos , Ensayo de Inmunoadsorción Enzimática , Anticuerpos contra la Hepatitis B/inmunología , Antígenos del Núcleo de la Hepatitis B/inmunología , Humanos , Epítopos Inmunodominantes/inmunología , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Albúmina Sérica Bovina/químicaRESUMEN
A set of four peptides from the HCV NS4-protein C-terminal region (aa 1921-1940) were obtained by solid-phase synthesis using activated esters and symmetrical anhydrides of Boc-amino acids. Peptide 1921-1940 has demonstrated a positive reaction in ELISA with individual anti-HCV-positive sera from patients with acute and chronic hepatitis C (80% and 56%, respectively). We analysed the antigenic properties of the peptide 1921-1940 and its fragments and suggested at least two antibody recognizing sites to be contained in this region.
Asunto(s)
Antígenos Virales/inmunología , Hepacivirus/química , Fragmentos de Péptidos/síntesis química , Proteínas no Estructurales Virales/química , Ensayo de Inmunoadsorción Enzimática , Fragmentos de Péptidos/inmunologíaRESUMEN
Hepatitis delta virus (HDV), a recently discovered infectious agent, participates in severe, often lethal forms of acute and chronic hepatitis and liver cirrhosis. Based on theoretical analysis of secondary structure, hydrophilicity and acrophilicity data, several regions of HDV antigen, presumably containing B-epitopes, have been revealed and the corresponding peptides have been synthesized by the solid phase method. All the peptides obtained reacted with the respective antipeptide rabbit sera. The peptides and their conjugates with BSA or KLH were used for ELISA with individual and pooled anti-HD-positive sera from patients with chronic delta hepatitis. The high antigenicity of the peptide 65-80 shows that one of the antigenically active regions of HDAg is situated between these amino acid residues and that the peptide may be used for detection of anti-HD antibodies in patients blood sera.
Asunto(s)
Cápside/metabolismo , Virus de la Hepatitis Delta/metabolismo , Péptidos/síntesis química , Proteínas del Núcleo Viral/metabolismo , Secuencia de Aminoácidos , Animales , Antígenos Virales/inmunología , Ensayo de Inmunoadsorción Enzimática , Virus de la Hepatitis Delta/inmunología , Humanos , Inmunización , Técnicas para Inmunoenzimas , Masculino , Datos de Secuencia Molecular , Péptidos/inmunología , ConejosRESUMEN
We synthesized the 24-41, 30-36, 31-36, 24-30 fragments of the preS1-region of the hepatitis B (subtype ayw) envelope. The peptides were prepared by the solid phase synthesis on perfluorpolyethylene polymer grafted with polystyrene. The peptide chains were elongated from C-terminus using activated esters and symmetrical anhydrides of Boc-amino acids, cleaved off the solid phase by HBr or TFMSA in TFA, purified by gel filtration, and, after conjugation with protein carriers, inoculated into test animals. The resultant antibodies were shown to react with peptides. The blood sera from patients with acute hepatitis B reacted with the conjugates of peptides 24-41, 30-36, 31-36 in the immunoenzymic solid phase assay. The monoclonal antibodies for the preS1-polypeptide were shown to react with peptides 24-41, 30-36, 31-36 and with their conjugates. The results obtained were proved by the data of the epitope-mapping with overlapping hexapeptides.
Asunto(s)
Epítopos/metabolismo , Hepatitis B/metabolismo , Péptidos/síntesis química , Proteínas del Envoltorio Viral/metabolismo , Secuencia de Aminoácidos , Cromatografía en Gel , Hepatitis B/inmunología , Técnicas para Inmunoenzimas , Datos de Secuencia Molecular , Proteínas del Envoltorio Viral/inmunologíaAsunto(s)
Antígenos Virales/inmunología , Cápside/inmunología , Virus de la Hepatitis Delta/inmunología , Fragmentos de Péptidos/inmunología , Proteínas del Núcleo Viral/inmunología , Antígenos Virales/síntesis química , Hepatitis D/inmunología , Humanos , Epítopos Inmunodominantes/inmunología , Fragmentos de Péptidos/síntesis química , Conformación Proteica , Relación Estructura-ActividadRESUMEN
A set of plasmids was constructed, that carries the hybrid operons with an artificial region for translation initiation of the second cistron. The SD-sequence situated close to the termination signal of the previous cistron facilitates the reinitiation of translation. Both HBcAg and beta-galactosidase coding cistrons are functionally active. The analysis of expression efficacy shows: 1) The second cistron possesses its own initiation region; ii) the opportunity of translation reinitiation increases of the protein synthesis level. The correlation between the translation initiation efficacy and the structure of the initiator codon was investigated. AUG and UUG provide comparable protein synthesis levels, AUG being 1.5-3 times more effective. Probably, there exists a different efficacy of recognition of initiator codons by ribosomes for the systems with independent and connected initiation of translation. The influence of mRNA secondary structure in the translation initiation region on expression is discussed.
Asunto(s)
Escherichia coli/enzimología , Antígenos del Núcleo de la Hepatitis B/genética , Operón , beta-Galactosidasa/genética , Secuencia de Bases , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Microscopía Electrónica , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Plásmidos , Biosíntesis de Proteínas , ARN Mensajero/genéticaRESUMEN
We have synthesized the 24-41 fragment of the preS region of the hepatitis B (subtype ayw) envelope. The peptide was prepared by the solid phase synthesis on perfluoropoly-ethylene polymer grafted with polystyrene. The peptide chain was elongated from C-terminus using pentafluorophenyl- and p-nitrophenyl esters of Boc-amino acids. The peptide was cleaved off the solid phase by HBr in CF3COOH, purified by gel filtration, and, after conjugation with serum albumin (BSA), inoculated into mice. The resultant antibodies were shown to react with the peptide. The blood sera from patients with acute hepatitis B reacted with the peptide-BSA conjugates in the immunoenzymic solid phase assay.
Asunto(s)
Virus de la Hepatitis B/inmunología , Fragmentos de Péptidos/síntesis química , Proteínas del Envoltorio Viral/síntesis química , Secuencia de Aminoácidos , Animales , Epítopos/síntesis química , Epítopos/inmunología , Antígenos de Superficie de la Hepatitis B/inmunología , Humanos , Inmunización , Masculino , Ratones , Datos de Secuencia Molecular , Fragmentos de Péptidos/inmunología , Conformación Proteica , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
New plasmids containing partially deleted lacZ genes were obtained. These genes determine high-level synthesis of polypeptides of molecular mass 43-45 and 49-51 kD under the control of the lambda phage PR-promoter; inspite of the deletion, E. coli cells carrying new plasmids were found to possess beta-galactosidase activity. Use of these plasmids as new expression vectors is suggested.
Asunto(s)
Escherichia coli/genética , Operón Lac , Plásmidos , Secuencia de Bases , Enzimas de Restricción del ADN , Datos de Secuencia MolecularRESUMEN
A set of plasmids containing short DNA deletions in the N-part of cro-lacIZ coding zone as constructed using Bal31 nuclease. Development of models of mRNA secondary structure was carried out stepwise beginning from the 5'-end by taking into consideration the hairpins first formed during mRNA synthesis. Comparison of the results of mRNA secondary structure determination and protein production analysis demonstrated a correlation between the efficiency of translation initiation and the appearance of a single-stranded region upon disruption of the mRNA local secondary structure in the translation initiation zone generated by ribosomes. These results confirm the suggestion of the central role played by the Shine-Dalgarno sequence in the generation of a single-stranded region. Some plasmids from the set are supposed to determine protein synthesis by a translation reinitiation mechanism in the absence of Shine--Dalgarno interaction. In this case, correlation between reinitiation efficiency and local disruption of the mRNA secondary structure by the terminating ribosome was also observed. The terminating ribosome that forms the single-stranded region near the initiation codon fulfils the major function of the Shine--Dalgarno interaction. In addition, the possible effect of another mRNA secondary structure region on the translation initiation efficiency is discussed.
Asunto(s)
Genes Bacterianos , Conformación de Ácido Nucleico , Iniciación de la Cadena Peptídica Traduccional , ARN Bacteriano/genética , ARN Mensajero/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Hibridación de Ácido Nucleico , PlásmidosRESUMEN
On the basis of theoretical analysis of different mRNAs secondary structure it is suggested that the efficiency of procaryotic translation initiation depends to a great extent on the possibility to generate a single-stranded region around the initiation codon. The local disruption of the mRNA secondary structure is mostly determined by interaction according to Shine--Dalgarno of 16S rRNA with the complementary mRNA region. Other mechanisms of single-stranded region generation in the initiation zone of mRNA are discussed.
Asunto(s)
Conformación de Ácido Nucleico , Operón , Iniciación de la Cadena Peptídica Traduccional , ARN Bacteriano/genética , ARN Mensajero/genética , Secuencia de Bases , Codón , Escherichia coli/genética , Genes , Modelos Genéticos , Modelos Moleculares , Hibridación de Ácido Nucleico , Plásmidos , Serratia marcescens/genéticaRESUMEN
5'-Terminal nucleotide was degenerated in the fragments produced by means of hydrolysis with vestrictase Eco CK. Analysis of the nucleotide sequence, carried out in four DNA fragments after the enzymatic hydrolysis' enabled to detect the only one common 13 residues nucleotide sequence, which is apparently involved in the recognizing site for vestrictase Eco CK: (A/T) (A/T) N (A/T) CGCNCNNNG. This sequence was not found in several DNA molecules with well-known primary structure, stable to the action of this enzyme.