RESUMO
In spite of extensive research, no effective vaccine against hepatitis C virus (HCV) has been developed so far. DNA immunization is a potent technique of vaccine design strongly promoting the cellular arm of immune response. The genes encoding nonstructural HCV proteins (NS2-NS5B) are promising candidates for vaccine development. NS5A is a protein involved in viral pathogenesis, in the induction of immune response, and probably in viral resistance to interferon treatment. The objective of this study was to construct a DNA vaccine encoding NS5A protein and evaluate its immunogenicity. A plasmid encoding a full-size NS5A protein was produced using the pcDNA3.1 (+) vector for eukaryotic expression system. The expression of the NS5A gene was confirmed by immunoperoxidase staining of the transfected eukaryotic cells with anti-NS5A monoclonal antibodies. Triple immunization of mice with the plasmid vaccine induced a pronounced cellular immune response against abroad spectrum of NSSA epitopes as assessed by T-cell proliferation andsecretion of antiviral cytokines IFN-gamma and IL-2. In in vitro T-cell stimulation experiments, NS5A-derived antigens were modeled by synthetic peptides, recombinant proteins of various genotypes, and phages carrying exposed NS5A peptides. A novel immunomodulator Immunomax showed high adjuvant activity in DNA immunization. The data obtained indicate that the suggested DNA construct has a strong potential in the development of the gene vaccines against hepatitis C.
Assuntos
Hepacivirus/imunologia , Hepatite C/imunologia , Plasmídeos/farmacologia , Linfócitos T/imunologia , Vacinas de DNA/farmacologia , Vacinas contra Hepatite Viral/farmacologia , Proteínas não Estruturais Virais/imunologia , Animais , Linhagem Celular Tumoral , Proliferação de Células , Chlorocebus aethiops , Feminino , Hepacivirus/genética , Hepacivirus/metabolismo , Hepatite C/genética , Hepatite C/metabolismo , Hepatite C/prevenção & controle , Humanos , Imunidade Celular , Interferon gama/biossíntese , Interferon gama/imunologia , Interleucina-2/biossíntese , Interleucina-2/imunologia , Camundongos , Plasmídeos/genética , Plasmídeos/imunologia , Plasmídeos/metabolismo , Linfócitos T/metabolismo , Vacinas de DNA/genética , Vacinas de DNA/imunologia , Vacinas de DNA/metabolismo , Células Vero , Vacinas contra Hepatite Viral/genética , Vacinas contra Hepatite Viral/imunologia , Vacinas contra Hepatite Viral/metabolismo , Proteínas não Estruturais Virais/biossíntese , Proteínas não Estruturais Virais/genéticaRESUMO
The ovine foot rot is a severe infectious disease of sheep. Dichelobacter nodosus is an essential pathogen of this disease. An obligatory anaerobic gram-negative rod-shaped microorganism has slow rate of accumulating bacterial density and fastidious growth requirements. This causes obstacles to vaccine production and makes it difficult to diagnose the disease. The diagnosis in this case is more expensive. Fimbriae (or pili) are one of the major factors of virulence of D. nodosus. Their antigenic and immunogenic properties make them good vaccine components for elevated immunogenicity. Since the nucleotide sequence of the fimA gene encoding fimbrial subunit was determined, attempts to produce recombinant pili were undertaken. The production of the genetic-engineering fimbriae would allow the price of the vaccines to be reduced and their manufacture to be simplified. The vaccine immunogenicity is increased in this case. At first, E. coli was selected as an expression system, but morphogenetic expression of the pili was not achieved on its surface because of some differences in the biogenesis and structure of fimbriae from D. nodosus. Successful morphogenesis of the pili was achieved in Pseudomonas aeruoginosa, which had closest similarity in the structure of pili. The level of the immunity obtained after immunization of the sheep with recombinant pili was similar to the level of the immunity after native pili or whole cells of D. nodosus had been used. This review contains information regarding the recombinant strains of Pseudomonas aeruoginosa obtained using fimbriae of D. nodosus and expression of pilin genes in different bacterial systems.
Assuntos
Vacinas Bacterianas/imunologia , Dichelobacter nodosus/imunologia , Proteínas de Fímbrias/imunologia , Fímbrias Bacterianas/imunologia , Pseudomonas aeruginosa/imunologia , Doenças dos Ovinos/imunologia , Animais , Vacinas Bacterianas/genética , Dichelobacter nodosus/genética , Proteínas de Fímbrias/genética , Fímbrias Bacterianas/genética , Pododermatite Necrótica dos Ovinos/imunologia , Pododermatite Necrótica dos Ovinos/microbiologia , Pseudomonas aeruginosa/genética , Ovinos , Doenças dos Ovinos/genética , Doenças dos Ovinos/microbiologiaRESUMO
Recombinant nucleocapsid (rN) protein N of porcine reproductive and respiratory syndrome virus (PRRSV) was prepared, by using the E. coli expressiom system. Insertion of a polyhistidine marker into the structure of the protein allowed the latter to be purified by metal-chelate affinity chromatography. The purity of protein was confirmed by PAAG electrophoresis and its immunospecificity was verified by immunoblotting using rN-specific monoclonal antibodies. The protein was used as an antigen to develop indirect ELISA of PRRSV antibodies. ELISA was shown to be highly sensitive and specific.
Assuntos
Síndrome Respiratória e Reprodutiva Suína/diagnóstico , Vírus da Síndrome Respiratória e Reprodutiva Suína/imunologia , Animais , Anticorpos Antivirais/sangue , Especificidade de Anticorpos , Ensaio de Imunoadsorção Enzimática/métodos , Proteínas do Nucleocapsídeo/imunologia , Síndrome Respiratória e Reprodutiva Suína/sangue , Kit de Reagentes para Diagnóstico , Proteínas Recombinantes/imunologia , Sensibilidade e Especificidade , Testes Sorológicos , SuínosRESUMO
The mechanisms of induction of heat shock protein synthesis in E. coli have been studied. For this purpose plasmids in which htpR gene expression is controlled by the PR-promoter of bacteriophage lambda and by the Trp-promoter have been constructed. An effective induction of heat shock proteins requires both an increased content of htpR protein and additional cofactors formed in the cell under heat shock conditions.
Assuntos
Bacteriófago lambda/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Regulação Viral da Expressão Gênica , Proteínas de Choque Térmico/biossíntese , Autorradiografia , Escherichia coli/metabolismo , Plasmídeos , Regiões Promotoras GenéticasRESUMO
The genomic library of Staphylococcus aureus genes on the plasmid vector pSL5 has been constructed. The library contains a 2.5 kb HindIII DNA fragment including the gene for enterotoxin A. The entA gene on the high copy number plasmids in the Escherichia coli cells deficient in proteolysis determines the synthesis of enterotoxin A in the amounts comparable to the ones in the parent strain Staphylococcus aureus FRI 722(H).
Assuntos
Enterotoxinas/genética , Regulação Bacteriana da Expressão Gênica , Genes Bacterianos , Staphylococcus aureus/genética , Bacillus subtilis/genética , Escherichia coli/genética , Biblioteca Gênica , Plasmídeos , Mapeamento por RestriçãoRESUMO
The porcine reproductive and respiratory syndrome (PRRS) is a contagious viral pathology caused by PRRS virus. There are 2 types of the above virus--the European and American ones. Distribution patterns of the PRRS virus were studied for Russia and Byelorussia. Above 700 porcine sera obtained from 32 households of 21 Russia's administrative regions and from 19 households of 6 Byelorussia's administrative regions were tested for presence of antibodies to the PRRS virus. Simultaneously, the samples were tested for virus presence by polymerase chain reaction (PCR). It was proven serologically that the PRRS virus is widespread in the territories of Russia and Byelorussia. Noteworthily, all field isolates found in Russia and Byelorussia belong to the European type. Not a single viral isolate of the American PRRS type was found. The nucleocapsid (N) recombinant protein was obtained on the basis of the Russian field isolate of the PRRS virus by using the E. coli. expression system. Finally, it was shown as possible to use the recombinant protein in indirect immune enzyme assay for the sake of detecting the antibodies to the PRRS virus.
Assuntos
Proteínas do Nucleocapsídeo/genética , Síndrome Respiratória e Reprodutiva Suína/virologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/genética , Animais , Anticorpos Antivirais/sangue , Técnica Indireta de Fluorescência para Anticorpo , Variação Genética , Síndrome Respiratória e Reprodutiva Suína/sangue , Síndrome Respiratória e Reprodutiva Suína/epidemiologia , Vírus da Síndrome Respiratória e Reprodutiva Suína/classificação , Vírus da Síndrome Respiratória e Reprodutiva Suína/imunologia , República de Belarus/epidemiologia , Federação Russa/epidemiologia , Estudos Soroepidemiológicos , SuínosRESUMO
Gene ent-A has been cloned on phage vector pSL5 with the use of the gene library of S. aureus FR1722(H). It is located within DNA fragment Hind III having 2,500 nucleotide pairs.
Assuntos
Clonagem Molecular/métodos , Enterotoxinas/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/genética , Genes Bacterianos , Staphylococcus aureus/genética , Colífagos/genética , DNA Bacteriano/genética , Biblioteca Gênica , Vetores Genéticos/genética , Plasmídeos/genética , Fagos de Staphylococcus/genética , Transfecção/genéticaRESUMO
The selection and subsequent synthesis, according to the nucleotide sequences of S. aureus genes responsible for the expression of enterotoxins A and B, of highly specific primers for polymerase chain reaction Pcr were carried out with the use of the program "Primer". The optimum temperature conditions of polymerase chain reaction for all pairs of primers were selected. The method for the rapid determination of the enterotoxigenic properties of S. aureus strains by means of Pcr was proposed. The enterotoxigenic properties of several S. aureus strains were determined, which revealed that 3 clinical isolates had the gene of enterotoxin A in their genome, while laboratory strain FRI-722(H) carried the genes of enterotoxins A and B.