RESUMO
Recombinant glyceraldehyde-3-phosphate dehydrogenase of the cestode parasite Echinococcus multilocularis was expressed in Escherichia coli and in Salmonella typhimurium. The potential of different forms of the recombinant antigen to protect BALB/c mice against oral challenge infections with E. multilocularis eggs was evaluated. Oral or intraperitoneal immunisation with live attenuated S. typhimurium as a carrier for recombinant glyceraldehyde-3-phosphate dehydrogenase of the E. multilocularis resulted in significant protection, reducing the number of developing metacestodes up to 79.8%. The sera of protected animals did not contain detectable amounts of antibody against glyceraldehyde-3-phosphate dehydrogenase of E. multilocularis. By contrast, although anti-glyceraldehyde-3-phosphate dehydrogenase of E. multilocularis antibodies were detectable in the sera, immunisation with E. coli-expressed recombinant glutathione-S-transferase-fusion protein or with glyceraldehyde-3-phosphate dehydrogenase of E. multilocularis fused to a 6HIS-tag failed to protect the animals against oral challenge infections. These data emphasise that antigen delivery systems play a critical role in vaccination and the induction of protective immunity against helminth parasites.
Assuntos
Equinococose/imunologia , Echinococcus/imunologia , Gliceraldeído-3-Fosfato Desidrogenases/imunologia , Imunização , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Clonagem Molecular , Equinococose/parasitologia , Equinococose/prevenção & controle , Echinococcus/enzimologia , Escherichia coli/enzimologia , Escherichia coli/genética , Feminino , Gliceraldeído-3-Fosfato Desidrogenases/biossíntese , Gliceraldeído-3-Fosfato Desidrogenases/genética , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Salmonella typhimurium/enzimologia , Salmonella typhimurium/genética , Homologia de Sequência de Aminoácidos , Estatísticas não ParamétricasRESUMO
A cloned cDNA, generated from mRNA isolates of phosphate-derepressed H. polymorpha cells, was identified to harbour an incomplete sequence of the coding region for a repressible acid phosphatase. The cDNA fragment served as a probe to screen a plasmid library of H. polymorpha genomic DNA. A particular clone, p606, of a 1.9-kb insert contained a complete copy of the PHO1 gene. Sequencing revealed the presence of a 1329-nucleotide open reading frame encoding a protein of 442 amino acids with a calculated M(r) of 49400. The encoded protein has an N-terminal 17-amino-acid secretory leader sequence and seven potential N-glycosylation sites. The leader cleavage site was confirmed by N-terminal sequencing of the purified enzyme. The nucleotide sequence is 48.9% homologous, the derived amino acid sequence 36% homologous to its Saccharomyces cerevisiae counterpart. The derived amino acid sequence harbours a consensus sequence RHGXRXP, previously identified as a sequence involved in active-site formation of acid phosphatases. The PHO1 promoter and the secretion leader sequence present promising new tools for heterologous gene expression.
Assuntos
Fosfatase Ácida/genética , Sequência Consenso , Regulação Fúngica da Expressão Gênica , Pichia/enzimologia , Homologia de Sequência de Aminoácidos , Fosfatase Ácida/química , Fosfatase Ácida/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Antifúngicos/biossíntese , Sequência de Bases , Western Blotting , Cromatografia em Gel , Cromatografia por Troca Iônica , Clonagem Molecular , DNA Complementar/química , Eletroforese em Gel de Poliacrilamida , Escherichia coli/química , Regulação Enzimológica da Expressão Gênica , Biblioteca Gênica , Dados de Sequência Molecular , Pichia/genética , RNA Mensageiro/química , Coelhos , Mapeamento por Restrição , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
Sequences of three cDNAs of Schistosoma japonicum are presented. Two of them code for the antigens Sj32 (hemoglobinase) and cathepsin B. The third clone represents a mutant derivative of the cathepsin B gene. The DNA and amino acid sequences are compared to their homologues from S. mansoni. The mutant cathepsin B, in which the Cys29 in the active center is replaced by Ser, is discussed with respect to a possible function.
Assuntos
Antígenos de Helmintos/genética , Catepsina B/genética , Cisteína Endopeptidases/genética , DNA Complementar/química , Proteínas de Helminto , Schistosoma japonicum/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Clonagem Molecular , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosRESUMO
The ura3 gene of Hansenula polymorpha was cloned, sequenced and used to generate a ura3 mutant from the wild-type strain of this yeast via integrative mutagenesis. The Tn5 neomycin-resistance marker (neo) under control of the ADH1 promoter from Saccharomyces cerevisiae served as a transformation marker. The results show that gene replacement can be achieved in H. polymorpha, a yeast with a high level of non-homologous integration.
Assuntos
Genes Fúngicos/genética , Marcadores Genéticos , Orotidina-5'-Fosfato Descarboxilase/genética , Pichia/genética , Transformação Genética/genética , Clonagem Molecular , Deleção de Genes , Mutagênese Sítio-Dirigida , Pichia/enzimologia , Análise de Sequência de DNARESUMO
An expression system has been developed for the methylotrophic yeast Hansenula polymorpha and used to co-express both the L (preS1-S2-S) and S hepatitis B surface antigens (HBsAg) under the control of strong methanol-inducible promoters derived from the methanol oxidase and from the formate dehydrogenase genes. A unique feature of this H. polymorpha expression system is the possibility of integrating up to 100 copies of an expression cassette via a multimeric integration mechanism. Several multimeric integrants containing various numbers of L and S expression cassettes were constructed to give a spectrum of strains characterized by different L to S ratios. The expression level of S antigen was 5-8% of the total soluble cell protein. Analysis by sucrose and CsCl density gradient centrifugation and by particle-specific immunoassays demonstrated that the synthesized HBsAg spontaneously assembled into composite subviral particles containing both S and L proteins. Only a minor portion of the L protein was found to be glycosylated. These H polymorpha-derived composite particles can be used for the production of a hepatitis B virus vaccine with the potential for improved immunogenicity due to the presence of a wider spectrum of epitopes and negligible glycosylation.
Assuntos
Regulação Fúngica da Expressão Gênica , Antígenos de Superfície da Hepatite B/biossíntese , Pichia/genética , Sequência de Bases , Western Blotting , Meios de Cultura , DNA Fúngico/química , Fermentação , Vetores Genéticos , Antígenos de Superfície da Hepatite B/genética , Antígenos de Superfície da Hepatite B/imunologia , Vírus da Hepatite B/genética , Vírus da Hepatite B/imunologia , Immunoblotting , Dados de Sequência Molecular , Pichia/metabolismo , Plasmídeos , Regiões Promotoras Genéticas , Precursores de Proteínas/biossíntese , Precursores de Proteínas/genética , Precursores de Proteínas/imunologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Mapeamento por RestriçãoRESUMO
We have introduced the glucoamylase gene (GAM1) from Schwanniomyces occidentalis into the genome of the methylotrophic yeast Hansenula polymorpha to study the potential of this organism as a host for high-level expression of a heterologous gene encoding a secretory protein. Transformants of H. polymorpha containing GAM1 under control of the formate dehydrogenase (FMD) promoter are stable and efficiently secrete an active glucoamylase that is faithfully processed and modified. Yields of up to 1.4g/l of active enzyme were obtained at cell densities of 100-130 grams dry weight per liter.
Assuntos
Clonagem Molecular/métodos , Glucana 1,4-alfa-Glucosidase/genética , Pichia/genética , Saccharomycetales/enzimologia , Sequência de Aminoácidos , Fermentação , Genes Fúngicos , Glucana 1,4-alfa-Glucosidase/biossíntese , Glucana 1,4-alfa-Glucosidase/metabolismo , Dados de Sequência Molecular , Pichia/enzimologia , PlasmídeosRESUMO
mRNA of 23-day-old maize endosperm was translated both in wheat germ extracts and rabbit reticulocyte lysates. A protein with an apparent molecular weight of 88,000 comigrates in dodecylsulfate/polyacrylamide electrophoresis with sucrose synthase. This protein is precipitated with an antiserum against sucrose synthase and shows the same protease digestion pattern as the enzyme. It is not synthesized with mRNA extracted from sh/sh mutant kernels lacking sucrose synthase. By these criteria, the protein is the translation product in vitro of sucrose synthase mRNA. The separation of mRNA in methylmercury-hydroxide--agarose gels and subsequent translation indicates a length of sucrose synthase mRNA of 2800 nucleotides which is compatible with the coding length necessary for a protein with a molecular weight of 88,000 plus untranslated sequences.
