RESUMO
Pfs48/45 is an important transmission-blocking vaccine candidate antigen of the human malaria parasite Plasmodium falciparum. This study was aimed at synthesis of recombinant Pfs48/45 containing conformation-constrained epitopes of the native antigen in yeast. Since in the yeast Saccharomyces cerevisiae induction of gene-expression led to prematurely terminated transcripts, an entirely synthetic gene of higher GC content was assembled. Replacement of the AT rich natural gene by the synthetic gene relieved the observed premature transcription termination. Nevertheless, recombinant protein expression could not be detected. In contrast, in the yeast Pichia pastoris low levels of recombinant Pfs48/45 were produced upon induction of synthetic gene expression. The recombinant protein was shown to be disulphide-bridge constrained, but was not recognised by transmission-blocking antibodies and did not induce transmission-blocking sera in mice.
Assuntos
Glicoproteínas de Membrana/genética , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , DNA de Protozoário/genética , Genes Sintéticos , Humanos , Malária/parasitologia , Glicoproteínas de Membrana/biossíntese , Glicoproteínas de Membrana/imunologia , Glicoproteínas de Membrana/isolamento & purificação , Dados de Sequência Molecular , Pichia/genética , Pichia/metabolismo , Proteínas de Protozoários/biossíntese , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/isolamento & purificação , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , TransfecçãoRESUMO
A precondition for the development of a transmission blocking vaccine based on the sexual stage-specific surface antigen Pfs48/45 of Plasmodium falciparum is its heterologous synthesis in a native state. Here we describe the production of recombinant Pfs48/45 in Escherichia coli. Two recombinant proteins, of which one is a glutathione-S-transferase fusion protein, were produced. Enzyme-linked immunosorbent assays showed that at least a subfraction of the recombinant proteins had a conformation capable of binding transmission blocking monoclonal antibodies. However, despite the fact that both proteins were very immunogenic, they did not induce transmission blocking immunity in mice or rabbits. Immunological studies with congenic mouse strains demonstrated that immune responses could be boosted with gametocyte extracts and were not restricted to a particular class II major histocompatibility complex haplotype.
Assuntos
Vacinas Antimaláricas/imunologia , Malária Falciparum/imunologia , Plasmodium falciparum/imunologia , Proteínas de Protozoários/imunologia , Proteínas Recombinantes de Fusão/imunologia , Animais , Anticorpos Monoclonais/imunologia , Anticorpos Antiprotozoários/sangue , Especificidade de Anticorpos , Escherichia coli , Vetores Genéticos , Humanos , Camundongos , Camundongos Congênicos , CoelhosRESUMO
With the aim of developing transmission-blocking vaccines based on the sexual stage-specific surface antigen Pfs48/45 of the human malaria parasite Plasmodium falciparum, the gene encoding Pfs48/45 was incorporated into the genome of a recombinant vaccinia virus. In virus-infected mammalian tissue culture cells, recombinant Pfs48/45 antigen (rPfs48/45) is posttranslational modified to produce a highly N-glycosylated polypeptide. The rPfs48/45 protein was radiolabeled with ethanolamine, consisting of a further posttranslational modification in the form of a glycosylphosphatidylinositol anchor at its carboxy-terminal end. The rPfs48/45 was not detected on the surface of the infected cells; instead, it remained within the secretion pathway of mammalian cells irrespective of the duration of infection or culture temperature. Studies with monoclonal antibodies specific for disulfide band-dependent epitopes of Pfs48/45 revealed that recombinant Pfs48/45 is not folded in its authentic conformation even if N-glycosylation was chemically inhibited. Infection of mice and rabbits with recombinant virus elicited Pfs48/45-specific antibodies; however, the antisera failed to block parasite transmission in a standard mosquito membrane-feeding assay.
Assuntos
Antígenos de Protozoários/biossíntese , Vacinas Antimaláricas , Plasmodium falciparum/imunologia , Proteínas de Protozoários/biossíntese , Vacinas Sintéticas , Animais , Antígenos de Protozoários/imunologia , Antígenos de Protozoários/metabolismo , Antígenos de Superfície/biossíntese , Antígenos de Superfície/imunologia , Antígenos de Superfície/metabolismo , Linhagem Celular , Técnica Indireta de Fluorescência para Anticorpo , Vacinas Antimaláricas/biossíntese , Vacinas Antimaláricas/metabolismo , Malária Falciparum/prevenção & controle , Camundongos , Camundongos Endogâmicos BALB C , Testes de Precipitina , Processamento de Proteína Pós-Traducional , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/metabolismo , Coelhos , Vacinas Sintéticas/biossíntese , Vacinas Sintéticas/metabolismo , Vaccinia virus/fisiologiaAssuntos
Glicoproteínas de Membrana/química , Plasmodium falciparum/imunologia , Plasmodium/imunologia , Proteínas de Protozoários/química , Sequência de Aminoácidos , Animais , Antígenos de Superfície/química , Antígenos de Superfície/genética , Sequência de Bases , DNA de Protozoário/química , Imunofluorescência , Humanos , Glicoproteínas de Membrana/genética , Dados de Sequência Molecular , Pan troglodytes , Plasmodium/genética , Plasmodium falciparum/genética , Reação em Cadeia da Polimerase , Proteínas de Protozoários/genética , Homologia de Sequência de AminoácidosAssuntos
Glicoproteínas de Membrana/genética , Plasmodium falciparum/genética , Proteínas de Protozoários/genética , Sequência de Aminoácidos , Animais , Sequência de Bases , Mapeamento de Epitopos , Variação Genética , Glicoproteínas de Membrana/imunologia , Dados de Sequência Molecular , Plasmodium falciparum/imunologia , Polimorfismo Conformacional de Fita Simples , Proteínas de Protozoários/imunologia , Vacinas ProtozoáriasRESUMO
1. We have compared the response to griseofulvin of rats and mice and, in mice, the effect of griseofulvin itself with that of two of its analogues. The severity of protoporphyria shows a correlation with the accumulation of both types of N-alkylated porphyrins previously described after treatment with this drug, namely N-methylproptoporphyrin and the N-griseofulvin protoporphyrin adduct. 2. Both N-alkylporphyrins are chiral, are labelled from 5-amino[4-14C]laevulinate, and their liver accumulation can be inhibited by pretreatment with a suicide substrate of cytochrome P-450, which also prevents porphyria. 3. These findings suggest that cytochrome P-450 is involved in the mechanism of griseofulvin-induced protoporphyria by generating N-methylprotoporphyrin. The N-griseofulvin protoporphyrin adduct may also originate from cytochrome P-450, but more work is necessary to elucidate whether it acts as the precursor for N-methylprotoporphyrin.