RESUMO
The cancer-testis antigen, NY-ESO-1, has been engineered into a bacterial expression plasmid which incorporates a His6-tag. The plasmid was transfected into E. coli strain BL21 and Master and Working cell banks generated from this expression system. Three 15-litre fermentations were performed under cGMP (code of Good Manufacturing Practice) conditions and the crude NY-ESO-1 tagged protein isolated as solubilised inclusion bodies. A three-step cGMP chromatography process (immobilised metal affinity, anion exchange, and hydrophobic interaction) was utilised to purify the protein. The purified NY-ESO-1 is being used in early stage human cancer vaccine trials in Australia and the U.S.A.
Assuntos
Antígenos de Neoplasias/biossíntese , Antígenos de Neoplasias/isolamento & purificação , Vacinas Anticâncer/biossíntese , Vacinas Anticâncer/isolamento & purificação , Proteínas de Membrana/biossíntese , Proteínas de Membrana/isolamento & purificação , Engenharia de Proteínas/métodos , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/uso terapêutico , Austrália , Vacinas Anticâncer/genética , Indústria Farmacêutica/normas , Guias como Assunto , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/uso terapêutico , Engenharia de Proteínas/normas , Garantia da Qualidade dos Cuidados de Saúde/normas , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/isolamento & purificação , Padrões de ReferênciaRESUMO
Culture conditions have been developed which enable the asexual erythrocytic stage of Plasmodium falciparum to be grown in deep culture and produce parasite yields comparable to shallow stationary culture. The major benefit of deep culture is that large volumes of parasites can be grown in a single vessel (up to 8 litres) and thus advantage can be taken of semi-automatic approaches to medium replacement. Other benefits are that large quantities of viable parasites can be matured without multiple medium replacements and synchronized parasite culture can be maintained easily over an extended time.
Assuntos
Parasitologia/métodos , Plasmodium falciparum/crescimento & desenvolvimento , Animais , Meios de Cultura , Eritrócitos/parasitologia , Hematócrito , HumanosRESUMO
The precursor to the major merozoite surface antigens of Plasmodium falciparum appears to be encoded by two distinctly different (dimorphic) alleles able to undergo limited recombination. We analyzed 18 previously uncharacterized P. falciparum isolates to test the dimorphic model. All but one, a Thailand isolate, conformed to the dimorphic model, and this isolate conformed to the dimorphic model in all but variable block 2. Sequence analysis revealed that block 2 of isolate CSL2 was a third form. Hence, the dimorphic model is not strictly correct. Recombination between alleles was found only within two conserved blocks near the 5' end of the gene.
Assuntos
Antígenos de Protozoários/genética , Plasmodium falciparum/genética , Precursores de Proteínas/genética , Alelos , Animais , Sequência de Bases , DNA/metabolismo , Dados de Sequência Molecular , Hibridização de Ácido Nucleico , Plasmodium falciparum/imunologiaRESUMO
A 26 kDa protein, present in trophozoites and schizonts of Plasmodium falciparum, has been identified as the target of a monoclonal antibody that weakly inhibits parasite growth in vitro. The antigen has been purified to homogeneity by immuno-affinity chromatography and electrophoresis. The sequence of 19 amino acids at the N-terminus of the protein has been determined.
Assuntos
Anticorpos Monoclonais/imunologia , Antígenos de Protozoários/isolamento & purificação , Plasmodium falciparum/imunologia , Sequência de Aminoácidos , Animais , Antígenos de Protozoários/análise , Antígenos de Protozoários/imunologia , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Imunofluorescência , Imunoensaio , Dados de Sequência Molecular , Plasmodium falciparum/crescimento & desenvolvimentoRESUMO
An in situ enzyme immunoassay to viral membrane antigen was developed to enable the specific estimation of antibodies to varicella zoster (VZ) virus. The technique was compared with a modified fluorescent antibody to membrane antigen (FAMA) procedure and with the complement fixation (CF) test by parallel assay of 352 plasma samples. The enzyme immunoassay (EIA) procedure showed very good correlation with the modified FAMA procedure, and both were far more specific than the CF test. This specificity was achieved by the use, in the EIA, of VZ virus-infected cells grown and fixed in situ with glutaraldehyde. Thus the only virus antigens accessible to antibody were the VZ-specific antigens expressed at the cell membrane, cross-reactions with herpes simplex virus antibodies thereby being avoided.