RESUMEN
Adenoviral dodecahedron (Dd) is a virus-like particle composed of twelve pentameric penton base (Pb) proteins, responsible for adenovirus cell penetration. It is generated spontaneously in the baculovirus system upon expression of the Pb gene of adenovirus serotype 3. This particle shows remarkable cell penetration ability with 2,00,000-3,00,000 Dd internalized into one cell in culture, conceivably delivering several millions of foreign cargo molecules to the target cell. We have used it in the past for delivery of small drugs as well as a vaccination platform, in which Dd serves as a particulate vaccine delivery system. Since development of new biomedicals depends strongly on the cost of their expression and purification, we attempted, albeit unsuccessfully, to obtain Dd expression in bacteria. We therefore retained its expression in the baculovirus/insect cells system but introduced significant improvements in the protocols for Dd expression and purification, leading to considerable savings in time and improved yield.
Asunto(s)
Adenoviridae/metabolismo , Centrifugación/métodos , Cromatografía en Gel/métodos , Proteínas Virales/aislamiento & purificación , Adenoviridae/genética , Clonación Molecular , Escherichia coli/genética , Genes Virales , Sacarosa , Proteínas Virales/genética , Proteínas Virales/metabolismoRESUMEN
We propose a novel influenza vaccine composed of the adenovirus dodecahedron (Dd) as delivery platform carrying an internal influenza matrix protein M1. To attach the antigen to the vector we used WW domains interacting with Dd. Successful internalization of the Dd-M1WW complex was observed using biochemical and cell biology techniques. We show here that the complex of Dd with antigen is a potent activator of human myeloid dendritic cells (MDC), and that it is efficiently presented by MDC to M1-specific CD8+ T lymphocytes. These results show that proposed vaccine model is feasible and that adenovirus dodecahedron is a potent delivery platform for foreign antigens to human cells.
Asunto(s)
Adenoviridae/inmunología , Presentación de Antígeno/inmunología , Vacunas contra la Influenza/inmunología , Proteínas de la Matriz Viral/inmunología , Secuencia de Aminoácidos , Linfocitos T CD8-positivos/inmunología , Clonación Molecular , Células Dendríticas/inmunología , Células HeLa , Humanos , Subtipo H1N1 del Virus de la Influenza A/inmunología , Vacunas contra la Influenza/biosíntesis , Gripe Humana/prevención & control , Datos de Secuencia Molecular , Proteínas Recombinantes/inmunología , Vacunas Sintéticas/inmunologíaRESUMEN
Human prolactin was expressed in insect culture cells by recombinant baculoviruses carrying prolactin gene cDNA placed under the transcriptional control of polyhedrin gene promoter of Autographa californica nuclear polyhedrosis virus. Preliminary results of recombinant human prolactin expression as extracellular as well as intracellular product of baculovirus expression system were presented at the FEBS Meeting in Nice, France, in 1999 (Abstracts, p. 288). In the present work prolactin was expressed as a hexahistidine-tagged fusion protein and recombinant protein was purified by metal affinity resin. Yields varied between approximately 20 and 35 mg/liter of medium. This recombinant prolactin was biologically active in Nb2 lymphoma cell proliferation assay and after simple purification could substitute for pituitary-derived prolactin.
Asunto(s)
Líquido Intracelular/metabolismo , Prolactina/biosíntesis , Prolactina/genética , Spodoptera/genética , Spodoptera/metabolismo , Animales , Baculoviridae/genética , Bioensayo , Sistema Libre de Células/química , Sistema Libre de Células/metabolismo , Células Cultivadas , Clonación Molecular , Sustancias de Crecimiento/biosíntesis , Sustancias de Crecimiento/genética , Sustancias de Crecimiento/aislamiento & purificación , Sustancias de Crecimiento/fisiología , Humanos , Cuerpos de Inclusión/genética , Cuerpos de Inclusión/metabolismo , Peso Molecular , Nucleopoliedrovirus/genética , Prolactina/aislamiento & purificación , Prolactina/fisiología , Desnaturalización Proteica , Renaturación de Proteína , Ratas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Solubilidad , Spodoptera/citología , Spodoptera/virología , Células Tumorales CultivadasAsunto(s)
Baculoviridae/genética , Baculoviridae/patogenicidad , Insectos/virología , Animales , Secuencia de Bases , Línea Celular , Mapeo Cromosómico , ADN Viral/análisis , ADN Viral/biosíntesis , Regulación Viral de la Expresión Génica , Interacciones Huésped-Parásitos , Sistemas de Lectura AbiertaRESUMEN
Brain extracts from day 1-4 last instar larvae of Galleria mellonella (Lepidoptera) stimulate RNA synthesis in cultured silk glands from day 3 last instar larvae. When the fibroin-synthesizing posterior parts of silk glands were incubated for 3 h in vitro in the presence of brain extract (0.1 brain equivalent), [3H]-uridine incorporation into RNA was stimulated more than twofold. The stimulating effect of brain extract showed a dose response relationship. It is suggested that the heat-resistant and protease-sensitive brain factor is a peptide.
Asunto(s)
Hormonas de Insectos/fisiología , Proteínas de Insectos , Mariposas Nocturnas/fisiología , Sistemas Neurosecretores/fisiología , Proteínas/metabolismo , Animales , Hormonas de Insectos/química , ARN/metabolismo , Seda , Extractos de TejidosRESUMEN
Synthesis of the low molecular mass silk proteins of 24 and 30 kDa in the last larval instar of Galleria mellonella starts between 24 and 48 h; synthesis of the former protein significantly preceding that of the latter. Posterior silk glands (PSG) from day-1 last instar larvae are transiently sensitive in vitro to exogenous 20-hydroxyecdysone (20-HE), which stimulates transcription of the 24 kDa protein gene and induces transcription of the gene for the 30 kDa protein. The glands from the day -3 last instar larvae are insensitive to this hormone. The brain extract acts directly on RNA synthesis in silk gland in vitro at the concentration of 0.1 brain equivalent per gland. This factor is protease sensitive and thermostable.
Asunto(s)
Ecdisterona/farmacología , Proteínas de Insectos , Mariposas Nocturnas/metabolismo , Biosíntesis de Proteínas , Animales , Encéfalo/fisiología , Larva/efectos de los fármacos , Larva/crecimiento & desarrollo , Larva/metabolismo , Peso Molecular , Mariposas Nocturnas/efectos de los fármacos , Mariposas Nocturnas/crecimiento & desarrollo , Proteínas/efectos de los fármacos , Proteínas/genética , Seda , Transcripción GenéticaRESUMEN
An in vitro sensitive bioassay for the Galleria mellonella brain allatotropic hormone (ATTH) was developed. This assay measures the rate of juvenile hormone (JH) synthesis in corpora cardiacacorpora allata complex (CC-CA) stimulated in vitro by ATTH released from the brain during short-term in vitro incubation, or by ATTH extracted from the tissue with methanol. CC-CA of the late VIth instar (VI3) larvae were used for assessment of ATTH. The maximum activation of test CC-CA by ATTH occurred at a concentration of 2 brain equivalents (per 100 ul medium). The highest ATTH activity was exhibited by the brains of chilled VII1 larvae: ATTH extracted from freshly dissected brains, or ATTH released from these brains during 6 h in vitro incubation, activated JH synthesis in the CC-CA nearly five or four times, respectively. The brain of VII1 hydroprenetreated larvae were ATTH inactive.
Asunto(s)
Hormonas de Insectos/análisis , Lepidópteros/química , Animales , Bioensayo , Química Encefálica , Corpora Allata/química , Ácidos Grasos Insaturados/farmacología , Técnicas In Vitro , Hormonas Juveniles/biosíntesis , Larva , Lepidópteros/metabolismo , Factores de TiempoRESUMEN
Hydrolytic rates of juvenile hormones (JHs) I, II and III by the corpora cardiaca-corpora allata complex (CC-CA) and by the haemolymph of Galleria mellonella remain in the same order (III greater than I greater than II in CC-CA and I greater than III greater than II in haemolymph) throughout the last larval instar. Haemolymph hydrolytic activity shows peak at the end of feeding when 80 pmol JH I versus 15 pmol JH II is degraded per 1 microliter and minute; hydrolysis rapidly declines in the apolysing insects. Hydrolytic rates in CC-CA reach a maximum of 240 fmol/pair per min for JH III and 85 fmol/pair per min for JH II in pharate pupae. Brain implantations or chilling of freshly ecdysed last instar larvae, which are known to elevate JH titer and induce supernumerary larval molt, do not affect JH hydrolysis. The results indicate that the dominance of JH II in Galleria may be at least partly controlled by preferential hydrolysis of homologs I and III.