RESUMEN
A new aminopeptidase was isolated from the biomass of the flagellate Astasia longa by precipitation with ammonium sulfate, gel filtration, and affinity chromatography on Arginine-Silochrome in 41% yield and with purification degree 490. The enzyme is irreversible inhibited by mercury chloride, EDTA, o-phenanthroline and, partially, bestatin and zinc chloride. It has an optimum pH 8.5 toward the hydrolysis of a synthetic chromogenic substrate Ala-pNA. The enzyme molecular mass is 45 kDa, isoelectric point 5.5, and temperature optimum 45 degrees C. The enzyme most effectively hydrolyzes p-nitroanilides of alanine, arginine, and leucine; it is classified as metalloaminopeptidase.
Asunto(s)
Aminopeptidasas/aislamiento & purificación , Euglena longa/enzimología , Aminopeptidasas/antagonistas & inhibidores , Aminopeptidasas/química , Compuestos de Anilina/química , Concentración de Iones de Hidrógeno , Hidrólisis , Punto Isoeléctrico , Especificidad por SustratoRESUMEN
The paper deals with the design of recombinant vector make-ups, with the expression of Echinococcus hybrid proteins, and with the study of their immunogenic properties. Theoretical rationale is given for the choice of the parasitic gene superexpression system (E. coli cells of SG 13009 strain--recombinant plasmid pQE/EgF). The authors show that the use ofpolymerase chain reaction with oligonucleotide primers homologous to the structures of unique genes coding for Echinococcus antigen is promising for the run of the preparative quantities of fragments of these genes. They consider the basic stages of obtaining hybrid EgF antigen: the isolation of genomic DNA from Echinococcus protoscolexes; the run of preparative quantities of an EgF DNA fragment; the obtaining of vector pQE plasmid DNA; the design of a recombinant make-up; the screening of positive clones; the recombinant plasmid expression of hybrid protein and its purification. The commission tests of EgF antigen in enzyme immunoassay using 93 human serum samples revealed the following: the sensitivity and specificity were 83.8 and 77.4%, respectively. The recombinant protein of EgF was found to exert a significant protective action on the development of E. multilocularis larvocysts in non-inbred albino mice.
Asunto(s)
Anticuerpos Antihelmínticos/sangre , Antígenos Helmínticos/biosíntesis , Equinococosis/diagnóstico , Echinococcus granulosus/inmunología , Echinococcus multilocularis/inmunología , Ensayo de Inmunoadsorción Enzimática/métodos , Animales , Antígenos Helmínticos/genética , Equinococosis/prevención & control , Escherichia coli/metabolismo , Vectores Genéticos , Humanos , Ratones , Plásmidos , Ingeniería de Proteínas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/inmunología , Sensibilidad y Especificidad , Vacunación , Vacunas/administración & dosificación , Vacunas Sintéticas/administración & dosificaciónAsunto(s)
Genes de Helminto , Glicoproteínas/genética , Proteínas del Helminto/genética , Epítopos Inmunodominantes/genética , Trichinella/genética , Animales , Secuencia de Bases , Glicoproteínas/química , Glicoproteínas/inmunología , Proteínas del Helminto/química , Proteínas del Helminto/inmunología , Epítopos Inmunodominantes/química , Ratones , Datos de Secuencia Molecular , Peso Molecular , Reacción en Cadena de la Polimerasa , Ratas , Trichinella/inmunologíaRESUMEN
A fraction of nucleoli is isolated from zooflagellates (Crithidia oncopelti) nuclei, its DNA-dependent RNA polymerase activity is studied at different temperature, ionic strength and Mg2+, Mn2+ and antibiotic concentrations. The effect of some factors and alpha-amantine on RNA polymerase activity of exonucleolar chromatin was studied as a control. A comparison of heat denaturation of nucleoli and chromatin RNA polymerase activities within the temperature range 30--55 degrees C has revealed a higher thermosensitivity of nucleoli RNA polymerase. Substitution of Mg2+ with equivalent amount of Mn2+ results in a considerable decrease of rRNA synthesis in nucleoli. Nucleoli RNA polymerase activity in the presence of Mg2+ is sensitive to the elevation of ionic strength from 0.12 to 1.30 u; chromatin RNA polymerase activity in the presence of Mn2+ is maximal at high ionic strength (1.30 mu). alpha-Amantine and cycloheximide at high concentrations (10 and 200 mkg/ml) practically do not affect RNA polymerase activity of nucleoli. Nucleoli RNA polymerase of zooflagellates (Crithidia oncopelti) is similar to the A-form of the enzyme in higher eukaryotes.