RESUMO
To evaluate the persistence of the immune response in a population of healthy volunteers that had been vaccinated with a new Hansenula polymorpha recombinant hepatitis B vaccine in a previous clinical study 4 years before, we measured the titre of anti-HBs. All, but one of the evaluated volunteers remained protected. None of them had experienced any adverse event related to the vaccine from the moment of immunization, to the present. The vaccine proved to be immunogenic and to confer long-term protection in this group.
Assuntos
Anticorpos Anti-Hepatite A/biossíntese , Vacinas contra Hepatite B/imunologia , Pichia/imunologia , Adolescente , Adulto , Feminino , Anticorpos Anti-Hepatite A/análise , Vacinas contra Hepatite B/efeitos adversos , Humanos , Esquemas de Imunização , Testes de Função Hepática , Masculino , Fatores de Tempo , Vacinas Sintéticas/imunologiaRESUMO
In order to isolate very strong promoters from bacteria and bacteriophage a plasmid named pProm was constructed. It possesses an origin (ORI) for replication in Gram-negative bacteria, an ORI for replication in Gram-positive bacteria, a promoterless ampicillin resistance gene with a multiple cloning site (MCS) in the position formerly occupied by the ampicillin promoter, a tetracycline resistance gene for selection in Gram-negative bacteria and a chloramphenicol resistance gene for selection in Gram-positive bacteria. Insertion in the MCS of DNA fragments of Staphylococcus aureus bacteriophages resulted in isolation of several clones very resistant to ampicillin. The DNA fragments inserted in these recombinant plasmids were sequenced and all of them contained putative promoter motifs. Direct measurement of the penicillinase activity indicated that one of the isolated promoters could be included within a group of the stronger known prokaryotic promoters. According to these results pProm is a powerful tool to perform studies on promoter strength and for industrial applications.
Assuntos
Escherichia coli/genética , Vetores Genéticos , Plasmídeos , Regiões Promotoras Genéticas , Transformação Bacteriana , Sequência de Bases , Clonagem Molecular , Replicação do DNA , DNA Bacteriano/química , DNA Bacteriano/genética , Bactérias Gram-Negativas/genética , Bactérias Gram-Positivas/genética , Dados de Sequência Molecular , Penicilinase/genética , Penicilinase/metabolismo , Origem de Replicação , Mapeamento por Restrição , Fagos de Staphylococcus/genética , Staphylococcus aureus/virologiaRESUMO
A Trypanosoma cruzi antigen gene was closed into a fusion vector based on the IgG binding domain of Staphylococcus aureus protein A. This vector transformed into Escherichia coli or Staphylococcus aureus and produced about 12 mg fusion protein/l culture. In E. coli, the product remained intracellular while in S. aureus it was excreted into the growth medium. The hybrid protein was purified by IgG Sepharose affinity chromatography. The presence of a cleavage site for enterokinase between protein A and the T. cruzi antigen in the fusion protein allowed the efficient release of the unfused antigen by enzymatic treatment. Further affinity chromatography through IgG Sepharose resulted in the production of the T. cruzi antigen free of protein A.
RESUMO
Two heat-stable inhibitors (a and b) of phosphoprotein phosphatases I and II from Mucor rouxii were isolated from mycelium of the fungus. They were partially purified from extracts by heating, DEAE-cellulose chromatography, and Sephadex G-75 gel filtration. The molecular weights of inhibitors a and b, estimated by gel filtration, are 5,000 and 20,000 respectively. Inhibitor a acts similarly on both enzymes while inhibitor b is relatively more active on enzyme II. Storage of inhibitor b at -20 degrees C for several weeks resulted in a partial conversion to a lower-molecular-weight form with properties similar to those of inhibitor a.
Assuntos
Mucor/análise , Fosfoproteínas Fosfatases/antagonistas & inibidores , Fenômenos Químicos , Química , Cromatografia em Gel , Temperatura Alta , Peso Molecular , Mucor/enzimologiaRESUMO
Soluble preparations from mycelium of the dimorphic fungus Mucor rouxii contained detectable amounts of phosphoprotein phosphatase activity. This cytosolic phosphatase activity exhibited a molecular weight below 80,000 and could be resolved into two different forms (enzymes I and II) by chromatography on DEAE-cellulose followed by gel filtration on Sephacryl S-300. Enzyme I (Mr 64,000) was mainly a histone phosphatase activity, absolutely dependent on divalent cations, with a K0.5 for MnCl2 of 2mM. Enzyme II (Mr 40,000) was active with histone and phosphorylase. Its activity was independent or slightly inhibited by Mn2+. This enzyme was strongly inhibited by 50 mM NaF or 1 mM ATP. When partially purified enzymes I and II were separately treated with ethanol, the catalytic properties of enzyme II were apparently not affected while those of enzyme I were drastically changed. The activity with histone, which was originally dependent on Mn2+, became independent or slightly inhibited by the cation. The treatment was accompanied by a notable increase in phosphorylase phosphatase activity which was strongly inhibited by Mn2+. Treated enzyme I eluted from DEAE-cellulose and Sephacryl S-300 columns at a position similar to that of enzyme II.