RESUMEN
Rabies continues to poses serious threats to the public health in many countries. The development of novel inexpensive, safe and effective vaccines has become a high priority for rabies control worldwide. We previously generated a novel recombinant rabies vaccine by cloning rabies virus glycoprotein into a chimpanzee adenoviral vector, termed ChAd68-Gp. The present study evaluated the immune responses and protection afforded by this vaccine in beagle dogs. The results demonstrated that intramuscular immunization with both low-dose and high-dose of ChAd68-Gp induced strong immune responses and provided complete protection in beagles even at low-dose. However, when administered orally, high-dose vaccination was protective while low-dose vaccination was ineffective. Further investigation indicated that the low-pH value of gastric juice in the stomach of beagles might decompose the adenovirus. Therefore, suitable formulation for adenovirus-based oral vaccine should be considered and developed. The chimpanzee adenovirus-vectored rabies vaccine ChAd68-Gp warrants extensive test for clinical application.
Asunto(s)
Adenovirus de los Simios/genética , Anticuerpos Neutralizantes/biosíntesis , Anticuerpos Antivirales/biosíntesis , Vacunas Antirrábicas/administración & dosificación , Virus de la Rabia/efectos de los fármacos , Rabia/prevención & control , Proteínas del Envoltorio Viral/administración & dosificación , Adenovirus de los Simios/inmunología , Administración Oral , Animales , Perros , Femenino , Vectores Genéticos/química , Vectores Genéticos/inmunología , Concentración de Iones de Hidrógeno , Sueros Inmunes/administración & dosificación , Inyecciones Intramusculares , Masculino , Ratones , Ratones Endogámicos BALB C , Rabia/mortalidad , Rabia/patología , Rabia/virología , Vacunas Antirrábicas/genética , Vacunas Antirrábicas/inmunología , Virus de la Rabia/genética , Virus de la Rabia/inmunología , Virus de la Rabia/patogenicidad , Análisis de Supervivencia , Vacunación/métodos , Vacunas Sintéticas , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunologíaRESUMEN
Subtilisin DFE is a fibrinolytic enzyme produced by Bacillus amyloliquefaciens DC-4. The promoter and signal peptide-coding sequence of alpha-amylase gene from B. amyloliquefaciens was cloned and fused to the sequence coding for pro-peptide and mature peptide of subtilisin DFE. This hybrid gene was inserted into the Escherichia coli/Bacillus subtilis shuttle plasmid vector, pSUGV4. Recombinant subtilisin DFE gene was successfully expressed in B. subtilis WB600 with a fibrinolytic activity of 200 urokinase units ml(-1).
Asunto(s)
Bacillus subtilis/genética , Proteínas Bacterianas/genética , Regulación Bacteriana de la Expresión Génica/genética , Regiones Promotoras Genéticas/genética , Subtilisina/genética , alfa-Amilasas/genética , Clonación Molecular , Expresión Génica , PlásmidosRESUMEN
White rot fungus Trametes gallica was studied for the production of lignocellulolytic enzymes: cellulase, xylanase, laccase, manganese-dependent peroxidase (MnP), and lignin peroxidase (LiP). The results demonstrated that low-nitrogen (2.2 mM N) and surface stationary cultivation favored production of extracellular MnP. MnP activity reached 118.1 UL(-1) while T. gallica was grown in a low-nitrogen culture containing phenylalanine. However, laccase levels observed in high-nitrogen (22 mM N) agitated cultures were much greater than those seen in low-nitrogen. The N source experiments seemed to reveal that NH4+ plays an important role in inducing MnP and laccase of the fungus. Results showed that T. gallica produces a series of the lignocellulolytic enzymes, and needs high N to produce all the enzymes during solid-state fermentation of wheat straw. This paper also presents a modified zymogram procedure to detect xylanase and laccase of T. gallica in polyacrylamide gel. Xylanase in crude enzyme of T. gallica was displayed by contacting protein gel strips with xylan substrate gels and by staining with iodine. By immersing the protein gel strips in o-tolidine solution, the blue-green zones representing laccase activity were visualized against a colorless background.
Asunto(s)
Basidiomycota/enzimología , Celulasa/metabolismo , Endo-1,4-beta Xilanasas/metabolismo , Lacasa/metabolismo , Peroxidasas/metabolismo , Celulasa/biosíntesis , Medios de Cultivo , Electroforesis en Gel de Poliacrilamida , Endo-1,4-beta Xilanasas/biosíntesis , Inducción Enzimática , Fermentación , Lacasa/biosíntesis , Nitrógeno/metabolismo , Peroxidasas/biosíntesis , Fenilalanina/metabolismo , Polisacáridos/metabolismo , Compuestos de Amonio Cuaternario/metabolismo , Coloración y Etiquetado/métodos , Triticum/metabolismoRESUMEN
Bacillus amyloliquefaciens DC-4, which produces a strongly fibrinolytic enzyme, was isolated from douchi, a traditional Chinese soybean-fermented food. A fibrinolytic enzyme (subtilisin DFE) was purified from the supernatant of B. amyloliquefaciens DC-4 culture broth and displayed thermophilic, hydrophilic and strong fibrinolytic activity. Subtilisin DFE was demonstrated to be homogeneous by SDS-PAGE and isoelectric focusing electrophoresis, and has molecular mass of 28000 Da and a pI of 8.0. The optimal reaction pH value and temperature were 9.0 and 48 degrees C, respectively. Subtilisin DFE not only hydrolyzed fibrin but also several synthetic substrates, particularly Suc-Ala-Ala-Pro-Phe-pNA, and phenylmethylsulfony fluoride can completely inhibit its fibrinolytic activity. These results indicated that subtilisin DFE is a subtilisin-family serine protease, similar to nattokinase from Bacillus natto. The first 24 amino acid residues of the N-terminal sequence of subtilisin DFE were AQSVPYGVSQIKAPALHSQGFTGS, which is identical to that of subtilisin K-54, and different from that of NK and CK. Results from subtilisin DFE gene sequence analysis showed that subtilisin DFE is a novel fibrinolytic enzyme.
