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1.
Vet Microbiol ; 114(3-4): 187-95, 2006 May 31.
Artículo en Inglés | MEDLINE | ID: mdl-16384666

RESUMEN

Rabbit hemorrhagic disease (RHD) is a contagious and highly lethal viral disease of rabbits that spreads rapidly and infects animals by nasal, conjunctival and oral routes. Therefore, this experiment was undertaken to study the immune response generated after intranasal (i.n.) vaccination with the recombinant VP60 capsid protein from rabbit hemorrhagic disease virus (RHDV) expressed at high levels in Pichia pastoris. Groups of BALB/c mice were immunized with three doses of purified VP60 protein (Group 1), VP60 formulated within the cell debris fraction of the transformed yeast (Group 2) and placebo (Group 3) by intranasal route. Mice were also intramuscularly injected with purified VP60 protein (Group 4). A rapid antibody response specific against rabbit hemorrhagic disease virus was observed in all the experimental groups, except in Group 3, as detected by ELISA. The highest titers were found 60 days after the first immunization. Mice from Group 1 showed the highest IgG response (p<0.05) and the most balanced profile of IgG1, IgG2a and IgG2b subclasses. IgA titers specific to the virus were found only in animals from this group, which also developed the highest specific lymphocyte proliferative response. Interferon-gamma (IFN-gamma) and interleukin-12 (IL-12) gene expression was also detected after an ex vivo-specific stimulation of mice from Groups 1 and 4. These data demonstrated the capacity of VP60 protein expressed in P. pastoris to elicit a potent humoral and cell-mediated immune response following an intranasal immunization scheme.


Asunto(s)
Anticuerpos Antivirales/sangre , Citocinas/biosíntesis , Virus de la Enfermedad Hemorrágica del Conejo/inmunología , Proteínas Estructurales Virales/inmunología , Vacunas Virales/inmunología , Administración Intranasal , Animales , Anticuerpos Antivirales/biosíntesis , Infecciones por Caliciviridae/prevención & control , Infecciones por Caliciviridae/veterinaria , Citocinas/genética , Relación Dosis-Respuesta Inmunológica , Ensayo de Inmunoadsorción Enzimática/métodos , Ensayo de Inmunoadsorción Enzimática/veterinaria , Femenino , Regulación Viral de la Expresión Génica , Inmunidad Celular , Inmunoglobulina G/biosíntesis , Inmunoglobulina G/sangre , Ratones , Ratones Endogámicos BALB C , Pichia/genética , Pichia/inmunología , Pichia/virología , Conejos , Distribución Aleatoria , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/inmunología , Factores de Tiempo , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/inmunología , Proteínas Estructurales Virales/administración & dosificación , Vacunas Virales/administración & dosificación
2.
J Biotechnol ; 117(3): 215-24, 2005 May 25.
Artículo en Inglés | MEDLINE | ID: mdl-15862351

RESUMEN

The VP60 capsid protein from rabbit hemorrhagic disease virus (RHDV) (Spanish isolate AST/89) was cloned and expressed in Pichia pastoris. The transformed yeast was grown at high cell density and an expression level of about 1.5 g VP60L(-1) culture was obtained. The protein was detected associated with the cell debris fraction of the recombinant yeast after mechanical disruption. It was purified by a simple method and was obtained N-glycosylated with purity of approximately 70% as deduced from densitometry scan analysis. The recombinant product was antigenically similar to the native capsid protein as determined with polyclonal antibodies obtained from rabbits vaccinated with VP60 protein purified from native virus. The immunogenicity of VP60 protein purified from P. pastoris was demonstrated by ELISA in a vaccination experiment conducted with two groups of rabbits subcutaneously immunized. Animals vaccinated with VP60 in Freund's incomplete adjuvant developed a significant (p<0.01) virus-specific antibody response while the group injected with placebo remained seronegative. Preliminary results showed that the antigen administered within the cell debris fraction of the transformed yeast protected rabbits immunized by the oral route against an intramuscular challenge with 100 LD50 (16,000 hemagglutination units) of homologous virus.


Asunto(s)
Pichia/genética , Proteínas Estructurales Virales/inmunología , Proteínas Estructurales Virales/metabolismo , Vacunas Virales/inmunología , Animales , Antígenos Virales/genética , Antígenos Virales/inmunología , Clonación Molecular , Ensayo de Inmunoadsorción Enzimática , Adyuvante de Freund/inmunología , Regulación Viral de la Expresión Génica , Virus de la Enfermedad Hemorrágica del Conejo , Inyecciones Subcutáneas , Masculino , Pichia/crecimiento & desarrollo , Pichia/metabolismo , Conejos , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Estructurales Virales/genética , Proteínas Estructurales Virales/aislamiento & purificación , Vacunas Virales/administración & dosificación , Vacunas Virales/genética
3.
Arch Biochem Biophys ; 432(2): 205-11, 2004 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-15542059

RESUMEN

Bm95 is an antigen isolated from Boophilus microplus strains with low susceptibility to antibodies developed in cattle vaccinated with the recombinant Bm86 antigen (Gavac, HeberBiotec S.A., Cuba). It is a Bm86-like surface protein, which by similarity contains seven EGF-like domains and a lipid-binding GPI-anchor site at the C-terminal region. The primary structure of the recombinant (rBm95) protein expressed in Pichia pastoris was completely verified by LC/MS. The four potential glycosylation sites (Asn 122, 163, 329, and 363) are glycosylated partially with short N-glycans, from Man(5)GlcNAc(2) to Man(9)GlcNAc(2) of which, Man(8-9)GlcNAc(2) were the most abundant. O-Glycopeptides are distributed mostly towards the protein N-terminus. While the first N-glycosylated site (Asn(122)) is located between EGF-like domains 2 and 3, where the O-glycopeptides were found, two other N-glycosylated sites (Asn(329) and Asn(363)) are located between EGF-like domains 5 and 6, a region devoid of O-glycosylated Ser or Thr.


Asunto(s)
Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Polisacáridos/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Garrapatas/metabolismo , Vacunas/química , Vacunas/genética , Secuencia de Aminoácidos , Animales , Sitios de Unión , Clonación Molecular , Glicosilación , Datos de Secuencia Molecular , Pichia/genética , Pichia/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Relación Estructura-Actividad , Garrapatas/genética
4.
Exp Appl Acarol ; 34(3-4): 375-82, 2004.
Artículo en Inglés | MEDLINE | ID: mdl-15651533

RESUMEN

Boophilus microplus has developed resistance against a range of chemical acaricides which has stimulated the development of alternative methods such as vaccination against ticks. In Cuba, the Bm86-based recombinant vaccine Gavac has been successfully used in a number of controlled laboratory and field trials in cattle against B. microplus. In this paper, we have evaluated Gavac in a large scale field trial wherein 588,573 dairy cattle were vaccinated with the aim to reduce the number of acaricidal treatments. It was found that the number of acaricidal treatments could be reduced by 87% over a period of 8 years (1995--2003). Prior to the introduction of the vaccine, 54 clinical cases of babesiosis and six fatal cases were reported per 1000 animals. Six years later, the incidence of babesiosis was reduced to 1.9 cases per 1000 cattle and mortality reduced to 0.18 per 1000. The national consumption of acaricides in Cuba could be reduced by 82% after the implementation of the integrated anti-B. microplus control program.


Asunto(s)
Control de Insectos/métodos , Ixodidae/fisiología , Glicoproteínas de Membrana/inmunología , Proteínas Recombinantes/inmunología , Infestaciones por Garrapatas/veterinaria , Vacunas/inmunología , Animales , Babesiosis/prevención & control , Bovinos , Enfermedades de los Bovinos/parasitología , Enfermedades de los Bovinos/prevención & control , Cuba , Insecticidas , Ixodidae/inmunología , Infestaciones por Garrapatas/prevención & control , Factores de Tiempo
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