RESUMEN
Bluetongue virus (BTV), a complex double-stranded segmented RNA virus, has been found to initiate cellular autophagy for its own benefit. Here, with a view to understanding the underlying mechanisms, we first systematically dissected the exact signaling network in BTV-induced autophagy. We found that the activity of mTOR, a crucial pivot, was inhibited by BTV1 infection, subsequently leading to downstream p70S6K suppression and autophagy initiation. We then explored the upstream regulators of mTOR and analyzed their activities via a series of assays. We found BTV1-induced autophagy to be independent of the ERK1/2 signaling pathway. However, the BTV1-induced inhibition of PI3K/Akt was found to be partially responsible for mTOR inactivation and subsequent autophagy initiation. Furthermore, we found unexpectedly that AMPK seemed to play a more important role in BTV1-induced autophagy. Elevated [Ca(2+)]cyto-mediated activation of CaMKKß exactly managed the activation of AMPK, which then positively regulated autophagy through suppressing mTOR. We must emphasize that TSC2 is a fatal mediator between upstream Akt or AMPK and downstream mTOR through its phosphorylation. Taken together, our data suggested that the BTV1-induced inhibition of the Akt-TSC2-mTOR pathway and the upregulation of the AMPK-TSC2-mTOR pathway both contributed to autophagy initiation and further favored virus replication.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Autofagia , Virus de la Lengua Azul/fisiología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Calcio/metabolismo , Células Cultivadas , Cricetinae , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismo , Proteína 2 del Complejo de la Esclerosis Tuberosa , Proteínas Supresoras de Tumor/metabolismo , Replicación ViralRESUMEN
Bluetongue virus (BTV) is an important pathogen of wild and domestic ruminants. We have previously reported that BTV1 infection induced autophagy for its own benefit, but how this occurs remains unclear. Here, the classical autophagy features including autophagsomes formation, GFP-LC3 dots and LC3-II conversation were shown in BTV1-infected cells, we also found the endoplasmic reticulum (ER) stress was triggered by BTV1 infection, which was demonstrated by the increased transcription level of the ER stress marker GRP78 and the expanded morphology of ER. During ER stress, PERK and eIF2α phosphorylation increased along with BTV1 infection, consistent with the elevated LC3 level, indicating that the PERK pathway of the unfolded protein response (UPR) was activated. In addition, both the blockage of PERK by GSK2656157 or knockdown of eIF2α by siRNA reduced the level of LC3, which suggested that the PERK-eIF2α pathway contributed to autophagy induced by BTV1. Furthermore, inactivation of PERK or silencing of eIF2α both significantly reduced the expression of VP2 protein and the viral yields in the supernatants. In sum, these data suggest that ER stress mediates autophagy via the PERK-eIF2α pathway and contributes to BTV1 replication, thus offering new insight into the molecular mechanisms of the BTV-host interaction.
Asunto(s)
Virus de la Lengua Azul/patogenicidad , Factor 2 Eucariótico de Iniciación/metabolismo , eIF-2 Quinasa/metabolismo , Adenina/análogos & derivados , Adenina/farmacología , Animales , Autofagia , Virus de la Lengua Azul/genética , Virus de la Lengua Azul/fisiología , Proteínas de la Cápside/genética , Proteínas de la Cápside/fisiología , Células Cultivadas , Chaperón BiP del Retículo Endoplásmico , Estrés del Retículo Endoplásmico , Factor 2 Eucariótico de Iniciación/antagonistas & inhibidores , Factor 2 Eucariótico de Iniciación/genética , Técnicas de Silenciamiento del Gen , Proteínas de Choque Térmico/metabolismo , Indoles/farmacología , Modelos Biológicos , Transducción de Señal , Respuesta de Proteína Desplegada , Replicación Viral , eIF-2 Quinasa/antagonistas & inhibidoresRESUMEN
The NS2 protein of Bluetongue virus (BTV) is an important non-structural protein and plays important roles in viral replication and assembly. In this study, one monoclonal antibody (mAb), 4D4, was raised against BTV8 NS2. Phage display technology was used and identified the consensus binding motif SNYD recognized by mAb 4D4. To define the minimal region required for antibody binding, a panel of synthetic peptides encompassing SNYD derived from the BTV8 NS2 was then used to more specifically define the 4D4 epitope as (149)RSNYDV(154). Furthermore, amino acid sequence alignments of different BTV serotypes and other orbiviruses suggested that this epitope is highly conserved among the BTV serotypes. The mAb reagent generated in this study may be applied to the development of BTV diagnosis and surveillance programs and the epitope defined here can lead to important insights into how BTV might interact with the sheep's immune system.
Asunto(s)
Virus de la Lengua Azul/clasificación , Virus de la Lengua Azul/inmunología , Epítopos de Linfocito B/inmunología , Biblioteca de Péptidos , Proteínas no Estructurales Virales/inmunología , Secuencia de Aminoácidos , Animales , Línea Celular , Cricetinae , Ensayo de Inmunoadsorción Enzimática , Alineación de SecuenciaRESUMEN
The VP2 protein of bluetongue virus (BTV) is an important structural protein and is the principal antigen responsible for BTV serotype specificity. In this study, we mapped the reactivity of two BTV16-specific monoclonal antibodies (MAbs) and identified two novel serotype-specific linear B cell epitopes on the BTV16 VP2 protein. By screening a series of peptides derived from the BTV16 VP2 protein and expressed as mannose-binding protein fusions, we determined that the linear epitopes recognized by the VP2-specific MAbs 3 G10 and 2B4 were located within the peptides 34EWSGHDVTEIPNRRMF49 and 540KNEDPYVKRTVKPIRA555, respectively. To define the minimal region required for antibody binding within these peptide regions, a series of progressively shorter peptides were synthesized and evaluated for 3 G10 and 2B4 binding. This work defined the motifs 34EWSGHDVTEIPNRRMF49 and 543DPYVKRTVK555 as the minimal linear peptides required for 3 G10 and 2B4 binding, respectively. Alignment of amino acid sequences from a number of BTV16 strains isolated from different regions indicated that these two epitopes are highly conserved among BTV16 strains. Furthermore, these two epitopes are not conserved among other BTV serotypes or prototype members of the genus Orbivirus in the family Reoviridae, as shown by sequence alignments. The MAb reagents and linear epitopes defined here provide the basis for the development of epitope-based serotype-specific differential diagnostic tools and may be useful in the design of epitope-based vaccines.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Virus de la Lengua Azul/inmunología , Proteínas de la Cápside/inmunología , Mapeo Epitopo , Epítopos de Linfocito B/inmunología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Virus de la Lengua Azul/clasificación , Unión Proteica , Alineación de Secuencia , Homología de Secuencia de Aminoácido , SerotipificaciónRESUMEN
Bluetongue virus (BTV) VP5 protein is an important antigenic protein which is centrally involved in serotype determination and the virus entry process. Very little is known about the B-cell epitopes on the BTV VP5 protein recognized by humoral immune responses. In this study, we generated five BTV16 VP5 protein-specific monoclonal antibodies (MAbs), named 3B11, 2B10, 1H7, 4A6 and 3G9, and defined the linear epitopes recognized by MAbs using a series of peptides expressed as maltose-binding protein (MBP)-fusion polypeptides. Three novel linear B-cell epitopes were identified: 3B11 and 3G9 recognized the motif ITANTREIQHIKEE; 2B10 recognized the motif LSGID; and 4A6 recognized the motif STMVKEYRQKIDALKA. Exact sequences corresponding to the three motifs identified were found in the BTV16 VP5 protein ((310)ITANTREIQHIKEE(323), (265)LSGID(269) and (188)STMVKEYRQKIDALKA(203)). These motifs represent the minimal linear peptide sequence required for MAb reactivity, as binding of each MAb was abolished when additional amino acids were removed from the amino and carboxy termini of the peptide. Amino acid sequence alignment indicated that three epitopes were totally conserved among different BTV16 strains. The MAbs generated along with identified epitopes will be useful for examining VP5 protein function and the development of epitope-based marker vaccines against BTV.
Asunto(s)
Proteínas de la Cápside/inmunología , Epítopos de Linfocito B/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Anticuerpos Antivirales/inmunología , Especificidad de Anticuerpos , Línea Celular Tumoral , Mapeo Epitopo , Epítopos de Linfocito B/análisis , Femenino , Cobayas , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Mieloma Múltiple/virología , Fragmentos de Péptidos/inmunología , Proteínas Recombinantes/inmunología , Homología de Secuencia de AminoácidoRESUMEN
VP7 is a major group-specific protein of the bluetongue virus (BTV), and is therefore a candidate for use as a diagnostic reagent. In this study, BALB/c mice were immunized with BTV16, and the lymphocyte hybridoma technique and indirect ELISA screening method were employed to obtain two strains of hybridoma cells secreting specific monoclonal antibodies (MAbs) to BTV16. Eukaryotic recombinant plasmids coding for 10 segments of BTV16 separately were transfected into BHK-21 cells, respectively, followed by immunofluorescence, showing that two MAbs only reacted with BTV-VP7. Western blot analysis showed the same result. Indirect immunofluorescence results indicated that two of the MAbs present different response spectrums with BTV1~24 serotypes. These results indicate that these MAbs may be good candidates for a specific diagnostic method and functional exploration of the VP7 protein.
Asunto(s)
Anticuerpos Monoclonales de Origen Murino/química , Virus de la Lengua Azul/inmunología , Proteínas del Núcleo Viral/inmunología , Animales , Anticuerpos Monoclonales de Origen Murino/inmunología , Anticuerpos Antivirales/química , Anticuerpos Antivirales/inmunología , Especificidad de Anticuerpos , Línea Celular , Cricetinae , Femenino , Hibridomas , Ratones , Ratones Endogámicos BALB C , Carga ViralRESUMEN
West Nile virus (WNV) non-structural protein 1 (NS1) elicits protective immune responses during infection of animals. WNV NS1-specific antibody responses can provide the basis for serological diagnostic reagents, so the antigenic sites in NS1 that are targeted by host immune responses need to be identified and the conservation of these sites among the Japanese encephalitis virus (JEV) serocomplex members also needs to be defined. The present study describes the mapping of linear B-cell epitopes in WNV NS1. We screened eight NS1-specific mAbs and antisera (polyclonal antibodies; pAbs) from mice immunized with recombinant NS1 for reactivity against 35 partially overlapping peptides covering the entire WNV NS1. The screen using mAbs identified four WNV-specific (including Kunjin virus) epitopes, located at aa 21-36, 101-116, 191-206 and 261-276 in WNV NS1. However, using pAbs, only three WNV-specific epitopes were identified, located at positions 101-116, 191-206 and 231-246. Two of these epitopes (aa 21-36 and 261-276) had different reactivity with mAbs and pAbs. The knowledge and reagents generated in this study have potential applications in differential diagnostics and epitope-based marker vaccine development for WNV and viruses of the JEV serocomplex.
Asunto(s)
Virus de la Encefalitis Japonesa (Especie)/inmunología , Encefalitis Japonesa/virología , Mapeo Epitopo , Epítopos de Linfocito B/inmunología , Proteínas no Estructurales Virales/inmunología , Fiebre del Nilo Occidental/virología , Virus del Nilo Occidental/inmunología , Secuencia de Aminoácidos , Animales , Anticuerpos Antivirales/inmunología , Línea Celular , Virus de la Encefalitis Japonesa (Especie)/química , Virus de la Encefalitis Japonesa (Especie)/genética , Encefalitis Japonesa/inmunología , Epítopos de Linfocito B/química , Epítopos de Linfocito B/genética , Femenino , Humanos , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Alineación de Secuencia , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genética , Fiebre del Nilo Occidental/inmunología , Virus del Nilo Occidental/química , Virus del Nilo Occidental/genéticaRESUMEN
BACKGROUND: The West Nile virus (WNV) nonstructural protein 1 (NS1) is an important antigenic protein that elicits protective antibody responses in animals and can be used for the serological diagnosis of WNV infection. Although previous work has demonstrated the vital role of WNV NS1-specific antibody responses, the specific epitopes in the NS1 have not been identified. RESULTS: The present study describes the identification of two linear B-cell epitopes in WNV NS1 through screening a phage-displayed random 12-mer peptide library with two monoclonal antibodies (mAbs) 3C7 and 4D1 that directed against the NS1. The mAbs 3C7 and 4D1 recognized phages displaying peptides with the consensus motifs LTATTEK and VVDGPETKEC, respectively. Exact sequences of both motifs were found in the NS1 ((895)LTATTEK(901) and (925)VVDGPETKEC(934)). Further identification of the displayed B cell epitopes were conducted using a set of truncated peptides expressed as MBP fusion proteins. The data indicated that (896)TATTEK(901) and (925)VVDGPETKEC(934) are minimal determinants of the linear B cell epitopes recognized by the mAbs 3C7 and 4D1, respectively. Antibodies present in the serum of WNV-positive horses recognized the minimal linear epitopes in Western blot analysis, indicating that the two peptides are antigenic in horses during infection. Furthermore, we found that the epitope recognized by 3C7 is conserved only among WNV strains, whereas the epitope recognized by 4D1 is a common motif shared among WNV and other members of Japanese encephalitis virus (JEV) serocomplex. CONCLUSIONS: We identified TATTEK and VVDGPETKEC as NS1-specific linear B-cell epitopes recognized by the mAbs 3C7 and 4D1, respectively. The knowledge and reagents generated in this study may have potential applications in differential diagnosis and the development of epitope-based marker vaccines against WNV and other viruses of JEV serocomplex.
Asunto(s)
Epítopos de Linfocito B/inmunología , Biblioteca de Péptidos , Proteínas no Estructurales Virales/inmunología , Virus del Nilo Occidental/inmunología , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Anticuerpos Antivirales/sangre , Western Blotting , Epítopos de Linfocito B/genética , Enfermedades de los Caballos/inmunología , Caballos , Tamizaje Masivo , Ratones , Ratones Endogámicos BALB C , Proteínas no Estructurales Virales/genética , Fiebre del Nilo Occidental/inmunología , Fiebre del Nilo Occidental/veterinaria , Virus del Nilo Occidental/genéticaRESUMEN
BACKGROUND: The West Nile virus (WNV) capsid (C) protein is one of the three viral structural proteins, encapsidates the viral RNA to form the nucleocapsid, and is necessary for nuclear and nucleolar localization. The antigenic sites on C protein that are targeted by humoral immune responses have not been studied thoroughly, and well-defined B-cell epitopes on the WNV C protein have not been reported. RESULTS: In this study, we generated a WNV C protein-specific monoclonal antibody (mAb) and defined the linear epitope recognized by the mAb by screening a 12-mer peptide library using phage-display technology. The mAb, designated as 6D3, recognized the phages displaying a consensus motif consisting of the amino acid sequence KKPGGPG, which is identical to an amino acid sequence present in WNV C protein. Further fine mapping was conducted using truncated peptides expressed as MBP-fusion proteins. We found that the KKPGGPG motif is the minimal determinant of the linear epitope recognized by the mAb 6D3. Western blot (WB) analysis demonstrated that the KKPGGPG epitope could be recognized by antibodies contained in WNV- and Japanese encephalitis virus (JEV)-positive equine serum, but was not recognized by Dengue virus 1-4 (DENV1-4)-positive mice serum. Furthermore, we found that the epitope recognized by 6D3 is highly conserved among the JEV serocomplex of the Family Flaviviridae. CONCLUSION: The KKPGGPG epitope is a JEV serocomplex-specific linear B-cell epitope recognized by the 6D3 mAb generated in this study. The 6D3 mAb may serve as a novel reagent in development of diagnostic tests for JEV serocomplex infection. Further, the identification of the B-cell epitope that is highly conserved among the JEV serocomplex may support the rationale design of vaccines against viruses of the JEV serocomplex.
Asunto(s)
Anticuerpos Monoclonales/análisis , Proteínas de la Cápside/química , Secuencia Conservada , Virus de la Encefalitis Japonesa (Especie)/química , Epítopos de Linfocito B/química , Biblioteca de Péptidos , Virus del Nilo Occidental/química , Secuencia de Aminoácidos , Proteínas de la Cápside/genética , Proteínas de la Cápside/inmunología , Virus de la Encefalitis Japonesa (Especie)/genética , Virus de la Encefalitis Japonesa (Especie)/inmunología , Encefalitis Japonesa/inmunología , Encefalitis Japonesa/virología , Mapeo Epitopo , Epítopos de Linfocito B/genética , Epítopos de Linfocito B/inmunología , Humanos , Datos de Secuencia Molecular , Alineación de Secuencia , Fiebre del Nilo Occidental/inmunología , Fiebre del Nilo Occidental/virología , Virus del Nilo Occidental/genética , Virus del Nilo Occidental/inmunologíaRESUMEN
With an aim at detecting the ultra-low concentration of avian influenza virus (AIV), a highly sensitive hybrid assay based on immunology and polymerase chain reaction was developed. The TopYield microtiter plates were coated with ten-fold serial dilutions of H5N1 subtype AIV ranging from 10 EID(50 )ml(-1)~10(-4) EID(50) ml(-1),which was recognized by mouse anti-AIV H5 monoclonal antibody (MAb) that was directly linked with reporter DNA using a heterobifunctional cross-linker. After extensive washing, the reporter DNA including a BamH I-restriction site was released by a specific enzymatic restriction, then transferred to PCR tubes, amplified, and used as the signal for detection of AIV. Under the optimized condition, MAb-based immuno-PCR (IPCR) method could measure 100 µl of AIV H5N1 with 10(-4 )EID(50) ml(-1).To evaluate the sensitivity of IPCR, the same concentration and volume of AIV H5N1 were detected by conventional RT-PCR and sandwich ELISA. The results showed that IPCR had an approximately 1,000-fold improvement over the conventional ELISA, and a 100-fold enhancement compared with RT-PCR in detection sensitivity. To further evaluate the specificity of IPCR for AIV H5 subtype, the tracheal swab specimens, taken from chickens which were infected with H9N2, and the allantoic fluid from eggs inoculated by AIV H3N2, H7N1, H9N2, were detected by IPCR. To mimic clinical samples, pharyngeal-tracheal swab specimens were collected from healthy chickens and spiked with H5N1, H5N2, H5N3 for analysis by immuno-PCR. The results demonstrated that IPCR was a highly sensitive and specific assay for AIV H5, and could be applied to clinical detection for low amount of AIV H5 subtype. This MAb-based immuno-PCR method provided a platform capable of mass screening of clinical samples for AIV H5 subtype and could serve as a model for other immuno-PCR assays.
Asunto(s)
Aves/virología , Inmunoensayo/métodos , Subtipo H5N1 del Virus de la Influenza A/genética , Subtipo H5N1 del Virus de la Influenza A/aislamiento & purificación , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Animales , Anticuerpos Antivirales/inmunología , Ensayo de Inmunoadsorción Enzimática , Subtipo H5N1 del Virus de la Influenza A/inmunología , Gripe Aviar/virología , Límite de Detección , Sensibilidad y EspecificidadRESUMEN
AIM: The goal of this project was to develop a rat model for neural stem cell (NSC) transplantation studies in which NSCs were modified with brain-derived neurotrophic factor (BDNF) genes that may permit extensive and reliable analysis of the transplants. METHODS: NSCs were cultured and purified by limiting dilution assay in vitro and infected with recombinant retrovirus pLXSN-BDNF (BDNF-NSCs) and retrovirus pLXSN (p-NSCs). The expression of BDNF genes in transgenic and control NSC groups was measured by FQ-PCR and ELISA assays. NSCs were then transplanted into the subretinal space of normal rat retinas in four groups, which included NSCs alone, BDNF-NSCs, phosphate buffered saline (PBS) control, and normal control. Survival, migration, and differentiation of donor cells in host retinas were observed with optical coherence tomography (OCT), Heidelberg retina angiograph (HRA), and immunohistochemistry, respectively. RESULTS: The results obtained by FQ-PCR demonstrated that the copy numbers of BDNF gene templates from BDNF-NSCs were the highest among the four groups (P<0.05). Consistent with the results of FQ-PCR, BDNF protein level from the supernatant of the BDNF-NSCs group was much higher than that of the other two groups (P<0.05) as suggested by the ELISA assays. HRA and OCT showed that graft cells could successfully survive. Immunohistochemical analysis revealed that transplanted BDNF-NSCs could migrate in the host retinas and differentiate into glial cells and neurons three months after transplantation. CONCLUSION: BDNF promotes NSCs to migrate and differentiate into neural cells in the normal host retinas.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/genética , Retina/metabolismo , Trasplante de Células Madre/métodos , Animales , Diferenciación Celular , Movimiento Celular , Células Cultivadas , Ensayo de Inmunoadsorción Enzimática , Femenino , Terapia Genética/métodos , Vectores Genéticos , Inmunohistoquímica , Modelos Animales , Neuronas/citología , Reacción en Cadena de la Polimerasa/métodos , Ratas , Ratas Sprague-Dawley , Retroviridae/genética , TransgenesRESUMEN
BACKGROUND: Neural stem cells (NSCs) transplantation and gene therapy have been widely investigated for treating the cerebullar and myelonic injuries, however, studies on the ophthalmology are rare. The aim of this study was to investigate the migration and differentiation of brain-derived neurotrophic factor (BDNF) gene transgenic NSCs transplanted into the normal rat retinas. METHODS: NSCs were cultured and purified in vitro and infected with recombinant retrovirus pLXSN-BDNF and pLXSN respectively, to obtain the BDNF overexpressed NSCs (BDNF-NSCs) and control cells (p-NSCs). The expression of BDNF genes in two transgenic NSCs and untreated NSCs were measured by fluorescent quantitative polymerase chain reaction (FQ-PCR) and enzyme-linked immunosorbent assay (ELISA). BDNF-NSCs and NSCs were infected with adeno-associated viruses-enhanced green fluorescent protein (AAV-EGFP) to track them in vivo and served as donor cells for transplantation into the subretinal space of normal rat retinas, phosphated buffer solution (PBS) served as pseudo transplantation for a negative control. Survival, migration, and differentiation of donor cells in host retinas were observed and analyzed with Heidelberg retina angiograph (HRA) and immunohistochemistry, respectively. RESULTS: NSCs were purified successfully by limiting dilution assay. The expression of BDNF gene in BDNF-NSCs was the highest among three groups both at mRNA level tested by FQ-PCR (P < 0.05) and at protein level measured by ELISA (P < 0.05), which showed that BDNF was overexpressed in BDNF-NSCs. The results of HRA demonstrated that graft cells could survive well and migrate into the host retinas, while the immunohistochemical analysis revealed that transplanted BDNF-NSCs differentiated into neuron more efficiently compared with the control NSCs 2 months after transplantation. CONCLUSIONS: The seed cells of NSCs highly secreting BDNF were established. BDNF can promote NSCs to migrate and differentiate into neural cells in the normal host retinas.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/metabolismo , Neuronas/citología , Retina/citología , Retina/metabolismo , Animales , Factor Neurotrófico Derivado del Encéfalo/genética , Diferenciación Celular/fisiología , Movimiento Celular/fisiología , Células Cultivadas , Embrión de Mamíferos/citología , Ensayo de Inmunoadsorción Enzimática , Inmunohistoquímica , Ratas , Trasplante de Células MadreRESUMEN
OBJECTIVE: To express the fusion protein of glutathione S-transferase (GST) and human Id-2 in E. coli and prepare the polyclonal antibodies against Id-2. METHODS: The coding sequence of Id-2 gene was amplified by RT-PCR from the total RNA of breast cancer tissue. The recombinant plasmid was identified by PCR, restriction endonuclease digestion analysis and sequencing. The fusion protein GST-Id-2 expressed in E. coli following IPTG induction was purified by glutathione-agarose affinity chromatography and used to immunize rabbits to prepare the polyclonal antibodies against GST-Id-2. RESULTS: PCR, restriction endonuclease digestion and sequence analyses showed that the Id-2 gene had been correctly inserted into pGEX-6P-1 vector, and the GST-Id-2 fusion protein expressed had a relative molecular mass of approximately 40,000 as shown by SDS-PAGE. The polyclonal antibodies obtained from the rabbit sera were found to specifically react with purified Id-2 by Western blotting, ELISA and agar gel immunodiffusion (AGP). CONCLUSION: The prepared polyclonal antibodies against Id-2 allow effective Id-2 detection and facilitate further investigation of the structure and antigen epitope of Id-2.
Asunto(s)
Neoplasias de la Mama/genética , Proteína 2 Inhibidora de la Diferenciación/biosíntesis , Proteínas Recombinantes/biosíntesis , Animales , Anticuerpos Monoclonales/biosíntesis , Anticuerpos Monoclonales/inmunología , Escherichia coli/genética , Escherichia coli/metabolismo , Femenino , Humanos , Sueros Inmunes/biosíntesis , Proteína 2 Inhibidora de la Diferenciación/genética , Proteína 2 Inhibidora de la Diferenciación/inmunología , Conejos , Proteínas Recombinantes/genéticaRESUMEN
AIM: To develop a quantitative ELISA by measuring interferon (IFN-gamma) of equine lymphocytes. METHODS: Sandwich ELISA for equine IFN-gamma was developed using mAb A5 as a capture antibody and biotinylated mAb SB10 as a detection antibody. RESULTS: The detection limit of the sandwich ELISA for equine IFN-gamma was 1 microg/L and did not show cross-reactivity with recombinant equine IL-18. Equine IFN-gamma was detected by ELISA in culture medium of the peripheral blood mononuclear cells (PBMCs) stimulated with ConA or PMA/Ionomycin. CONCLUSION: This method can be used to help understand the role of this cytokine in various equine diseases and develop specific cell-mediated immunity assay.
Asunto(s)
Anticuerpos Monoclonales , Citocinas/aislamiento & purificación , Ensayo de Inmunoadsorción Enzimática/métodos , Interferón gamma/aislamiento & purificación , Interleucina-18/aislamiento & purificación , Límite de Detección , Animales , Anticuerpos Monoclonales/aislamiento & purificación , Bioensayo , Citocinas/inmunología , Caballos , Interferón gamma/análisis , Interferón gamma/inmunología , Interleucina-18/inmunología , Ionomicina/química , Activación de Linfocitos , Pruebas de Neutralización , Proteínas Recombinantes , Sefarosa/análogos & derivados , Sefarosa/química , Linfocitos T ReguladoresRESUMEN
Japanese encephalitis virus (JEV), a mosquito-borne flavivirus, causes an acute infection of the central nervous system resulting in encephalitis of humans and many kinds of animals. NS5, the largest and most conserved flavivirus protein, is homologous to methyltransferase and RNA-dependent RNA polymerase. RNA interference is an effective anti-viral strategy to inhibit viral replication in vitro. In this study, four short hairpin RNA (shRNA) expression vectors (pS4.1-NS5-201, pS4.1-NS5-455, pS4.1-NS5-699, and pS4.1-NS5-804) targeting the NS5 gene of JEV were employed to target and destroy JEV transcripts. The four shRNAs expression plasmids were individually co-transfected into 293T cells with the plasmid pNS5-EGFP expressing NS5 fused to enhanced green fluorescent protein. The expression level of NS5 was evaluated by fluorescence microscopy, flow cytometry, real time RT-PCR, and Western blot. The four shRNA expression plasmids were also transfected into BHK-21 cells to examine their inhibition of viral replication by indirect immunofluorescence, real time RT-PCR, and Western blot. The results provided strong evidence that shRNAs targeting the NS5 gene could specifically and efficiently inhibit JEV replication. Three out of four plasmids were highly efficient at inhibiting viral replication, including pS4.1-NS5-455, pS4.1-NS5-699, and pS4.1-NS5-804. This was especially true for pS4.1-NS5-699, which reduced the levels of virus RNA and protein the most. Our data suggest that shRNAs could be used as a tool to inhibit JEV replication in vivo.
Asunto(s)
Virus de la Encefalitis Japonesa (Especie)/genética , Interferencia de ARN , ARN Interferente Pequeño/genética , Proteínas no Estructurales Virales/genética , Replicación Viral , Animales , Secuencia de Bases , Línea Celular , Virus de la Encefalitis Japonesa (Especie)/fisiología , Citometría de Flujo , Técnica del Anticuerpo Fluorescente Indirecta , Vectores Genéticos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Datos de Secuencia Molecular , ARN Bicatenario/síntesis química , ARN Bicatenario/genética , ARN Interferente Pequeño/síntesis química , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , Proteínas no Estructurales Virales/metabolismoRESUMEN
Coxsackievirus B3 (CVB3) was thought to be the most common causative agent of life-threatening viral myocarditis. Coxsackievirus B3 strain CC (CVB3-CC) was isolated in China; however, no sequence data are available. The 1A and 3D regions of CVB3-CC were sequenced and phylogenetic analysis was done with reference to ten other CVB3 strains and all 36 prototype strains of human enterovirus B (HEV-B). Sequence analysis showed that the 1A gene region of CVB3-CC consisted of 207 nucleotides, encoding 69 amino acids; and the 3D gene region was comprised of 1386 nucleotides, encoding 462 amino acids. Variation analysis showed that the 3D gene of CVB3 strain CC varied the least among the two regions. Phylogenetic tree analysis of the 1A and 3D regions indicated that CVB3-CC clustered together with CVB3 Nancy strain suggesting that there may be a close evolutionary relationship between the two strains. Incongruity was observed between the non-structural protein gene and the structural protein gene trees, according to the topological structure, indicating that recombination was occurred among these strains.
Asunto(s)
Secuencia de Bases , Enterovirus Humano B/genética , Variación Genética , Filogenia , Análisis de Secuencia de ADN , Genoma Viral/genética , Humanos , Datos de Secuencia MolecularRESUMEN
In the early 1970s, the Chinese Equine Infectious Anemia Virus (EIAV) vaccine, EIAV(DLA), was developed through successive passages of a wild-type virulent virus (EIAV(L)) in donkeys in vivo and then in donkey macrophages in vitro. EIAV attenuation and cell tropism adaptation are associated with changes in both envelope and long terminal repeat (LTR). However, specific LTR changes during Chinese EIAV attenuation have not been demonstrated. In this study, we compared LTR sequences from both virulent and attenuated EIAV strains and documented the diversities of LTR sequence from in vivo and in vitro infections. We found that EIAV LTRs of virulent strains were homologous, while EIAV vaccine have variable LTRs. Interestingly, experimental inoculation of EIAV(DLA) into a horse resulted in a restriction of the LTR variation. Furthermore, LTRs from EIAV(DLA) showed higher Tat transactivated activity than LTRs from virulent strains. By using chimeric clones of wild-type LTR and vaccine LTR, the main difference of activity was mapped to the changes of R region, rather than U3 region.
Asunto(s)
Variación Genética , Caballos/virología , Virus de la Anemia Infecciosa Equina/patogenicidad , Macrófagos/virología , Monocitos/virología , Regiones Promotoras Genéticas , Secuencias Repetidas Terminales/genética , Animales , Secuencia de Bases , Células Cultivadas , Equidae , Anemia Infecciosa Equina/fisiopatología , Anemia Infecciosa Equina/virología , Regulación Viral de la Expresión Génica , Genes tat , Enfermedades de los Caballos/fisiopatología , Enfermedades de los Caballos/virología , Virus de la Anemia Infecciosa Equina/genética , Virus de la Anemia Infecciosa Equina/metabolismo , Datos de Secuencia Molecular , Filogenia , Análisis de Secuencia de ADN , Secuencias Repetidas Terminales/fisiología , Activación Transcripcional , Vacunas ViralesRESUMEN
To identify the epitope of SARS-CoV spike protein specific neutralizing monoclonal antibody (MAb) 2C5. The antibody was used as target and three rounds of bio-panning were conducted with phage-display peptide library. After the third panning, 20 phage-plague clones were randomly picked and analyzed for the binding ability with the MAb 2C5 by ELISA. The display sequence analysis demonstrated that among the twenty phage clones, eight clones displayed the same seven-peptide TPEQQFT. All these eight phage-clones showed strongest binding activity with 2C5 in phage ELISA analysis. Furthermore, phages displaying peptide TPEQQFT could specifically inhibit the binding of MAb 2C5 with SARS-CoV spike protein. The results demonstrated that TPEQQFT is a mimic epitope peptide containing neutralizing MAb 2C5. This study may provide information for further structural and functional analysis of spike protein and development vaccine for severe acute respiratory syndrome.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Glicoproteínas de Membrana/inmunología , Biblioteca de Péptidos , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/inmunología , Proteínas del Envoltorio Viral/inmunología , Secuencia de Aminoácidos , Ensayo de Inmunoadsorción Enzimática , Epítopos , Glicoproteínas de Membrana/química , Datos de Secuencia Molecular , Glicoproteína de la Espiga del Coronavirus , Proteínas del Envoltorio Viral/químicaRESUMEN
AIM: To evaluate the relationship between the transcriptional level of IFN-gamma mRNA in peripheral blood mononuclear cells (PBMC) and immune protective response driven by inoculated horses with donkey leukocyte attenuated vaccine of EIAV(DLV), and to elucidate the immune mechanism of DLV. METHODS: A real-time PCR method was established for quantitative detection of IFN-gamma mRNA level from horse PBMCs. Twelve horses were divided into vaccination group, healthy control group, challenging control group and EIAV natural infection group. The transcriptional level and distribution of IFN-gamma mRNA in PBMCs were analyzed. The temperature and other parameters of the inoculated horses were monitored or a daily basis. Horses inoculated with DLV vaccines were challenged post-inoculated 8 months, and the change of transcriptional level of IFN-gamma mRNA during pre/post-challenge was evaluated. RESULTS: Compared with the healthy control and natural infection group. The transcriptional level of IFN-gamma mRNA was much higher in vaccination group (P<0.01), and kept increasing even after challenging with virulent EIAV strains. In the challenging control group, the transcriptional level of IFN-gamma mRNA fluctuated with the development of disease and reduced markedly during febrific episodes. CONCLUSION: This study for the first time reveals that EIAV-attenuated vaccine could induce high level transcription of IFN-gamma mRNA, with is associated closely with immune protective response induced by DLV. This finding provides some clues to elucidate the mechanism of immunity protection of DLV at the molecular level.
Asunto(s)
Caballos/inmunología , Caballos/virología , Virus de la Anemia Infecciosa Equina/inmunología , Interferón gamma/genética , Leucocitos Mononucleares/metabolismo , Transcripción Genética/inmunología , Vacunas Virales/inmunología , Animales , Clonación Molecular , ADN Complementario/genética , Caballos/metabolismo , Inmunización , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Temperatura , Vacunación , Vacunas Atenuadas/inmunologíaRESUMEN
OBJECTIVE: To investigate the effect of cilazapril on endothelial cell function and fibrinolysis system in the canine atrial fibrillation (AF) models. METHODS: All canines were divided into three groups: (1) Control group, without atrial pacing; (2) Atrial pacing group, in which atrial fibrillation was established by rapid atrial pacing at 400 bpm for 6 weeks; (3) Atrial pacing together with cilazapril group, in which cilazapril was given before and after atrial pacing. Nitric oxide (NO) of atrial endocardium was measured with NO-specific microelectrode. The expression of plasminogen activator inhibitor-1 (PAI-1) and tissue-type plasminogen activator (tPA) protein in atrium was determined by Western Blot analysis and immunohistochemical staining. Plasma levels of von Willebrand Factor (vWF), PAI-1 and tPA were analyzed by enzyme-linked immunoadsorbent assay. RESULTS: NO production from atrial endocardium was significantly increased in atrial pacing together with cilazapril group than atrial pacing group [(42.6 +/- 9.9) nmol/L vs (23.4 +/- 5.8) nmol/L, P < 0.05], whereas the plasma levels of vWF were decreased [(75.4 +/- 12.8)% vs (125.9 +/- 20.6)%, P < 0.05]. Compared to controls, the expression of atrium tPA protein in atrial pacing together with cilazapril group was significantly upregulated (4052 +/- 857 vs 1936 +/- 421, P < 0.05) and PAI-1 protein was downregulated (2487 +/- 542 vs 3164 +/- 827, P < 0.05). Cilazapril also significantly increased tPA antigen and decreased PAI-1 antigen in plasma. CONCLUSION: Cilazapril can favorably improve endothelial function and resume the balance of fibrinolysis system in AF, which might be of beneficial to hypercoagulated state in AF.