RESUMEN
It is still a huge challenge to find a new strategy for rationally designing covalent drugs because most of them are discovered by serendipity. Considering that the effect of covalent drugs is closely associated with the kinetics of the reaction between drug molecule and its target protein, here we first demonstrate an example of the kinetic effect of pi-stacking of drug molecules on covalent antimicrobial drug design. When PEGylated 7-aminocephalosporanic acid (PEG-ACA) is used as a substrate drug, pi-stacking of the ACA group via the self-assembly of PEG-ACA on the surface of gold nanoparticles (i.e. Au@ACA) exhibits antibacterial activity against E. coli fourfold higher than a PEG-ACA monomer does. The reason can be reasonably attributed to the kinetic rate enhancement for the covalent reaction between Au@ACA and penicillin binding proteins. We believe that the self-assembly of functional groups onto the surface of gold nanoparticles represents a new strategy for covalent drug design.
Asunto(s)
Nanopartículas del Metal , Antibacterianos , Escherichia coli , Oro , CinéticaRESUMEN
Understanding the interplay between Influenza viruses and host cells is key to elucidating the pathogenesis of these viruses. Several host factors have been identified that exert antiviral functions; however, influenza viruses continue to replicate utilizing host cell machinery. Herein, we review the mechanisms of action of two host-derived proteins on conferring cellular resistance to the influenza virus; (1) the interferon inducible trans-membrane proteins, 1, 2 and 3, a recently identified family of early restriction factors; and (2) retinoic acid inducible gene I, a key mediator of antiviral immunity. These data may contribute to the design of novel and efficient anti-influenza treatments.
Asunto(s)
Antígenos de Diferenciación/metabolismo , Interacciones Huésped-Patógeno , Proteínas de la Membrana/metabolismo , Infecciones por Orthomyxoviridae/metabolismo , Infecciones por Orthomyxoviridae/virología , Orthomyxoviridae/fisiología , Replicación Viral , Animales , Antígenos de Diferenciación/genética , Resistencia a la Enfermedad , Interacciones Huésped-Patógeno/genética , Interacciones Huésped-Patógeno/inmunología , Humanos , Inmunidad , Proteínas de la Membrana/genética , Familia de Multigenes , Infecciones por Orthomyxoviridae/genética , Infecciones por Orthomyxoviridae/inmunología , Receptores Virales/metabolismo , Transducción de SeñalRESUMEN
The spike (S) protein of porcine transmissible gastroenteritis virus (TGEV) is located within the viral envelope and is the only structural protein that possesses epitopes capable of inducing virus-neutralizing antibodies. Among the four N-terminal antigenic sites A, B, C, and D, site A and to a lesser extent site D (S-AD) induce key neutralizing antibodies. Recently, we expressed S-AD (rS-AD) in recombinant form. In the current study, we used the rS-AD as an immobilized target to identify peptides from a phage-display library with application for diagnosis. Among the 9 phages selected that specifically bound to rS-AD, the phage bearing the peptide TLNMHLFPFHTG bound with the highest affinity and was subsequently used to develop a phage-based ELISA for TGEV. When compared with conventional antibody-based ELISA, phage-mediated ELISA was more sensitive; however, it did not perform better than semi-quantitative RT-PCR, though phage-mediated ELISA was quicker and easier to set up.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática/métodos , Gastroenteritis Porcina Transmisible/diagnóstico , Péptidos/aislamiento & purificación , Péptidos/metabolismo , Glicoproteína de la Espiga del Coronavirus/metabolismo , Virus de la Gastroenteritis Transmisible/aislamiento & purificación , Animales , Gastroenteritis Porcina Transmisible/virología , Biblioteca de Péptidos , Unión Proteica , Sensibilidad y Especificidad , PorcinosRESUMEN
Three phage-displayed peptides designated H, S and F that recognize porcine aminopeptidase N (pAPN), the cellular receptor of porcine transmissible gastroenteritis virus (TGEV) were able to inhibit cell infection by TGEV. These same peptides had no inhibitory effects on infection of Vero cells by porcine epidemic diarrhea virus (PEDV). However, when PEDV, TGEV and porcine pseudorabies virus were incubated with peptide H (HVTTTFAPPPPR), only infection of Vero cells by PEDV was inhibited. Immunofluoresence assays indicated that inhibition of PEDV infection by peptide H was independent of pAPN. Western blots demonstrated that peptide H interacted with PEDV spike protein and that pre-treatment of PEDV with peptide H led to a higher inhibition than synchronous incubation with cells. These results indicate direct interaction with the virus is necessary to inhibit infectivity. Temperature shift assays demonstrated that peptide H inhibited pre-attachment of the virus to the cells.
Asunto(s)
Antivirales/metabolismo , Antígenos CD13/metabolismo , Péptidos/metabolismo , Virus de la Diarrea Epidémica Porcina/efectos de los fármacos , Virus de la Diarrea Epidémica Porcina/fisiología , Internalización del Virus/efectos de los fármacos , Animales , Antivirales/aislamiento & purificación , Chlorocebus aethiops , Herpesvirus Suido 1/efectos de los fármacos , Herpesvirus Suido 1/fisiología , Biblioteca de Péptidos , Péptidos/aislamiento & purificación , Virus de la Gastroenteritis Transmisible/efectos de los fármacos , Virus de la Gastroenteritis Transmisible/fisiología , Células VeroRESUMEN
Coronaviruses belong to the family Coronaviridae, which primarily cause infection of the upper respiratory and gastrointestinal tract of hosts. Transmissible gastroenteritis virus (TGEV) is an economically significant coronavirus that can cause severe diarrhea in pigs. Silver nanomaterials (Ag NMs) have attracted great interests in recent years due to their excellent anti-microorganism properties. Herein, four representative Ag NMs including spherical Ag nanoparticles (Ag NPs, NM-300), two kinds of silver nanowires (XFJ011) and silver colloids (XFJ04) were selected to study their inhibitory effect on TGEV-induced host cell infection in vitro. Ag NPs were uniformly distributed, with particle sizes less than 20 nm by characterization of environmental scanning electron microscope and transmission electron microscope. Two types of silver nanowires were 60 nm and 400 nm in diameter, respectively. The average diameter of the silver colloids was approximately 10 nm. TGEV infection induced the occurring of apoptosis in swine testicle (ST) cells, down-regulated the expression of Bcl-2, up-regulated the expression of Bax, altered mitochondrial membrane potential, activated p38 MAPK signal pathway, and increased expression of p53 as evidenced by immunofluorescence assays, real-time PCR, flow cytometry and Western blot. Under non-toxic concentrations, Ag NPs and silver nanowires significantly diminished the infectivity of TGEV in ST cells. Moreover, further results showed that Ag NPs and silver nanowires decreased the number of apoptotic cells induced by TGEV through regulating p38/mitochondria-caspase-3 signaling pathway. Our data indicate that Ag NMs are effective in prevention of TGEV-mediated cell infection as a virucidal agent or as an inhibitor of viral entry and the present findings may provide new insights into antiviral therapy of coronaviruses.
Asunto(s)
Nanopartículas del Metal/química , Nanoestructuras/química , Plata/química , Virus de la Gastroenteritis Transmisible/patogenicidad , Animales , Antivirales/efectos adversos , Antivirales/química , Antivirales/farmacología , Línea Celular , Supervivencia Celular/efectos de los fármacos , Citometría de Flujo , Técnica del Anticuerpo Fluorescente Indirecta , Porcinos , Virus de la Gastroenteritis Transmisible/efectos de los fármacos , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Porcine parvovirus (PPV), a member of the genus Parvovirus, family Parvoviridae, is a significant causative agent in porcine reproductive failure, causing serious economic losses in the swine industry. Previous phylogenetic studies based on the NS1 or VP2 genes indicated that current PPV strains diverged 30 years ago and that VP2 was under neutral or positive selection. Our analysis of NS1, VP2 and complete ORFs indicated that the most recent common ancestor of PPV strains existed about 250 years ago and that the 127-nt repeat in the 3'NTR was present in viruses of some subclades that evolved about 80 years ago. Nucleotide substitution rates of NS1 and VP2 genes were 3.03 × 10(-5) and 1.07 × 10(-4), respectively. Both the NS1 and VP2 proteins were under purifying selection and recombination did not contribute to the genetic diversity of PPV. As expected, surface amino acids are hydrophilic and make up the majority of mutations in the VP2 protein; residues in VP2 interfaces were substituted gradually, often in conjunction with complementary substitutions in the neighboring VP2.
Asunto(s)
Evolución Molecular , Variación Genética , Parvovirus Porcino/clasificación , Parvovirus Porcino/genética , Filogenia , Proteínas Virales/genética , Animales , Biología Computacional , Tasa de Mutación , Selección Genética , Análisis de Secuencia de ADN , PorcinosRESUMEN
The membrane (M) protein is one of the major structural proteins of coronavirus particles. In this study, the M protein of transmissible gastroenteritis virus (TGEV) was used to biopan a 12-mer phage display random peptide library. Three phages expressing TGEV-M-binding peptides were identified and characterized in more depth. A phage-based immunosorbent assay (phage-ELISA) capable of differentiating TGEV from other coronaviruses was developed using one phage, phTGEV-M7, as antigen. When the phage-ELISA was compared to conventional antibody-based ELISA for detecting infections, phage-ELISA exhibited greater sensitivity. A chemically synthesized, TGEV-M7 peptide (pepTGEV-M7; HALTPIKYIPPG) was evaluated for antiviral activity. Plaque-reduction assays revealed that pepTGEV-M7 was able to prevent TGEV infection in vitro (p<0.01) following pretreatment of the virus with the peptide. Indirect immunofluorescence and real-time RT-PCR confirmed the inhibitory effects of the peptide. These results indicate that pepTGEV-M7 might be utilized for virus-specific diagnostics and treatment.
Asunto(s)
Gastroenteritis Porcina Transmisible/virología , Péptidos/metabolismo , Virus de la Gastroenteritis Transmisible/metabolismo , Proteínas de la Matriz Viral/metabolismo , Secuencia de Aminoácidos , Animales , Antivirales/química , Antivirales/farmacología , Proteínas M de Coronavirus , Gastroenteritis Porcina Transmisible/diagnóstico , Gastroenteritis Porcina Transmisible/tratamiento farmacológico , Ligandos , Datos de Secuencia Molecular , Péptidos/química , Péptidos/genética , Porcinos , Virus de la Gastroenteritis Transmisible/efectos de los fármacos , Virus de la Gastroenteritis Transmisible/genética , Proteínas de la Matriz Viral/genéticaRESUMEN
The objective of the present study was to gain new insights into the evolution, homologous recombination, and selection pressures imposed on the porcine torovirus (PToV), by examining the changes in the hemagglutinin-esterase (HE) gene. The most recent common ancestor of PToV was estimated to have emerged 62 years ago based upon HE gene sequence data obtained from PToV isolates originating from Spain, South Korea, Netherlands, Hungary, and Italy and using the HE gene of Bovine torovirus isolates Niigata1 (AB661456) and Niigata3 (AB661458) as outgroups. The HE gene sequence data segregated all the PToV isolates into two well-supported monophyletic groups; however, various isolates from Spain, Italy, and South Korea did not segregate geographically suggesting very recent translocation of the viruses to these localities. Evidence of recombination was observed between two South Korean isolates that partitioned into two distinct subclades. Data further suggest that most of the nucleotides in the HE gene are under negative selection; however, changes within codon 237 showed an evidence of positive selection.
Asunto(s)
Evolución Molecular , Hemaglutininas Virales/genética , Recombinación Homóloga , Enfermedades de los Porcinos/virología , Infecciones por Torovirus/veterinaria , Torovirus/genética , Proteínas Virales de Fusión/genética , Animales , Secuencia de Bases , Hemaglutininas Virales/química , Italia , Datos de Secuencia Molecular , Países Bajos , Conformación de Ácido Nucleico , Filogenia , República de Corea , Selección Genética , España , Porcinos , Torovirus/química , Torovirus/clasificación , Infecciones por Torovirus/virología , Proteínas Virales de Fusión/químicaRESUMEN
BACKGROUND: Transmission of SARS-associated coronavirus (SARS-CoV) is now well controlled, nevertheless, it is important to develop effective methods to identify this virus from other pathogens. OBJECTIVES: The purpose of this study was to identify potential ligands and develop a novel diagnostic test to SARS-CoV using phage display technology. STUDY DESIGN: The SARS-CoV spike 1 (S1) protein containing the receptor binding region (RBD) was used as an immobilized target followed by incubation with a 12-mer phage display random peptide library. After four rounds of biopanning, 10 monoclonal phages with specific binding activity to the S1-RBD protein were obtained and subjected to binding and diagnostic assays. RESULTS: DNA sequencing showed that two phage displayed peptides HHKTWHPPVMHL (phage-H) and SQWHPRSASYPM (phage-S) that were specific ligands to the S1 protein. Moreover, the selected phage-H and phage-S were capable of differentiating SARS-CoV from other coronaviruses in indirect enzyme-linked immunosorbent assays. CONCLUSION: The peptides identified in this study are useful reagents for detection of SARS-CoV.
Asunto(s)
Bacteriófagos/genética , Péptidos/genética , Síndrome Respiratorio Agudo Grave/virología , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/genética , Glicoproteína de la Espiga del Coronavirus/genética , Secuencia de Aminoácidos , Animales , Bacteriófagos/metabolismo , Técnicas de Visualización de Superficie Celular/métodos , Pruebas Diagnósticas de Rutina/métodos , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Ligandos , Ratones , Biblioteca de Péptidos , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/metabolismo , Síndrome Respiratorio Agudo Grave/diagnóstico , Glicoproteína de la Espiga del Coronavirus/metabolismo , Porcinos/genética , Porcinos/metabolismoRESUMEN
Porcine epidemic diarrhoea virus (PEDV) is one of the important pathogens that may cause severe diarrhoea in piglets. In this study, the nucleocapsid (N) gene of a Chinese PEDV isolate designated HLJBY was cloned. The phylogeny of PEDV strains was investigated by constructing a phylogenetic tree based on the N protein sequences. The results indicate that there are two major groups of Chinese PEDVs, a Japanese PEDV group and a Korean PEDV group. High-level expression of the N protein was achieved in Escherichia coli. The immunoreactivity between PEDV particles or the bacterially expressed N protein and rabbit anti-PEDV serum was confirmed by immunofluorescence assays and Western blot. Both PEDV N protein and the polyclonal antibody generated in this study are valuable diagnostic reagents for PEDV surveillance.
Asunto(s)
Filogenia , Virus de la Diarrea Epidémica Porcina , Animales , Infecciones por Coronavirus , Datos de Secuencia Molecular , Nucleocápside , Enfermedades de los Porcinos/epidemiologíaRESUMEN
BACKGROUND: The context and purpose of the study included 1) bacterial expression of viral protein 6 (VP6) of porcine rotavirus (PRV) and generation of rabbit polyclonal antiserum to the VP6 protein; 3) establishment of a discrimination ELISA to distinguish PRV from a panel of other porcine viruses. RESULTS: The VP6 gene of PRV isolate DN30209 amplified by reverse transcription-PCR was 1356 bp containing a complete open reading frame (ORF) encoding 397 amino acids. Sequence comparison and phylogenetic analysis indicated that PRV DN30209 may belong to group A of rotavirus. Bacterially expressed VP6 was expressed in E.coli and anti-VP6 antibody was capable of distinguishing PRV from Porcine transmissible gastroenteritis virus, Porcine epidemic diarrhea virus, Porcine circovirus type II, Porcine reproductive and respiratory syndrome virus, Porcine pseudorabies virus and Porcine parvovirus. CONCLUSIONS: PRV VP6 expressed in E. coli can be used to generate antibodies in rabbit; anti-VP6 serum antibody can be used as good diagnostic reagents for detection of PRV.
Asunto(s)
Anticuerpos Antivirales , Antígenos Virales/inmunología , Proteínas de la Cápside/inmunología , Rotavirus/clasificación , Rotavirus/aislamiento & purificación , Virología/métodos , Animales , Anticuerpos Antivirales/aislamiento & purificación , Antígenos Virales/genética , Proteínas de la Cápside/genética , Ensayo de Inmunoadsorción Enzimática/métodos , Escherichia coli/genética , Expresión Génica , Conejos , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Sensibilidad y Especificidad , PorcinosRESUMEN
Porcine transmissible gastroenteritis virus (TGEV) and porcine epidemic diarrhea virus (PDEV) can cause severe diarrhea in pigs. Development of effective vaccines against TGEV and PEDV is one of important prevention measures. The spike (S) protein is the surface glycoprotein of TGEV and PEDV, which can induce specific neutralization antibodies and is a candidate antigen for vaccination attempts. In this study, the open reading frames of the TGEV S1 protein and in addition of the S or S1 proteins of PEDV were inserted into the eukaryotic expression vector, pIRES, resulting in recombinant plasmids, pIRES-(TGEV-S1-PEDV-S1) and pIRES-(TGEV-S1-PEDV-S). Subsequently, 6-8 weeks old Kunming mice were inoculated with both DNA plasmids. Lymphocyte proliferation assay, virus neutralization assay, IFN-γ assay and CTL activity assay were performed. TGEV/PEDV specific antibody responses as well as kinetic changes of T lymphocyte subgroups of the immunized mice were analyzed. The results showed that the recombinant DNA plasmids increased the proliferation of T lymphocytes and the number of CD4+ and CD8+ T lymphocyte subgroups. In addition, the DNA vaccines induced a high level of IFN-γ in the immunized mice. The specific CTL activity in the pIRES-(TGEV-S1-PEDV-S) group became significant at 42 days post-immunization. At 35 days post-immunization, the recombinant DNA plasmids bearing full-length S genes of TGEV and PEDV stimulated higher levels of specific antibodies and neutralizing antibodies in immunized mice.
Asunto(s)
Virus de la Diarrea Epidémica Porcina/genética , Virus de la Diarrea Epidémica Porcina/inmunología , Virus de la Gastroenteritis Transmisible/genética , Virus de la Gastroenteritis Transmisible/inmunología , Vacunas de ADN/genética , Vacunas de ADN/inmunología , Proteínas Virales/genética , Proteínas Virales/inmunología , Vacunas Virales/genética , Vacunas Virales/inmunología , Animales , Anticuerpos Antivirales/sangre , Infecciones por Coronavirus/inmunología , Infecciones por Coronavirus/prevención & control , Infecciones por Coronavirus/veterinaria , ADN Recombinante/genética , ADN Recombinante/inmunología , ADN Viral/genética , ADN Viral/inmunología , Gastroenteritis Porcina Transmisible/inmunología , Gastroenteritis Porcina Transmisible/prevención & control , Genes Virales , Interferón gamma/sangre , Interleucina-4/sangre , Ratones , Plásmidos/genética , Plásmidos/inmunología , Porcinos , Enfermedades de los Porcinos/inmunología , Enfermedades de los Porcinos/prevención & control , Linfocitos T/inmunologíaRESUMEN
The enhanced antioxidant activity of surface-functionalized gold nanoparticles (AuNPs) synthesized by self-assembly has attracted great attention, but little is known about the mechanism behind the enhanced activity. To address this challenge, the antioxidant activity of Au@PEG3SA (i.e., surface-functionalization of spherical AuNPs with the antioxidant salvianic acidâ A) was used as an example to illustrate the mechanism of the enhanced activity. Evaluation of the antioxidant activity was performed in a radical-scavenging reaction between Au@PEG3SA and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical. As expected, the rate constant for the reaction of Au@PEG3SA with DPPH was about nine times greater than that for the salvianic acidâ A monomer. A comparative analysis of the spectral characteristics of Au@PEG3SA and the salvianic acidâ A monomer further imply that the enhancement of the antioxidative reaction kinetics may be ascribed to the variation in the transition state for the DPPH-radical scavenging reaction through π-π stacking interactions between and among adjacent groups on the surface of Au@PEG3SA. On the other hand, the kinetic enhancement of Au@PEG3SA on reactive-oxygen-species (ROS) scavenging can be observed in living cells and in vivo, which possibly provides new insight for the bioapplication of self-assembly of surface-functionalized AuNPs.
Asunto(s)
Antioxidantes/química , Oro/química , Nanopartículas del Metal/química , Antioxidantes/farmacología , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Humanos , Cinética , Lactatos/química , Nanopartículas del Metal/toxicidad , Especies Reactivas de Oxígeno/metabolismo , Propiedades de SuperficieRESUMEN
Incidence and mortality due to tuberculosis (TB) have been decreasing worldwide. Given that TB is a cosmopolitan disease, proper surveillance and evaluation are critical for controlling dissemination. Herein, mathematical modeling was performed in order to: 1) demonstrate a correlation between the incidence of TB in HIV-free patients in the US and Germany, and their corresponding mortality rates; 2) show the utility of the newly developed D-R algorithm for analyzing and predicting the incidence of TB in both countries; and 3) inform us on population death rates due to TB in HIV-negative patients. Using data published by the World Health Organization between 1990 and 2009, the relationship between incidence and mortality that could not be ascribed to HIV infection was evaluated. Using linear, quadratic and cubic curves, we found that a cubic function provided the best fit with the data in both the US (Y = 2.3588+2.2459X+61.1639X(2)-60.104X(3)) and Germany (Y = 1.9271+9.4967X+18.3824X(2)-10.350X(3)) where the correlation coefficient (R) between incidence and mortality was 0.995 and 0.993, respectively. Second, we demonstrated that fitted curves using the D-R model were equal to or better than those generated using the GM(1,1) algorithm as exemplified in the relative values for Sum of Squares of Error, Relative Standard Error, Mean Absolute Deviation, Average Relative Error, and Mean Absolute Percentage Error. Finally, future trends using both the D-R and the classic GM(1,1) models predicted a continued decline in infection and mortality rates of TB in HIV-negative patients rates extending to 2015 assuming no changes to diagnosis or treatment regimens are enacted.
Asunto(s)
Seronegatividad para VIH , Modelos Teóricos , Tuberculosis/epidemiología , Algoritmos , Alemania/epidemiología , Humanos , Incidencia , Tuberculosis/mortalidad , Estados Unidos/epidemiologíaRESUMEN
The objective of this study was to develop a reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for detection of type II porcine reproductive and respiratory syndrome virus (PRRSV). Based on sequence alignment, four primers were designed amplifying the M gene of type II PRRSV and were subsequently utilized in an RT-LAMP assay. The RT-LAMP product had a ladder-like pattern of bands and the optimal reaction condition for this assay was determined to be 40 min at 63°C. Comparative analysis indicated that the RT-LAMP method was more sensitive than a conventional RT-PCR assay and comparable to a real-time PCR assay. In addition, the RT-LAMP assay was capable of detecting type II PRRSV in field samples and differentiating type II PRRSV from seven other porcine viruses which are all associated frequently with similar clinical symptoms.
Asunto(s)
Técnicas de Amplificación de Ácido Nucleico/métodos , Síndrome Respiratorio y de la Reproducción Porcina/diagnóstico , Virus del Síndrome Respiratorio y Reproductivo Porcino/aislamiento & purificación , Medicina Veterinaria/métodos , Virología/métodos , Animales , Cartilla de ADN/genética , Genes Virales , Síndrome Respiratorio y de la Reproducción Porcina/virología , Sensibilidad y Especificidad , PorcinosRESUMEN
Porcine epidemic diarrhea virus (PEDV) is the causative agent of porcine epidemic diarrhea, a highly contagious enteric disease of swine. The Spike (S) protein is one of the main structural proteins of PEDV capable of inducing neutralizing antibodies in vivo. Herein, we generated three distinct DNA constructs in the eukaryotic expression plasmid pVAX1; one encoding the S protein [pVAX1-(PEDV-S)], the second encoding the N-terminal fragment (S1) [pVAX1-(PEDV-S1)] containing potent antigenic sites, and the third expressing the porcine interleukin-18 (pIL-18) [pVAX1-(IL-18)]. Immunofluorescence assays in BHK-21 cells demonstrated successful protein expression from all 3 constructs. Kunming mice were injected separately with each of these constructs or with a pVAX1-(PEDV-S1)/pVAX1-(IL-18) combination, an attenuated PEDV vaccine, or vector only control. Animals were examined for T lymphocyte proliferation, anti-PEDV antibodies, IFN-γ and IL-4 protein levels, and cytotoxic T cell function in mouse peripheral blood and spleen. In all cases, results showed that pVAX1-(PEDV-S) and the combination of pVAX1-(PEDV-S1) with pVAX1-(IL-18) induced the strongest responses; however, pIL-18 had no adjuvant effects when given in combination with pVAX1-(PEDV-S1).
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Interleucina-18/administración & dosificación , Glicoproteínas de Membrana/inmunología , Virus de la Diarrea Epidémica Porcina/inmunología , Vacunas de ADN/inmunología , Proteínas del Envoltorio Viral/inmunología , Adyuvantes Inmunológicos/genética , Animales , Anticuerpos Antivirales/sangre , Sangre/inmunología , Proliferación Celular , Interferón gamma/metabolismo , Interleucina-18/genética , Interleucina-4/metabolismo , Glicoproteínas de Membrana/genética , Ratones , Plásmidos , Virus de la Diarrea Epidémica Porcina/genética , Glicoproteína de la Espiga del Coronavirus , Bazo/inmunología , Linfocitos T/inmunología , Linfocitos T Citotóxicos/inmunología , Vacunas de ADN/administración & dosificación , Vacunas de ADN/genética , Proteínas del Envoltorio Viral/genéticaRESUMEN
Epidemiological data have suggested that drinking green tea is negatively associated with diabetes, and adipose oxidative stress may have a central role in causing insulin resistance, according to recent findings. The aim of this work is to elucidate a new mechanism for green tea's anti-insulin resistance effect. We used obese KK-ay mice, high-fat diet-induced obese rats, and induced insulin resistant 3T3-L1 adipocytes as models. Insulin sensitivity and adipose reactive oxidative species (ROS) levels were detected in animals and adipocytes. The oxidative stress assay and glucose uptake ability assay were performed, and the effects of EGCG on insulin signals were detected. Green tea catechins (GTCs) significantly decreased glucose levels and increased glucose tolerance in animals. GTCs reduced ROS content in both models of animal and adipocytes. EGCG attenuated dexamethasone and TNF-α promoted ROS generation and increased glucose uptake ability. EGCG also decreased JNK phosphorylation and promoted GLUT-4 translocation. EGCG and GTCs could improve adipose insulin resistance, and exact this effect on their ROS scavenging functions.
Asunto(s)
Tejido Adiposo/efectos de los fármacos , Catequina/farmacología , Resistencia a la Insulina , Té/química , Células 3T3-L1 , Animales , Catequina/química , Espectroscopía de Resonancia por Spin del Electrón , Femenino , Prueba de Tolerancia a la Glucosa , Insulina/metabolismo , Ratones , Ratones Endogámicos C57BL , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismoRESUMEN
The open reading frame (ORF) 5 of porcine reproductive and respiratory syndrome virus (PRRSV) encodes a major envelope glycoprotein designated GP5. The GP5 protein is a candidate for vaccinating against PRRSV infection. In this study, recombinant plasmids bearing the PRRSV GP5 gene (pVAX-GP5) or the porcine interleukin 15 gene (pVAX-IL15) were generated. Mice were vaccinated with these gene constructs singularly or in combination, and subsequent humoral and cellular immune responses were evaluated. Proliferation assays showed that the number of T lymphocytes in the peripheral blood and spleens of treated mice were elevated by pVAX-GP5 and significantly enhanced by combination therapy involving pVAX-IL15. Flow cytometry data showed that the numbers of CD4+ and CD8+ T cells were also higher in treated mice. Both pVAX-GP5 treatment alone and in combination with pVAX-IL15 resulted in elevated antibody levels as demonstrated by indirect ELISA. The pVAX-IL15 gene construct served as a molecular adjuvant in conjunction with the pVAX-GP5 to enhance the immune responses where intermediate doses of pVAX-IL15 were most effective.
Asunto(s)
Interleucina-15/metabolismo , Síndrome Respiratorio y de la Reproducción Porcina/inmunología , Virus del Síndrome Respiratorio y Reproductivo Porcino/inmunología , Porcinos/inmunología , Proteínas del Envoltorio Viral/metabolismo , Vacunas Virales/inmunología , Animales , Células Cultivadas , Inmunización , Interleucina-15/genética , Interleucina-15/inmunología , Ratones , Ratones Endogámicos , Sistemas de Lectura Abierta/genética , Plásmidos , Síndrome Respiratorio y de la Reproducción Porcina/prevención & control , Vacunas de ADN/administración & dosificación , Vacunas de ADN/inmunología , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunología , Vacunas Virales/administración & dosificaciónRESUMEN
The spike (S) protein is a key structural protein of coronaviruses including, the porcine transmissible gastroenteritis virus (TGEV). The S protein is a type I membrane glycoprotein located in the viral envelope and is responsible for mediating the binding of viral particles to specific cell receptors and therefore specific cell types. It is also an important immune target for the host in neutralizing the virus. Four antigenic sites A, B, C, and D that reside near the N-terminal domain have been defined in the S protein. Of these, the region encoding antigenic sites A and to a lesser extent D, herein defined as S-AD, are most critical in eliciting host neutralizing antibodies. Herein, we enzymatically amplified, cloned, and expressed the S-AD fragment from TGEV in the prokaryotic expression vector, pET-30a. Maximum protein expression was achieved at 30°C over a 5-h period post-induction. Rabbit polyclonal antiserum was generated using recombinant S-AD (rS-AD) protein. In contrast to prior studies showing no activity with bacterially produced S protein, results indicated that polyclonal serum recognized TGEV-infected cells and reduced infection by 100%. Furthermore, the truncated rS-AD peptide was able to bind to the surface of cells from swine testes in a competitive manner and completely inhibit viral infection.
Asunto(s)
Antígenos Virales/genética , Antígenos Virales/metabolismo , Gastroenteritis Porcina Transmisible/prevención & control , Expresión Génica , Virus de la Gastroenteritis Transmisible/fisiología , Proteínas Virales/metabolismo , Secuencias de Aminoácidos , Animales , Antígenos Virales/química , Línea Celular , Escherichia coli/genética , Escherichia coli/metabolismo , Gastroenteritis Porcina Transmisible/virología , Unión Proteica , Conejos , Porcinos , Virus de la Gastroenteritis Transmisible/química , Virus de la Gastroenteritis Transmisible/genética , Virus de la Gastroenteritis Transmisible/inmunología , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
The gene-encoding mature porcine interleukin-18 (pIL-18) was amplified by PCR and cloned into a prokaryotic expression vector pET-30a(+). The resulting recombinant plasmid pET-30a-pIL-18 was transformed into Escherichia coli. Expression of recombinant pIL-18 protein was induced by 1 mM isopropyl ß-D-thiogalactoside at 37°C. The purified recombinant protein was used to generate a hyperimmune antiserum in a rabbit. The anti-pIL-18 serum was evaluated for its specificity and titer through indirect enzyme-linked immunosorbent assay. The specific reactivity of the anti-pIL-18 antibody was further confirmed by Western blot and immunofluorescence assays. Moreover, the pIL-18 was able to stimulate the proliferation of pig peripheral blood mononuclear cells in vitro, indicating it may have certain biological activity.