RESUMEN
The VPl gene of enterovirus 71 (EV71) was synthesized, construct a recombinant plasmid pET15b/VP1 and expressed in E. coli BL21. The recombinant VP1 protein could specifically react with EV71-infected patient sera without the cross-reaction with serum antibodies of coxsackievirus A16 (CA16), A4, A5, B3 and B5 as well as echovirus 6. In acute and convalescent phases, IgM and IgG antibodies of 182 serum samples were detected by ELISA with recombinant VP1 protein as a coated antigen. The results showed that the sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of IgM antibodies in serum samples for the diagnosis of EV71 infection were 90.1, 98.4, 98.8 and 88.7%, respectively; similarly, those of IgG antibodies in serum samples were 82.4, 89.1, 91.5 and 78.1%, respectively. Five of 80 samples (6.25%) from CA16infected patients were detected positive by ELISA with recombinant VP1 protein in which indicated the cross reactions and 0 of 5 samples from patients infected with other enteroviruses including CA4, CA5, CB3, CB5 and echovirus 6. Therefore, the recombinant VP1 protein of EV7l may provide a theoretical reference for establishing an effective antibody screening of IgM for EV71-infected patients with clinically suspected hand, foot, and mouth disease (HFMD).
Asunto(s)
Preescolar , Femenino , Humanos , Lactante , Masculino , Anticuerpos Antivirales/sangre , Proteínas de la Cápside , Enterovirus Humano A/inmunología , Enfermedad de Boca, Mano y Pie/diagnóstico , Clonación Molecular , Proteínas de la Cápside/genética , Proteínas de la Cápside/inmunología , Enterovirus Humano A/genética , Ensayo de Inmunoadsorción Enzimática/métodos , Escherichia coli/genética , Expresión Génica , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Valor Predictivo de las Pruebas , Proteínas Recombinantes , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Sensibilidad y Especificidad , Pruebas Serológicas/métodosRESUMEN
BACKGROUND: Mitogen-activated protein kinase (MAPK) signaling pathway plays an important role in response to viral infection. The aim of this study was to explore the function and mechanism of MAPK signaling pathway in enterovirus 71 (EV71) infection of human rhabdomyosarcoma (RD) cells. METHODS: Apoptosis of RD cells was observed using annexin V-FITC/PI binding assay under a fluorescence microscope. Cellular RNA was extracted and transcribed to cDNA. The expressions of 56 genes of MAPK signaling pathway in EV71-infected RD cells at 8 h and 20 h after infection were analyzed by PCR array. The levels of IL-2, IL-4, IL-10, and TNF-α in the supernatant of RD cells infected with EV71 at different time points were measured by ELISA. RESULTS: The viability of RD cells decreased obviously within 48 h after EV71 infection. Compared with the control group, EV71 infection resulted in the significantly enhanced releases of IL-2, IL-4, IL-10 and TNF-α from infected RD cells (p < 0.05). At 8 h after infection, the expressions of c-Jun, c-Fos, IFN-i, MEKK1, MLK3 and NIK genes in EV71-infected RD cells were up-regulated by 2.08-6.12-fold, whereas other 19 genes (e.g. AKT1, AKT2, E2F1, IKK and NF-κB1) exhibited down-regulation. However, at 20 h after infection, those MAPK signaling molecules including MEKK1, ASK1, MLK2, MLK3, NIK, MEK1, MEK2, MEK4, MEK7, ERK1, JNK1 and JNK2 were up-regulated. In addition, the expressions of AKT2, ELK1, c-Jun, c-Fos, NF-κB p65, PI3K and STAT1 were also increased. CONCLUSION: EV71 infection induces the differential gene expressions of MAPK signaling pathway such as ERK, JNK and PI3K/AKT in RD cells, which may be associated with the secretions of inflammatory cytokines and host cell apoptosis.
Asunto(s)
Humanos , Enterovirus Humano A/genética , Proteínas Quinasas Activadas por Mitógenos/genética , Rabdomiosarcoma/virología , Citocinas/genética , Ensayo de Inmunoadsorción Enzimática , Enterovirus Humano A/enzimología , Enterovirus Humano A/fisiología , Regulación Neoplásica de la Expresión Génica , Proteínas Quinasas Activadas por Mitógenos/fisiología , Reacción en Cadena de la Polimerasa , Rabdomiosarcoma/enzimología , Rabdomiosarcoma/genética , Transducción de Señal/genética , Transducción de Señal/fisiología , Factores de Tiempo , Células Tumorales Cultivadas , Regulación hacia Arriba , Replicación ViralRESUMEN
Clinical isolates of carbapenem-resistant Klebsiella pneumoniae (K. pneumoniae) strains are being increased worldwide. Five pan-resistant K. pneumoniae strains have been isolated from respiratory and ICU wards in a Chinese hospital, and reveal strong resistance to all β-lactams, fluoroquinolones and aminoglycosides. Totally 27 β-lactamase genes and 2 membrane pore protein (porin) genes in 5 K. pneumoniae strains were screened by polymerase chain reaction (PCR). The results indicated that all of 5 K. pneumoniae strains carried blaTEM-1 and blaDHA-1 genes, as well as base deletion and mutation of OmpK35 or OmpK36 genes. Compared with carbapenem-sensitive isolates by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the resistant isolates markedly lacked the protein band of 34-40 kDa, which might be the outer membrane proteins of OmpK36 according to the electrophoresis mobility. In addition, the conjugation test was confirmed that blaDHA-1 mediated by plasmids could be transferred between resistant and sensitive strains. When reserpine (30 µg/mL) and carbonyl cyanide m-chlorophenylhydrazone (CCCP) (50 µg/mL) were added in imipenem and meropenem, the MICs had no change against K. pneumoniae strains. These results suggest that both DHA-1 β-lactamase and loss or deficiency of porin OmpK36 may be the main reason for the cefoxitin and carbapenem resistance in K. pneumoniae strains in our hospital.