RESUMO
Hepatitis B virus(HBV) infection remains a global problem, despite the effectiveness of the Hepatitis B vaccine in preventing infection. The resolution of Hepatitis B virus infection has been believed to be attributable to virus-specific immunity. In vivo direct evaluation of anti-HBV immunity in the liver is currently not possible. We have developed a new assay system that detects HBV clearance in the liver after the hydrodynamic transfer of a reporter gene and over-length, linear HBV DNA into hepatocytes, followed by bioluminescence imaging of the reporter gene (Fluc). We employed bioluminescence detection of luciferase expression in HBV-infected hepatocytes to measure the Hepatitis B core antigen (HBcAg)-specific immune responses directed against these infected hepatocytes. Only HBcAg-immunized, but not mock-treated, animals decreased the amounts of luciferase expression, HBsAg and viral DNA from the liver at day 28 after hydrodynamic infection with over-length HBV DNA, indicating that control of luciferase expression correlates with viral clearance from infected hepatocytes.
Assuntos
Genes Reporter/genética , Vírus da Hepatite B/genética , Vírus da Hepatite B/metabolismo , Luciferases de Vaga-Lume/genética , Animais , DNA Viral/genética , DNA Viral/metabolismo , Vetores Genéticos/genética , Antígenos do Núcleo do Vírus da Hepatite B/imunologia , Vírus da Hepatite B/imunologia , Vírus da Hepatite B/fisiologia , Hidrodinâmica , Injeções , Fígado/imunologia , Fígado/metabolismo , Fígado/virologia , Medições Luminescentes , Masculino , Camundongos , Modelos Animais , Imagem Molecular , Regiões Promotoras Genéticas/genética , Transfecção , Carga Viral , Replicação ViralRESUMO
BACKGROUND/AIMS: Interferon beta (IFN-ß) is the priming cytokine in the interferons (IFNs) response that plays essential roles in innate immune system. Only very few studies on IFN activation in animals have been reported before, therefore, we embarked to develop a novel method to dynamically examine IFN-ß activation in mouse liver by noninvasive molecular imaging. METHODS: Interferon beta promoter-directed firefly luciferase gene was integrated into chromosomes of hepatocytes by hydrodynamic injection. Mouse hepatitis virus type 3 (MHV-3) and polyinosinic-polycytidylic acid [poly(I:C)] were used to stimulate the activation of IFN-ß. Luciferase activity was used as an indicator of the IFN-ß promoter activity in vitro and in vivo. The expression level of IFN-ß in the sera and firefly luciferase in the liver was assessed by ELISA and bioluminescence imaging respectively. Western blot was used for detecting proteins expression. RESULTS: A rapid and elevated luciferase expression in the mouse liver induced by poly (I:C) and MHV-3 was detected by bioluminescence imaging. The detectable level of IFN-ß in the sera was not induced by MHV-3. Moreover, IFN-ß activation was significantly inhibited by the hepatitis C virus (HCV) NS3/4A protease in mouse liver. These results were consistent with IFN-ß production in the sera. Therefore, a novel visual method to monitor IFN-ß promoter activity was established in the current study. CONCLUSION: This novel sensitive method can be used for not only assessing IFN-ß activation or inhibition in the liver under different conditions, but also screening drug candidates of stimulating or inhibiting of IFN-ß production.
Assuntos
Regulação da Expressão Gênica/imunologia , Hepatócitos/metabolismo , Imunidade Inata/imunologia , Interferon beta/metabolismo , Fígado/imunologia , Imagem Molecular/métodos , Animais , Western Blotting , Primers do DNA/genética , Ensaio de Imunoadsorção Enzimática , Regulação da Expressão Gênica/efeitos dos fármacos , Interferon beta/sangue , Interferon beta/genética , Fígado/citologia , Luciferases de Vaga-Lume/metabolismo , Medições Luminescentes , Camundongos , Poli I-C/farmacologia , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas/genética , Proteínas não Estruturais Virais/farmacologiaRESUMO
AIM: To develop a sensitive assay for screening compounds against hepatitis C virus (HCV). METHODS: The proteolytic cleavage of NS3/4A on enhanced yellow fluorescent protein (eYFP)-mitochondrial antiviral signaling protein (MAVS) was examined by reporter enzyme secreted placental alkaline phosphatase (SEAP), which enabled us to perform ongoing monitoring of anti-HCV drugs through repeated chemiluminescence. Subcellular localization of eYFP-MAVS was assessed by fluorescence microscopy. Cellular localization and protein levels were examined by Western blotting. RESULTS: HCV NS3/4A protease cleaved eYFP-MAVS from mitochondria to block the activation of interferon (IFN)-ß promoter, thus resulting in downregulation of SEAP activity. The decrease in SEAP activity was proportional to the dose of active NS3/4A protease. Also this reporter assay was used to detect anti-HCV activity of IFN-α and cyclosporine A. CONCLUSION: Our data show that this reporter system is a sensitive and quantitative reporter of anti-HCV inhibitors. This system will constitute a new tool to allow the efficient screening of HCV inhibitors.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antivirais/farmacologia , Técnicas Biossensoriais , Hepacivirus/efeitos dos fármacos , Interferon beta/genética , Transdução de Sinais/efeitos dos fármacos , Proteínas não Estruturais Virais/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Fosfatase Alcalina/genética , Fosfatase Alcalina/metabolismo , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Western Blotting , Linhagem Celular Tumoral , Ciclosporina/farmacologia , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Genes Reporter , Hepacivirus/enzimologia , Hepacivirus/genética , Humanos , Interferon-alfa/farmacologia , Isoenzimas/genética , Isoenzimas/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Microscopia de Fluorescência , Regiões Promotoras Genéticas , Proteínas Recombinantes de Fusão/metabolismo , Replicon , Fatores de Tempo , Ativação Transcricional , Transfecção , Proteínas não Estruturais Virais/genéticaRESUMO
AIM: To express stably human CD81 gene on mouse hepatoma cell line Hepa 1-6 using liver specific promoter. METHODS: RNA were isolated from human HepG2 cells which could be infected with hepatitis C virus. RT-PCR was carried out using human CD81 gene specific primers. Amplified fragments were cloned into pGEM-T vector. Albumin promoter and enhancer which were liver tissue specific were ligated to the 5'end of human CD81 gene and SV40 polyA sequence was fused with 3'end of CD81. The fused CD81 gene was inserted into eukaryotic expression vector pcDNA3 to construct a recombinant vector pcDNA3-Alb p-CD81 which was then transfected into Hepa 1-6 cells through lipofectamine mediation. Human CD81 mRNA transcription and its protein expression were detected by RT-PCR and FACS, respectively. RESULTS: Sequence analysis showed that the cloned gene segment was human CD81 gene sequence. After transfection, transcripted human CD81 mRNA was obtained and human CD81 molecules were expressed stably on Hepa 1-6 cells. CONCLUSION: The obtained positive cell clones which stably express HCV receptor human CD81 lay the foundation for further study on interactions between HCV envelope proteins and human CD81, screening of HCV infection blocking drugs and development of HCV infection mouse model.