RESUMO
The present study details the design and demonstrates function for a series of reagents and methods to allow the detection of exposure to antigens specific for Porcine endogenous retrovirus (PERV). The detection of PERV is carried out by the means of a variety of immunological screening methods including, indirect immunofluorescence, Western blotting and enzyme linked immunosorbent assay (ELISA) for the detection of antibodies in serum specific for PERV gag and env antigens. Alternatively, PERV-specific antisera for gag and env can be used to detect viral antigen in serum or other samples. PERV env peptides with potential specificity for the known PERV types are also described. Antisera against the peptides can be used to detect PERV antigens directly or to characterise viral type. Using electron microscopy coupled with labelled PERV-gag-specific antisera it was possible to visualise PERV virions.
Assuntos
Retrovirus Endógenos/isolamento & purificação , Testes Imunológicos/métodos , Virologia/métodos , Sequência de Aminoácidos , Animais , Anticorpos Antivirais/análise , Anticorpos Antivirais/sangue , Antígenos Virais/análise , Antígenos Virais/genética , Antígenos Virais/imunologia , Western Blotting , Linhagem Celular , Retrovirus Endógenos/genética , Ensaio de Imunoadsorção Enzimática , Técnica Indireta de Fluorescência para Anticorpo , Produtos do Gene env/química , Produtos do Gene gag/química , Humanos , Soros Imunes/biossíntese , Microscopia Imunoeletrônica , Dados de Sequência Molecular , Alinhamento de Sequência , Testes Sorológicos , Suínos , Proteínas Virais/análise , Proteínas Virais/genética , Proteínas Virais/imunologiaRESUMO
Primary rat hepatocytes dedifferentiate rapidly losing theactivities of the drug metabolising enzymes involved in thedetoxification of xenobiotics in the liver. An alternativeapproach to using primary hepatocytes for toxicity testing isthe development of immortalised hepatocyte cell lines via thetransfection of primary hepatocytes with SV40 DNA. In order toassess the suitability of immortalised lines as an alternativeto primary cell cultures we have used RNA arbitrarily primedpolymerase chain reaction to compare gene expression inimmortalised rat hepatocyte cell lines with that in primary rathepatocytes. We have found that differences exist in the RNAtranscripts between fresh and immortalised hepatocyteshighlighting RNA arbitrarily primed polymerase chain reaction asa useful screening method for identifying immortalised lineswhich retain the most ;normal' phenotype in relation to theprimary cells from which they originated.
