RESUMO
To produce chitoorigosaccharides using chitosan, we attempted to construct Paenibacillus fukuinensis chitosanase-displaying yeast cells as a whole-cell biocatalyst through yeast cell-surface engineering. The localization of the chitosanase on the yeast cell surface was confirmed by immunofluorescence labeling of cells. The chitosanase activity of the constructed yeast was investigated by halo assay and the dinitrosalicylic acid method.
Assuntos
Bacillus/enzimologia , Proteínas de Bactérias/biossíntese , Biotecnologia/métodos , Membrana Celular/enzimologia , Glicosídeo Hidrolases/biossíntese , Proteínas Recombinantes/biossíntese , Saccharomyces cerevisiae/enzimologia , Proteínas de Bactérias/análise , Proteínas de Bactérias/genética , Catálise , Clonagem Molecular , Engenharia Genética , Glicosídeo Hidrolases/análise , Glicosídeo Hidrolases/genética , Oligossacarídeos/biossíntese , Plasmídeos/genética , Proteínas Recombinantes/análise , Proteínas Recombinantes/genéticaRESUMO
Outbreaks of koi herpesvirus (KHV) infection in carp are still a serious problem worldwide. KHV is closely related to other two cyprinid herpesviruses, pox herpesvirus (CHV) and haematopoietic necrosis herpesvirus (CyHV-2) in goldfish. In this study, two major KHV antigenic proteins (ORF62 and ORF68) were identified by immunoscreening using a KHV-specific polyclonal antibody, and then monoclonal antibodies were generated for immunodiagnostic studies. After screening hybridoma cells, one mAb against ORF68 (mAb-7C6) was obtained but no mAbs against ORF62. mAb-7C6 specifically reacted with a lysate of KHV-infected koi fin cells (KF-1 cells) but not with lysates of CHV- or CyHV-2-infected KF-1 cells in an immuno-blotting analysis. Similar results were shown in the following tests: (1) a indirect fluorescent antibody test using infected KF-1 cells and (2) an immunohistochemical investigation by fast red stain (infected liver) or FITC detection (infected spleen). These results suggested that mAb-7C6 specifically reacts with KHV ORF68 protein.