ABSTRACT
DNA methylation has been discovered in Drosophila only recently. Current evidence indicates that de novo methylation patterns in drosophila are maintained in a different way compared to vertebrates and plants. As the genomic role and determinants of DNA methylation are poorly understood in invertebrates, its link with several factors has been suggested. In this study, we tested for the putative link between DNA methylation patterns in Drosophila melanogaster and radiation or the activity of P transposon. Neither of the links was apparent from the results, however, we obtained some hints on a possible link between DNA methylation pattern and genomic heterogeneity of fly lineages.
Subject(s)
DNA Methylation , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Animals , DNA Transposable Elements , Genetic Heterogeneity , UkraineABSTRACT
Antibody responses to purified protein derivate PPD of tuberculin and to antigens MPB63 and MPB83 of Mycobacterium bovis were determined in bovine herd (94 adult animals). Statistical approach based on approximation by multiple Gaussians with Levenberg-Marquardt algorithm for analysis of antibody level distribution against antigens examined was provided. Our results confirm that indirect ELISA with recombinant MPB83 and MPB63 as well as conventional PPD could be used for test-systems development for detection of cow tuberculosis infection at the herd level.
Subject(s)
Antibodies, Bacterial/blood , Antigens, Bacterial/immunology , Cattle Diseases/diagnosis , Immunoenzyme Techniques/statistics & numerical data , Mycobacterium bovis/immunology , Statistical Distributions , Tuberculosis, Bovine/diagnosis , Animals , Cattle , Cattle Diseases/microbiology , Immunoenzyme Techniques/methods , Recombinant Proteins/immunology , Tuberculosis, Bovine/microbiologyABSTRACT
Diphtheria toxin's B subunit provides toxin interaction with its receptor on the cell surface and translocation of toxin's A subunit from endosome to cytozole of sensitive cells. Functional analogues of B subunit with fluorescent label are considered as perspective tools for studying the above mentioned processes. The aim of the work was to obtain fluorescent B subunit analogues and to detect the specificity of their interaction with Vero line cells. B subunit fluorescent analogues were obtained in two different ways. The first one was B subunit chemical conjugation with fluorescein isothiocyanate and the second one was genetic fusion of recombinant B subunit chain with enhanced green fluorescent protein chain. Specific interaction of B subunit fluorescent derivatives with Vero cells was studied by flow cytometry and confocal microscopy. Using competitive analysis it was shown that B subunit fluorescent analogues possessed different affinity for cells. The affinity of EGFP-SbB was higher than FITC-SbB. Our results indicate the possibility to use the fluorescent derivatives of B subunit as tools for identification of diphtheria toxin's receptor (HB-EGF) expression on the cell surface as well as for studying the interaction and penetration of diphtheria toxin to the cell.
Subject(s)
Diphtheria Toxin/metabolism , Green Fluorescent Proteins/metabolism , Intercellular Signaling Peptides and Proteins/metabolism , Recombinant Fusion Proteins/metabolism , Animals , Binding, Competitive , Cell Culture Techniques , Chlorocebus aethiops , Diphtheria Toxin/genetics , Flow Cytometry , Fluorescein-5-isothiocyanate/metabolism , Fluorescent Dyes/metabolism , Genetic Vectors , Green Fluorescent Proteins/genetics , Heparin-binding EGF-like Growth Factor , Microscopy, Confocal , Peptide Fragments/genetics , Peptide Fragments/metabolism , Plasmids , Protein Binding , Recombinant Fusion Proteins/genetics , Vero CellsABSTRACT
The aim of this work was to obtain the panel of recombinant single-chain Fv-antibodies against diphtheria toxin B subunit, the main diagnostic and pathogenic antigen of Corynebacterium diphtheriae. For this purpose we have constructed the immune library of murine immunoglobulin genes. A number of scFv specific to diphtheria toxin B subunit were acquired from the obtained library after one round of selection by phage-display. ScFv encoding DNA-fragments of eight clones were subcloned into plasmid pET-22b(+). It was shown that selected scFv were highly specific to diphtheria toxin B subunit, with affinity constant for different clones ranged from 10(7) to 10(9) M(-1).
