ABSTRACT
Diphtheria toxin is the main pathogenicity factor of causative agent of diphtheria Corynebacterium diphtheriae. Due to the small molecule size, it is of considerable interestfor the development of synthetic protein molecules with transporting function, e.g. immunotoxins. Expression and characterization of nontoxic recombinant fluorescent derivates of diphtheria toxin and its nontoxic mutant CRM 197 were described in this article. Obtained proteins may be applied in studies of receptor-binding and transporting functions of the toxin in cells, for determination of toxin receptor proHB-EGF expression level, immunization and antibody generation against the toxin and in development of diagnostic test-systems, detection of diphtheria toxin and antitoxic antibodies.
Subject(s)
Diphtheria Toxin/genetics , Luminescent Proteins/genetics , Protein Engineering , Recombinant Proteins/genetics , Antibodies, Bacterial/chemistry , Antibodies, Bacterial/immunology , Cloning, Molecular , Corynebacterium diphtheriae/genetics , Corynebacterium diphtheriae/pathogenicity , Diphtheria Toxin/chemistry , Humans , Immunization , Luminescent Proteins/biosynthesis , Luminescent Proteins/chemistry , Models, Molecular , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistryABSTRACT
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
The recombinant fluorescent derivative of diphtheria toxin (EGFP-SbB) obtained by the replacement of toxin A subunit by enhanced green fluorescent protein (EGFP) has been used for visualization of the interaction of diphtheria toxin (DT) with sensitive and insensitive cells. It was shown that EGFP-SbB could interact with cell surface of both toxin-sensitive monkey cells (Vero cell line) and toxin-resistant mouse cells (3T3 cell line). The affinity of this protein for receptors of Vero cells was three times higher as compared with 3T3 cells. It was demonstrated that fluorescent derivate was able to interact with receptors of both cell lines and to internalize into these cells. Internalization of EGFP-SbB into the cells was inhibited by endocytosis inhibitor phenyl arsine oxide. We suppose that diverse sensitivity to DT of monkey and mouse cells can be explained not only by differences in their receptor affinity for DT but also by the processes that occur after internalization of the toxin into the cells.
