ИСТИНА

Many viral and cellular proteins are difficult of access by isolation procedure and therefore it is difficult to obtain antibodies necessary for their immunochemical detection in situ. Modern genetic engineering allows the creation of nucleic acid vectors that can express the majority of proteins in bacteria. These proteins are generally obtained in sufficient quantities for immunization of laboratory animals. However, in bacteria cloned (often recombinant) viral proteins are often in the form of protein aggregates - "inclusion bodies (IB)". To dissolve these IBs one has to use protein denaturing agents. Subsequent renaturation of the recombinant protein generates a stochastic set of molecules with a different spatial structure having epitopes of the native molecule, its intermediate thermodynamic state, and a fully denatured molecule. The three-dimensional structure of the native and denatured coat protein in aqueous solution differs from the viral particle structure. Due to this obstacle, the antibodies to isolated viral proteins recognize poorly (or not) the native viruses. To unify the antigen detection and diagnostics of the infection with the hard to rich viruses, the method is proposed where protein antigen multiplied by the DNA cloning and having complex spatial structure is completely and irreversibly denatured. This treatment transforms three-dimensional protein epitopes in the linear ones. Then antibodies are raised to the irreversibly denatured antigen with linear epitopes. Before detection of the target viral or rare hard to rich cellular antigen, the specimen is irreversibly denatured under the same conditions for the denaturation of the antigen. The proposed strategy was examined with the difficult to access the beet yellows virus and showed good results as illustrated on the slides. It should mention here that the irreversible denaturation of the protein makes its antigenic power weaker. It means that thus prepared antigens may be used as vaccines for animals and humans.