Isolation of Rabies Virus Glycoprotein Using Three-Phase Extraction and Characteristics of its Antigenic Properties

Abstract

The aim of the work was to develop an approach to isolation of rabies virus glycoprotein applying threephase extraction and to characterize its antigenic properties.Materials and methods. Infectious activity of the rabies virus (production strain, “Ovine” GNKI) after long-term storage was restored on white BALB/c mice. The strain was used for cultivation on BHK-21 cells; the culture liquid was concentrated applying ultracentrifugation followed by separation by buoyant density in a sucrose gradient, selection of visually opalescent zones, phase concentration, chromatographic separation on an ENrich™ SEC650 column (Bio-Rad, USA) and selection of monomeric fractions with high serological activity according to the results of Western blotting.Results and discussion. We have demonstrated that preliminary mechanical destruction of brain suspension, extraction of the virus-containing material from the cell suspension through successive low-speed and high-speed centrifugation, separation of the sediment produced in sucrose gradient with further phase concentration and chromatographic separation of the precipitate allows to obtain monomeric antigenic preparations with high serological activity. This methodology has made it possible to obtain an antigen, which is rabies virus glycoprotein with a molecular weight of 67 kDa, and two of its isoforms, having molecular weights of 60 and 54 kDa. The described approach can be viewed as an option for isolation of the rabies virus specific antigen when improving laboratory diagnostics techniques. The resulting antigen is a monomeric discrete containing one fraction with a molecu lar weight of 67 kDa. The data obtained corroborate the high specificity of the antigen and its suitability for the design of enzyme immunoassay and immunochromatographic tests, production of specific immunoglobulins, the study of the antigen/antibody interaction, as well as for the assessment of the protective immunity intensity after vaccination.

Keywords

• rabies virus glycoprotein
• phase extraction
• western blotting
• gel filtration chromatography