Isolation of Rabies Virus Glycoprotein Using Three-Phase Extraction and Characteristics of its Antigenic Properties
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 o...
| Main Authors: | , , , , , , , |
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| Format: | Article |
| Language: | Russian |
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Federal Government Health Institution, Russian Research Anti-Plague Institute “Microbe”
2022-04-01
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| Series: | Проблемы особо опасных инфекций |
| Subjects: | |
| Online Access: | https://journal.microbe.ru/jour/article/view/1661 |
| _version_ | 1797220751986655232 |
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| author | M. A. Efimova R. M. Akhmadeev A. G. Galeeva A. R. Valeeva N. R. Miftakhov M. N. Mukminov K. S. Khaertynov Е. A. Shuralev |
| author_facet | M. A. Efimova R. M. Akhmadeev A. G. Galeeva A. R. Valeeva N. R. Miftakhov M. N. Mukminov K. S. Khaertynov Е. A. Shuralev |
| author_sort | M. A. Efimova |
| collection | DOAJ |
| description | 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. |
| first_indexed | 2024-03-08T13:41:36Z |
| format | Article |
| id | doaj.art-b452824914424eb5be2ad42ef49a3546 |
| institution | Directory Open Access Journal |
| issn | 0370-1069 2658-719X |
| language | Russian |
| last_indexed | 2024-04-24T12:54:32Z |
| publishDate | 2022-04-01 |
| publisher | Federal Government Health Institution, Russian Research Anti-Plague Institute “Microbe” |
| record_format | Article |
| series | Проблемы особо опасных инфекций |
| spelling | doaj.art-b452824914424eb5be2ad42ef49a35462024-04-05T16:47:17ZrusFederal Government Health Institution, Russian Research Anti-Plague Institute “Microbe”Проблемы особо опасных инфекций0370-10692658-719X2022-04-0101869310.21055/0370-1069-2022-1-86-931355Isolation of Rabies Virus Glycoprotein Using Three-Phase Extraction and Characteristics of its Antigenic PropertiesM. A. Efimova0R. M. Akhmadeev1A. G. Galeeva2A. R. Valeeva3N. R. Miftakhov4M. N. Mukminov5K. S. Khaertynov6Е. A. Shuralev7Federal Center for Toxicological, Radiation and Biological Safety; Kazan State Academy of Veterinary Medicine named after N.E. BaumanFederal Center for Toxicological, Radiation and Biological SafetyFederal Center for Toxicological, Radiation and Biological SafetyKazan State Medical Academy – affiliated branch of the Russian Medical Academy of Continuing Professional Education of the Ministry of Health of RussiaFederal Center for Toxicological, Radiation and Biological SafetyKazan State Medical Academy – affiliated branch of the Russian Medical Academy of Continuing Professional Education of the Ministry of Health of Russia; Kazan (Volga Region) Federal UniversityFederal Center for Toxicological, Radiation and Biological Safety; Kazan State Medical Academy – affiliated branch of the Russian Medical Academy of Continuing Professional Education of the Ministry of Health of RussiaKazan State Academy of Veterinary Medicine named after N.E. Bauman; Kazan State Medical Academy – affiliated branch of the Russian Medical Academy of Continuing Professional Education of the Ministry of Health of Russia; Kazan (Volga Region) Federal UniversityThe 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.https://journal.microbe.ru/jour/article/view/1661rabies virus glycoproteinphase extractionwestern blottinggel filtration chromatography |
| spellingShingle | M. A. Efimova R. M. Akhmadeev A. G. Galeeva A. R. Valeeva N. R. Miftakhov M. N. Mukminov K. S. Khaertynov Е. A. Shuralev Isolation of Rabies Virus Glycoprotein Using Three-Phase Extraction and Characteristics of its Antigenic Properties Проблемы особо опасных инфекций rabies virus glycoprotein phase extraction western blotting gel filtration chromatography |
| title | Isolation of Rabies Virus Glycoprotein Using Three-Phase Extraction and Characteristics of its Antigenic Properties |
| title_full | Isolation of Rabies Virus Glycoprotein Using Three-Phase Extraction and Characteristics of its Antigenic Properties |
| title_fullStr | Isolation of Rabies Virus Glycoprotein Using Three-Phase Extraction and Characteristics of its Antigenic Properties |
| title_full_unstemmed | Isolation of Rabies Virus Glycoprotein Using Three-Phase Extraction and Characteristics of its Antigenic Properties |
| title_short | Isolation of Rabies Virus Glycoprotein Using Three-Phase Extraction and Characteristics of its Antigenic Properties |
| title_sort | isolation of rabies virus glycoprotein using three phase extraction and characteristics of its antigenic properties |
| topic | rabies virus glycoprotein phase extraction western blotting gel filtration chromatography |
| url | https://journal.microbe.ru/jour/article/view/1661 |
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