Russian Journal of Biological Physics and Chemisrty Russian Journal of Biological Physics and Chemisrty АКТУАЛЬНЫЕ ВОПРОСЫ БИОЛОГИЧЕСКОЙ ФИЗИКИ И ХИМИИ 2499-9962 54084 БИООРГАНИЧЕСКАЯ, БИОФИЗИЧЕСКАЯ И МЕДИЦИНСКАЯ ХИМИЯ BIOORGANIC, BIOPHYSICAL AND MEDICINAL CHEMISTRY БИООРГАНИЧЕСКАЯ, БИОФИЗИЧЕСКАЯ И МЕДИЦИНСКАЯ ХИМИЯ SPECIFIC BIOLUMINESCENT DETECTION OF MICROORGANISMS USING STREPTAVIDIN-LUCIFERASE FUSION PROTEIN БИОЛЮМИНЕСЦЕНТАЯ СПЕЦИФИЧЕСКАЯ ДЕТЕКЦИЯ МИКРООРГАНИЗМОВ С ИСПОЛЬЗОВАНИЕМ ГИБРИДНОГО БЕЛКА СТРЕПТАВИДИН-ЛЮЦИФЕРАЗА Смирнова Д В Smirnova D V S_mir_nova@mail.ru Рубцова М Ю Rubtsova M Yu Угарова Н Н Ugarova N N Московский государственный университет им. М.В. Ломоносова ru Lomonosov Moscow State University ru Московский государственный университет им. М.В. Ломоносова ru Lomonosov Moscow State University ru Московский государственный университет им. М.В. Ломоносова ru Lomonosov Moscow State University ru 25 12 2016 25 12 2016 1 2 137 141 20 12 2016 20 12 2016 https://rusjbpc.ru/en/nauka/article/54084/view

Разработаны аналитические системы для детекции микроорганизмов с использованием гибридного белка люциферазы Luciola mingrelica со стрептавидином (His6-SA-Luc). Разработан гетерогенный иммуноанализ клеток Salmonella с использованием моноклональных биотинилированных антител, позволяющий определять концентрацию клеток в диапазоне 104-5×106 КОЕ/мл. Оптимизирован метод гибридизационного анализа специфических фрагментов ДНК клеток E. coli . с пределом обнаружения ДНК 10-10 М. Показано, что гибридный белок His6-SA-Luc является высокочувствительным реагентом при специфической детекции клеток микроорганизмов на основе биотин-стрептавидиновых взаимодействий как с использованием иммуноанализа, так и с использованием гибридизационного анализа специфических фрагментов ДНК клеток микроорганизмов.

The analytical systems for specific microorganism detection were developed using streptavidin- luciferase Luciola mingrelica fusion protein (His6-SA-Luc). Heterogeneous immunoassay is proposed for detection of Salmonella cells using monoclonal biotinylated antibodies in range 104-5×106 CFU/ml. We optimized hybridization analysis of a specific DNA fragment from E. coli. The detection limit for DNA was 10-10 M. We showed that His6-SA-Luc fusion protein is a highly sensitive and specific reagent for microorganism detection based on biotin- streptavidin interaction using or ELISA, or hybridization analysis of a specific DNA fragment from microbial cells.

 биолюминесценция ИФА стрептавидин-люцифераза гибрид Luciola mingrelica гибридизационный анализ Salmonella bioluminescence ELISA streptavidin-luciferase fusion Luciola mingrelica hybridization analysis Salmonella

Koksharov M.I., Ugarova N.N. Approaches to engineer stability of beetle luciferases. Computational and structural biotechnology journal, 2012, vol. 2, pp. 1-7. Koksharov M.I., Ugarova N.N. Approaches to engineer stability of beetle luciferases. Computational and structural biotechnology journal, 2012, vol. 2, pp. 1-7. Smirnova D.V., Koksharov M.I., Zorov I.N., Ugarova N.N. Luciferase-streptavidin fusion proteins: Preparation and properties. Moscow Univ. Chem. Bull., 2014, vol. 69, pp. 56-61. Smirnova D.V., Koksharov M.I., Zorov I.N., Ugarova N.N. Luciferase-streptavidin fusion proteins: Preparation and properties. Moscow Univ. Chem. Bull., 2014, vol. 69, pp. 56-61. Koksharov M.I., Ugarova N.N. Thermostabilization of firefly luciferase by in vivo directed evolution. Protein Eng. Des. Sel, 2011, vol. 24, pp. 835-844. Koksharov M.I., Ugarova N.N. Thermostabilization of firefly luciferase by in vivo directed evolution. Protein Eng. Des. Sel, 2011, vol. 24, pp. 835-844. Koksharov M.I., Ugarova N.N. Triple substitution G216N/A217L/S398M leads to the active and thermostable Luciola mingrelicafirefly luciferase. Photochem. Photobiol. Sci., 2011, vol. 10, pp. 931-938. Koksharov M.I., Ugarova N.N. Triple substitution G216N/A217L/S398M leads to the active and thermostable Luciola mingrelicafirefly luciferase. Photochem. Photobiol. Sci., 2011, vol. 10, pp. 931-938. Quiñones B., Swimley M.S., Narm K.-E., Patel R.N., Cooley M.B., Mandrell R.E. O-antigen and virulence profiling of Shiga toxin-producing Escherichia coli by a rapid and cost-effective DNA microarray colorimetric method. Front. Cell. Infect. Microbiol., 2012, vol. 2, pp.1-10. Quiñones B., Swimley M.S., Narm K.-E., Patel R.N., Cooley M.B., Mandrell R.E. O-antigen and virulence profiling of Shiga toxin-producing Escherichia coli by a rapid and cost-effective DNA microarray colorimetric method. Front. Cell. Infect. Microbiol., 2012, vol. 2, pp.1-10. Koksharov M.I., Smirnova D.V., Abbasova S.G., Ugarova N.N. A fusion protein of Luciola mingrelica luciferase with a biotin-binding domain: Production, properties, and application. Moscow Univ. Chem. Bull., 2011, vol. 66, pp. 241-246. Koksharov M.I., Smirnova D.V., Abbasova S.G., Ugarova N.N. A fusion protein of Luciola mingrelica luciferase with a biotin-binding domain: Production, properties, and application. Moscow Univ. Chem. Bull., 2011, vol. 66, pp. 241-246. Smith D.K., Kassam T., Singh B., Elliott J.F. Escherichia coli has two homologous glutamate decarboxylase genes that map to distinct loci. J. Bacteriol., 1992, vol. 174, pp. 5820-5826. Smith D.K., Kassam T., Singh B., Elliott J.F. Escherichia coli has two homologous glutamate decarboxylase genes that map to distinct loci. J. Bacteriol., 1992, vol. 174, pp. 5820-5826. Li W., Li J. High speed quantitative analysis of DNA methylation, by immobilization of DNA on the plastic carrier coated with polylysine, then incubating at a two-phase temperature and immunodetection of 5-methylcytosine structures as markers of DNA methylation, 2010, US 778579382. Li W., Li J. High speed quantitative analysis of DNA methylation, by immobilization of DNA on the plastic carrier coated with polylysine, then incubating at a two-phase temperature and immunodetection of 5-methylcytosine structures as markers of DNA methylation, 2010, US 778579382. Lomakina G.Y., Istrate A., Rudenko N.V., Ugarova N.N. Synthesis and application of firefly luciferase antibody conjugates in a bioluminescent immunoassay of Salmonella cells. Moscow Univ. Chem. Bull., 2014, vol. 69, pp. 49-55. Lomakina G.Y., Istrate A., Rudenko N.V., Ugarova N.N. Synthesis and application of firefly luciferase antibody conjugates in a bioluminescent immunoassay of Salmonella cells. Moscow Univ. Chem. Bull., 2014, vol. 69, pp. 49-55.