Comparative molecular-genetic analysis of mobile elements in natural strains of cholera agent

Smirnova, N. I.; Zadnova, S. P.; Agafonov, D. A.; Shashkova, Anna; Cheldyshova, N. B.; Cherkasov, Alexander

doi:10.1134/s1022795413090081

Cited by 9 publications

(6 citation statements)

References 18 publications

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“…компьютерный анализ нуклеотидных и амино-кислотных последовательностей и конструирование праймеров для амплификации клонируемого фраг-мента днк осуществляли с помощью пакета про-грамм Vector NTI Suit 9 (Invitrogen, сШа). полимеразную цепную реакцию (пцр) прово-дили как описано ранее [3]. продукты визуализи-ровали под уФ-светом и фотографировали на гель-документирующей системе VersaDoc фирмы (BioRad, сШа) с использованием программы Quantity One v.4.6.9 (Bio-Rad, сШа).…”

Section: Cirsunclassified

“…основной субъединицей тср является белок TcpA, кодируемый геном tcpA [2]. структурные гены ст и тср лока-лизованы в геноме возбудителя на двух мобильных элементах -профаге стхφ и острове патогенности VPI-1 (Vibrio pathogenicity island) [3].…”

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Construction of Recombinant Escherichia coli Strain – Producer of Basic Subunit of Toxin-Coregulated Pilus of Adhesion (TCPA) of Vibrio cholerae Biovar El Tor

Владимировна¹,

Витальевич²,

Алексеевна³

et al. 2017

Problemy Osobo Opasnykh Infektsii

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CIRS определяли в пцр, используя рассчитанные нами праймеры. Фрагменты днк выделяли из агарозного геля с помощью набора PCR Clear-Up-System. SDS-PAGE проводили по методу U.K.Laemmli. концентрацию белка в пробах измеряли методом M.M.Bradford. для очистки рекомбинантного белка TcpA применяли набор для аффиной хроматографии. результаты и выводы. сконструирован безопасный штамм E. coli -продуцент рекомбинантного белка TcpA -основной субъединицы токсин-коррегулируемых пилей адгезии возбудителя холеры эль тор. участок гена tcpA V. cholerae биовара эль тор был клонирован в составе векторной плазмиды pET302 по сайтам рестрикции XhoI-BamHI в штамм E. coli BL21(DE3)Star. в этой конструкции биосинтез протеина находится под транскрипционным контролем промотора фага т7 и индуцируется с помощью изопропил-β-тиогалактопиранозида (иптг). отработаны условия оптималь-ной продукции белка TcpA и схема его очистки с помощью аффинной хроматографии. показано, что TcpA при-сутствует в клетках кишечной палочки как в нативной форме, так и в виде телец включения. общая продукция белка TcpA составляет 60 мкг/мл. полученный очищенный белок TcpA может быть использован для изучения его иммуногенных и физико-химических свойств, а также для разработки иммунодиагностических препаратов с целью оценки уровня продукции TcpA у различных штаммов V. cholerae и определения антигенного состава холерных вакцинных препаратов.Ключевые слова: холерный вибрион, белок TcpA, клонирование генов, рекомбинантная плазмида, штамм-продуцент, аффинная хроматография.Корреспондирующий автор: Клавдиенко Елена Владимировна , e-mail: rusrapi@microbe.ru. E.V.Klavdienko, I.V.Tuchkov, T.A.Polunina, N.P.Guseva, N.I.Smirnova Construction of Recombinant escherichia coli Strain -Producer of Basic Subunit of Toxin-coregulated Pilus of Adhesion (TcPA) of Vibrio cholerae Biovar el tor Russian Research Anti-Plague Institute "Microbe", Saratov, Russian FederationObjective of the study is to construct recombinant E. coli strain -producer of TcpA protein of cholera agent El Tor, carrying tcpA CIRS gene in its genome, and use the strains for antigen production. Materials and methods. Utilized was non-toxigenic genovariant strain of Vibrio cholerae biovar El Tor from the "State Collection of Pathogenic Bacteria" at the premises of RusRAPI "Microbe", as well as commercial E. coli strains and plasmids for cloning (Invitrogen, USA). Chromosomal DNA from V. cholerae cells was extracted using Charge Smitch gDNA Mini Bacteria Kit applying nucleo-sorption. To extract plasmid DNA from E. coli cells PureLink Quick Plasmid DNA MiniprepKits were used. The presence of tcpA CIRS gene was assayed by PCR, using designed through our own efforts primers. DNA fragments were isolated from agarose gel with the help of PCR Clear-Up-System panel. SDS-PAGE was performed according to U.K.Laemmli method. Protein content of samples was measured by M.M.Bradford method. The panel for affinity chromatography was applied for recombinant TcpA protein purification. Results and conclusions. Constructed safe strain of E. ...

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Section: Cirsunclassified

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Construction of Recombinant Escherichia coli Strain – Producer of Basic Subunit of Toxin-Coregulated Pilus of Adhesion (TCPA) of Vibrio cholerae Biovar El Tor

Владимировна¹,

Витальевич²,

Алексеевна³

et al. 2017

Problemy Osobo Opasnykh Infektsii

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“…The other 30 strains, including one from seawater, have been imported into Russia and Ukraine from different countries endemic for cholera over the last two decades (1991-2012) and belonged to the above mentioned genovariants containing a DNA fragment of the classical cholera vibrio biovar in their genome. The totality of our data show that the repre sented strains of the genovariants differed from one another in the structure of their genome [19,21]. The ctxB1 allele was present in the genome of 90% of the studied genovariants.…”

Section: Resultsmentioning

confidence: 87%

“…Among them, 20 clinical strains were isolated in the initial period of the pandemic (1967-1990) on the territories of India and Russia and carried the ctxB3 allele in the CTXϕ Eltor prophage and the intact VSP II [19]. The other 30 strains, including one from seawater, have been imported into Russia and Ukraine from different countries endemic for cholera over the last two decades (1991-2012) and belonged to the above mentioned genovariants containing a DNA fragment of the classical cholera vibrio biovar in their genome.…”

Section: Resultsmentioning

confidence: 99%

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MLVA typing of clinical Vibrio cholerae strains isolated during different periods of the current cholera pandemic

Smirnova

Кульшань

Krasnov

2015

Mol. Genet. Microbiol. Virol.

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MLVA typing was performed for five variable loci of the 52 Vibrio cholerae El Tor biovar strains iso lated before the onset and in different periods of the seventh cholera pandemic, as well as for eight strains of the classical V. cholerae biovar-the causative agent of the preceding Asiatic cholera pandemics. It has been shown that the studied strains, which differ in their molecular genetic properties and are characterized by 38 MLVA types, form seven clonal clusters. Clusters I and II were formed, respectively, by V. cholerae strains of the classical biovar and prepandemic V. cholerae strains, whereas clusters III-VII were formed by V. cholerae El Tor biovar strains isolated in different periods of the latest pandemic. It has been shown that the MLVA typing allows us to clearly differentiate V. cholerae El Tor biovar strains into typical and genetically altered strains. The genetically altered strains are characterized by higher virulence compared with the typical strains, as well as the presence of the ctxB gene of the classical cholera vibrio in the genome. The study showed the possibility of differentiating genovariants isolated in different periods and having different structures of pandemicity island VSP II. Since the VSP II structure directly correlates with the epidemic potential of the strains, the possibility of differentiating isolates with intact and deleted VSP II using the MLVA method deserves further research. Based on the obtained results, we can propose a polyclonal origin of different strains of genovariants.

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Whole-Genome Sequencing of Vibrio cholerae O1 El Tor Strains Isolated in Ukraine (2011) and Russia (2014)

et al. 2017

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Comparative molecular-genetic analysis of mobile elements in natural strains of cholera agent

Cited by 9 publications

References 18 publications

Construction of Recombinant Escherichia coli Strain – Producer of Basic Subunit of Toxin-Coregulated Pilus of Adhesion (TCPA) of Vibrio cholerae Biovar El Tor

Construction of Recombinant Escherichia coli Strain – Producer of Basic Subunit of Toxin-Coregulated Pilus of Adhesion (TCPA) of Vibrio cholerae Biovar El Tor

MLVA typing of clinical Vibrio cholerae strains isolated during different periods of the current cholera pandemic

Whole-Genome Sequencing of Vibrio cholerae O1 El Tor Strains Isolated in Ukraine (2011) and Russia (2014)

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