Изучение экспрессии репликативных белков клостеровирусов in vitro и in vivo и разработка метода детекции вируса деградации плодов черешни
Диссертация
С другой стороны, изучение молекулярной биологии клостеровирусов, которые являются опасными патогенами сельскохозяйственных культур, имеет важное практическое значение, открывая возможности для разработки методов борьбы с вирусными инфекциями и создания современных методов их диагностики. Показано, что полимеразный домен ВЖС может экспрессироваться в составе слитного la/lb белка в бесклеточной… Читать ещё >
Содержание
- СПИСОК СОКРАЩЕНИЙ
- 1. ВВЕДЕНИЕ
- 2. ОБЗОР ЛИТЕРАТУРЫ
- 2. 1. Репликативные белки (+)РНК-содержащих вирусов и их функции
- 2. 1. 1. РНК-зависимые РНК полимеразы
- 2. 1. 2. РНК хеликазы
- 2. 1. 3. Ферменты кепирования и метилирования
- 2. 1. 4. Репликативные комплексы (+)РНК содержащих вирусов и их локализация
- 2. 2. Экспрессия репликативных белков / (+)РНК-содержащих вирусов
- 2. 2. 1. Сегментация вирусных геномов
- 2. 2. 2. Рибосомальный сдвиг рамки считывания
- 2. 2. 3. 1. Сдвиг рамки в направлении (-1)
- 2. 2. 3. 2. Сдвиг рамки в направлении (+1)
- 2. 2. 3. Супрессия терминаторно^о кодона
- 2. 2. 4. Посттрансляционный процессинг полипротеина-предшественника
- 2. 2. 5. 1. Пикорна-подобные вирусы
- 2. 2. 5. 2. Семейство флавивирусов
- 2. 2. 5. 3. Корона-подобные вирусы
- 2. 2. 5. 4. Синбис-подобные вирусы растений
- 2. 1. Репликативные белки (+)РНК-содержащих вирусов и их функции
- 2. 3. Семейство (+)РНК содержащих клостеровирусов
- 2. 3. 1. Общие сведения: типичные представители, особенности строения
- 2. 3. 2. Экология и диагностика клостеровирусных инфекций
- 2. 3. 3. Геномная организация представителей семейства клостеровирусов
- 2. 3. 4. Комбинация экспрессионных стратегий, используемых в процессе клостеровирусной инфекции
- 3. 1. Ферменты и химические соединения
- 3. 2. Вирусные изоляты и кДНК клоны
- 3. 3. Векторы для клонирования и бактериальные штаммы
- 3. 4. Олигонуклеотиды
- 3. 5. Общие молекулярно-биологические методы
- 3. 5. 1. Рестрикция
- 3. 5. 2. Электрофорез в агарозном геле и выделение ДНК
- 3. 5. 3. Лигирование ДНК
- 3. 5. 4. Трансформация бактериальных клеток плазмидной ДНК
- 3. 5. 5. Выделение плазмидной ДНК
- 3. 5. 6. Амплифицйрование фрагментов ДНК с помощью полимеразной цепной реакции (ПЦР)
- 3. 5. 7. Клонирование ПЦР фрагментов
- 3. 5. 8. Сплавление одноцепочечных праймеров и клонирование короткого фрагмента дцДНК
- 3. 5. 9. Секвенирование ДНК
- 3. 6. Пепскан-анализ моноклональных антител к хеликазному (ХЕЛ) и метилтрансферазному (МТ) доменам вируса желтухи свеклы (ВЖС)
- 3. 7. Метод иммунного мечения золотом (ИМЗ) тонких срезов растений
- 3. 8. Сайт-направленный мутагенез по методу Кюнкеля
- 3. 9. Транскрипция in vitro фреймшифтинг конструкций ВЖС и трансляция в экстрактах зародышей пшеницы
- 3. 10. Тестирование конструкций, содержащих ген бета-глюкуронидазы, в растениях
- 3. 11. Метод детекции вируса деградации плодов черешни (ВДПЧ) в растениях
- 4. 1. Изучение внутриклеточной локализации продуктов экспрессии репликативного гена 1а ВЖС в зараженных клетках растений
- 4. 1. 1. Картирование эпитопов для моноклональных антител в ХЕЛ и МТ доменах ВЖС
- 4. 1. 2. Анализ тонких срезов тканей растений, зараженных ВЖС, с помощью иммуноэлектронной микроскопии с моноклона л ьными антителами к МТ и ХЕЛ доменам
- 4. 2. Тестирование предполагаемого (+1) рибосомального сдвига рамки считывания при экспрессии OPTlb ВЖС в системах in vitro и in vivo
- 4. 2. 1. Получение экспрессирующих конструкций, содержащих вероятный сигнал сдвига рамки считывания ВЖС, и трансляция in vitro
- 4. 2. 2. Тестирование вероятного сигнала (+1) фреймшифтинга у ВЖС и ВДПЧ в системе in vivo с использованием инфекционной копии Xj^BK
- 4. 3. Отработка метода ревертазной ПЦР для детекции ВДПЧ и характеристика некоторых изолятов, распространенных в Европе
- 5. 1. Внутриклеточная локализация репликативных белков ВЖС и анализ состояния эпитопов для моноклональных антител в ХЕЛ и МТ белках
- 5. 2. Тестирование вероятного сигнала сдвига рамки считывания в системах in vitro и in vivo
- 5. 3. Новый метод диагностики ВДПЧ и его использование для различных вирусных изолятов
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