Создание новой эукариотической системы экспрессии бета-интерферона человека на основе рекомбинантного аденовируса птиц
Диссертация
Апробация работы. Результаты диссертационной работы были доложены и обсуждены на IV международной научной конференции студентов и молодых учёных «Живые системы и биологическая безопасность населения» (Москва, 2005), международной юбилейной научно-практической конференции, посвящённой 110-летию Курской биофабрики и агробиологической промышленности России (Курск, 2006), V международной научной… Читать ещё >
Содержание
- ПРИНЯТЫЕ СОКРАЩЕНИЯ
- 1. ОБЗОР ЛИТЕРАТУРЫ
- 1. 1. Вирусные системы доставки генов в клетки эукариот
- 1. 2. Общая характеристика аденовирусов и их использование в качестве векторов для переноса генетической информации
- 1. 2. 1. Общая характеристика и классификация аденовирусов
- 1. 2. 2. Аденовирусы как векторы для переноса генетической информации
- 1. 2. 2. 1. Векторы на основе аденовирусов человека
- 1. 2. 2. 2. Векторы на основе аденовирусов животных и птиц
- 1. 2. 3. Аденовирус птиц CELO, его особенности, использование в качестве вектора
- 1. 3. Общая характеристика бета-интерферона, механизмы действия, свойства и технология получения
- 1. 3. 1. Бета-интерферон и его функции
- 1. 3. 2. Производство бета-интерферона
- 2. 1. 1. Вирусы и бактериальные штаммы
- 2. 1. 2. Клеточные линии
- 2. 1. 3. Плазмидные векторы
- 2. 1. 4. Ферменты и другие реактивы
- 2. 1. 5. Лабораторные животные
- 2. 1. 6. Лабораторное оборудование 43 2.2. МЕТОДЫ
- 2. 2. 1. Выделение аналитических количеств плазмидной ДНК
- 2. 2. 2. Выделение препаративных количеств плазмидной
- 2. 2. 3. Гидролиз ДНК специфическими эндодезоксирибонуклеазами рестрикции
- 2. 2. 4. Обработка Фрагментом Кленова
- 2. 2. 5. Лигирование фрагментов ДНК
- 2. 2. 6. Трансформация клеток Е. соП
- 2. 2. 7. Приготовление компетентных клеток
- 2. 2. 8. Приготовление электрокомпетентных клеток Е. соН
- 2. 2. 9. Гомологичная рекомбинация в Е. соИ
- 2. 2. 10. Получение рекомбинантных аденовирусов
- 2. 2. 11. Накопление аденовирусов
- 2. 2. 12. Очистка и концентрирование аденовирусов
- 2. 2. 13. Титрование вирусов
- 2. 2. 14. Выделение ДНК из клеток, зараженных аденовирусом
- 2. 2. 15. Выделение вирионной ДНК
- 2. 2. 16. Окраска клеток метиленовым синим
- 2. 2. 17. Определение биологической активности бета-интерферона человека
- 2. 2. 18. Электрофорез белков в ПААГ-ДСН
- 2. 2. 19. Иммуноблотинг
- 2. 2. 20. Дегликозилирование рекомбинантного белка бета-интерферона человека
- 2. 2. 21. Количественное определение продукции белка бета-интерферона человека методом сэндвич-ИФА
- 2. 2. 22. Выделение бета-интерферона человека на №-ЫТА-агарозе
- 2. 2. 23. Выделение бета-интерферона человека на голубой сефарозе
- 3. 1. Исследование токсичности бета-интерферона человека для культуры клеток птиц
- 3. 2. Получение рекомбинантных аденовирусов птиц СЕЬО-ИФН-Р и СЕШ-ИФН-РЫб
- 3. 2. 1. Создание челночных плазмидных конструкций, содержащих исходный и модифицированный ген бета-интерферона человека в составе экспрессирующей кассеты под контролем CMV-промотора
- 3. 2. 2. Получение плазмиды, несущей геном рекомбинантного аденовируса СЕЬО-ИФН-? методом гомологичной рекомбинации в Е. col
- 3. 2. 3. Получение аденовируса СЕЬО-ИФН-? и CELO-ИФН
- 3. 3. Продукция рекомбинантного бета-интерферона человека in vitro
- 3. 3. 1. Экспрессия гена бета-интерферона входящего в состав генома аденовирусов СЕШ-ИФН-? и СЕЬО-ИФН-?his и накопление рекомбинантного белка бета-интерферона в культуре клеток LMH
- 3. 3. 2. Изучение динамики накопления и биологической активности рекомбинантного бета-интерферона человека продуцируемого в культуре клеток птиц
- 3. 4. Выделение рекомбинантного белка бета-интерферона из культуральной жидкости методом аффинной хроматографии
- 3. 4. 1. Выделение рекомбинантного белка бета-интерферона из культуральной жидкости на Ni-NTA-агарозе
- 3. 4. 2. Выделение рекомбинантного белка бета-интерферона из культуральной жидкости на голубой сефарозе
- 3. 5. Анализ физико-химических и антигенных свойств бета-интерферона человека, продуцируемого в клетках птиц
- 3. 6. Продукция рекомбинантного белка бета-интерферона человека in ovo
- 3. 6. 1. Продукция и накопление рекомбинантного белка бета-интерферона человека в куриных эмбрионах, инфицированных рекомбинантным аденовирусом CELO-ИФН-Р
- 3. 6. 2. Изучение биологической активности бета-интерферона продуцируемого в аллантоисной жидкости куриных эмбрионов
- 3. 7. Исследование возможности использования рекомбинантного аденовируса CELO-ИФН-р для генной терапии in vivo
SPF — specific pathogen free — свободный от вирусных и бактериальных контаминаций трис — гидроксиметиламинометан ЭДТА — этилендиаминтетраацетат CMV — цитомегаловирус человека ORF — открытая рамка считывания polyA — сигнал полиаденилирования ИФН-Р — бета-интерферон
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