Разработка рекомбинантных вакцин-кандидатов против весенней виремии карпа и исследование их иммуногенных и протективных свойств
Диссертация
Для повышения защитных свойств кандидатных ДНК-вакцин была проведена серия экспериментов, задачами которых являлись: подбор оптимальной дозы плазмидной ДНК, изменение температурного режима содержания рыбы в ходе исследования, оценка влияния неспецифических иммуностимуляторов (диэтиламиноэтилдекстрана, полиглюкина и ридостина) на эффективность созданных конструкций. В результате было выявлено, что… Читать ещё >
Содержание
- Список принятых сокращений
- Научная новизна и практическая ценность работы
- Публикации и апробация работы
- Глава 1. ОБЗОР ЛИТЕРАТУРЫ
- I. Весенняя виремия карпа
- 1. Историческая справка
- 2. Предрасполагающие факторы, группы риска
- 3. Клиническая картина
- 4. Возбудитель заболевания — вирус весенней виремии карпа
- 4. 1. Структура вируса
- 4. 2. Гликопротеин (G-белок) рабдовирусов
- 5. Существующие подходы к профилактике и лечению ВВК
- II. Вакцинология в аквакультуре
- 1. Традиционные инактивированные и атгенуированные вакцины для рыб
- 2. Рекомбинантные технологии в аквакультуре
- 2. 1. Субъединичные вакцины для рыб
- 2. 2. ДНК-вакцины 25 2.2.1 .Опыт применения ДНК-вакцин для рыб
- 2. 2. 2. Возможные системы доставки вакцины в организм рыбы
- 2. 2. 3. Исследование тканевого распределения плазмидной ДНК и продолжительности экспрессии рекомбинантног гена в организме рыб
- 2. 2. 4. Изучение механизмов представления АГ клеткам ИС после введения ДНК-вакцин
- 2. 2. 5. Иммунный ответ
- 2. 2. 6. Исследование безопасности ДНК-вакцин
- Глава 2. МАТЕРИАЛЫ И МЕТОДЫ
- 2. 1. Реактивы
- 2. 2. Ферменты 47 2.3.0лигонулеотиды
- 2. 4. Плазмиды
- 2. 5. Бактерии
- 2. 6. Культуральные среды
- 2. 7. Вирусы и клетки 48 2.9. Растворы 49 2.9. Методы
- 2. 9. 1. Выделение вирусной РНК
- 2. 9. 2. Синтез ДНК, комплементарной вирусной РНК
- 2. 9. 3. Амплификация фрагментов ДНК
- 2. 9. 4. Элюция фрагментов ДНК
- 2. 9. 5. Коструирование рекомбинантных ДНК
- 2. 9. 6. Трансформация клеток Е. Coli плазмидной ДНК
- 2. 9. 7. Выделение плазмидной ДЕК
- 2. 9. 8. Ферментативный гидролиз ДНК
- 2. 9. 9. Электрофоретическое фракционирование ДНК
- 2. 9. 10. Анализ первичной структуры ДНК 53 2.9.11.Электрофоретический анализ белка
- 2. 9. 12. Получение иммунной сыворотки
- 2. 9. 13. Иммуноферментный анализ
- 2. 9. 14. Иммуноблотинг белков с иммунными сыворотками
- 2. 9. 15. Приготовление препарата субъединичной вакцины для иммунизации 55 2.9.16.Оценка протективности экспериментальных вакцин 56 2.9.17.Оценка иммуногенности экспериментальных вакцин
- 3. 1. 1. Рекомбинантная конструкция pcDNA-G-a
- 3. 1. 2. Конструирование рекомбинантной плазмидной ДНК pQE-G для экспрессии гена гликопротеина вируса в E. coli — субъединичная вакцина
- 3. 1. 3. Исследование продукции рекомбинантного вирусного гликопротеина в клетках Е. col
- 3. 1. 4. Иммуногенность экспериментальных вакцин
- 3. 1. 5. Защитные свойства экспериментальных вакцин
- 3. 1. 5. 1. Эффективность экспериментальной ДНК-вакцины
- 3. 1. 5. 2. Эффективность субъединичной вакцины
- 3. 1. 6. Влияние изменения дозы вводимой плазмидной конструкции и неспецифических иммуностимуляторов на эффективность ДНК-вакцинации
- 3. 2. Генно-инженерные плазмидные ДНК — кандидатные вакцины, кодирующие гликопротеин референсного для России изолята вируса весенней виремии карпа
- 3. 2. 1. Конструирование рекомбинантной плазмидной ДНК pcDNA-G-3JI4 — кандидатной ДНК-вакцины
- 3. 2. 2. Конструирование рекомбинантной плазмидной ДНК pBACarpVax-G-3JI4 — кандидатная ДНК-вакцина
- 3. 2. 3. Продукция рекомбинантного вирусного гликопротеина в культуре клеток
- 3. 2. 4. Защитные свойства вакцин-кандидатов, кодирующих гликопротеин референсного для России изолята вируса весенней виремии карпа
- 3. 3. Генно-инженерные плазмидные ДНК — кандидатные вакцины, кодирующие нуклеопротеин россйского изолята вируса весенней виремии карпа
- 3. 4. Влияние повышенной температуры воды и ридостина на эффективность ДНК-вакцинации
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