Идентификация иммуноактивных участков белка Е вируса клещевого энцефалита штамма Софьин с помощью синтетических пептидов тема автореферата и диссертации по химии, 02.00.10 ВАК РФ

ВВЕДЕНИЕ.

ГЛАВА I. ОБЗОР ЛИТЕРАТУРЫ.

Иммунобиология вирусов семейства Flaviviridae.

1. Изучение антигенной структуры флавивирусов

1.1 исследование с помощью моноклональных антител.

1.2 гипотетические модели пространственной структуры гликопротеина Е.

1.3 рентгеноструктурный анализ белка Е.

1.4 исследование с использованием синтетических пептидов.

1.5 антителозависимое усиление инфекции.

1.6 исследование с помощью "escape''-мутантов.

2. Рецепторы и рецепторсвязывающие участки флавивирусов

2.1 рецепторсвязывающие участки белка Е.

2.2 рецепторы флавивирусов.

3. Проникновение флавивирусов в клетку путем слияния с клеточной мембраной.

4. Роль неструктурных белков в формировании противовирусного иммунитета.

ГЛАВА II. ОБСУЖДЕНИЕ РЕЗУЛЬТАТОВ.

1. Изучение антигенной структуры гликопротеина Е

1.1 В ыбор пептидов для синтеза.

1.2 Синтез пептидов.

1.3 Иммуногенные и антигенные свойства пептидов 1-ХШ.

2. Выявление фрагментов в последовательности белка Е, обладающих Т-хелперной активностью.

2.1 Выбор фрагментов.

2.2 Синтез пептидов.

2.3 Иммуногенные и антигенные свойства пептидов XIV-XX.

3. Установление последовательности, являющейся участком слияния вируса клещевого энцефалита с клеточной мембраной.

ГЛАВА III ЭКСПЕРИМЕНТАЛЬНАЯ ЧАСТЬ.

ВЫВОДЫ.

Вирус клещевого энцефалита относится к семейству Flaviviridae и является возбудителем серьезного заболевания человека. Восточные субтипы этого вируса, распространенные на территории России, к одному из которых относится исследуемый штамм Софьин, обладают наибольшей патогенностью и для них часты летальные исходы или длительные поражения нервной системы.

Разработка новых методов иммунопрофилактики данного заболевания, а также эффективных подходов к его диагностике требует знаний молекулярных основ функционирования вируса и механизмов формирования противовирусного иммунитета. Незаменимым инструментом в решении этих научных проблем являются синтетические фрагменты вирусных белков, моделирующие иммуноактивные и другие функционально важные участки вирусов. В литературе описано несколько работ по изучению вирусов семейства Flaviviridae с помощью синтетических пептидов, однако для вируса клещевого энцефалита таких исследований не проводилось.

Настоящая работа посвящена выявлению с помощью синтетических пептидов иммуноактивных участков поверхностного белка Е вируса клещевого энцефалита, необходимых для осуществления вирусом своих функций и для индукции противовирусного иммунитета. Данная работа включает: синтез фрагментов белка Е, входящих в состав В-эпитопов белка и вируса; получение противопептидных антител, способных нейтрализовать вирус; выявление фрагментов, индуцирующих образование антител в свободном виде, без конъюгации с белком-носителем и содержащих Т-хелперные эпитопы; установление участка белка, с помощью которого вирус проникает в клетку хозяина и антитела к которому обладают вируснейтрализующими свойствами. Причем вывод о локализации данного участка может быть распространен на все семейство флавивирусов.

Выявление в последовательности белка Е вируса клещевого энцефалита участков, ответственных за формирование противовирусного иммунитета и за проникновение вируса в клетку хозяина, позволяет внести вклад в общее понимание механизмов взаимодействия вируса с организмом хозяина и создает основу для разработки синтетических вакцин и диагностикумов нового поколения.

выводы

1. Синтезировано 20 пептидных фрагментов белка Е вируса клещевого энцефалита, выбранных на основании данных литературы и теоретических методов анализа В-эпитопов и Т-эпитопов белковых последовательностей.

2. Получены антитела к KLH-конъюгатам потенциальных В-эпитопов белка и показано, что антитела к пептидам 98-113 и 394-403 связываются с белком Е и являются вируснейтрализующими.

3. Установлено, что пептид 35-51 связывается с противобелковыми антителами и является частью конформационно зависимого В-эпитопа белка Е.

4. В ряду потенциальных Т-эпитопов белка выявлены пептиды 48-74, 90-113, 204-224, 275-302 и 377-403, иммуногенные в свободном виде без конъюгации с белком-носителем и содержащие Т-хелперные эпитопы.

5. Установлено, что пептиды 204-224, 275-302 и 377-403 способны эффективно связываться с противовирусной сывороткой человека и входят в состав В-эпитопов вируса. Наиболее высокую активность при связывании с противовирусными антителами проявляет пептид 275-302.

6. На основании исследований по ингибированию слияния вируса с макрофагами, липосомами и с применением специфических моноклональных антител установлено, что фрагмент 98-113 является участком слияния вируса клещевого энцефалита с эндосомальной мембраной клетки-хозяина.

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