Получение и изучение рекомбинантных Fab'-фрагментов антител к афлатоксинам тема диссертации и автореферата по ВАК РФ 03.01.04, кандидат биологических наук Калиниченко, Артем Андреевич

• Калиниченко, Артем Андреевич
• кандидат биологических наук
• 2010

Оглавление диссертации кандидат биологических наук Калиниченко, Артем Андреевич

ИСПОЛЬЗУЕМЫЕ СОКРАЩЕНИЯ

I. ВВЕДЕНИЕ

II. ОБЗОР ЛИТЕРАТУРЫ 7 И. 1 Общие сведения об афлатоксинах.

11.1.1 Открытие афлатоксинов.

И. 1.2 Химические и физические свойства афлатоксинов, их источники. 8 II. 1.3 Воздействие афлатоксинов на организм человека и животных.

11.2 Способы детоксикации афлатоксинов в пищевых продуктах и кормах

11.3 Способы детекции афлатоксинов в пищевых продуктах и кормах.

11.3.1 Аналитические методы.

11.3.2 Скрининговые методы.

11.4 Антитела против афлатоксинов: получение и использование.

11.5 Системы эксперессии антител.

II.5.1 Экспрессия в клетках E.coli.

II.5.1 Эксперессия в клетках дрожжей.

II.5.1 Эксперессия в культурах клеток животных.

И.5.1 Эксперессия в растениях.

III. МАТЕРИАЛЫ И МЕТОДЫ 43 Реактивы.

Клетки гибридом и МА.

Буферные растворы.

Микробиологические среды.

Олигонуклеотиды, использованные для ПЦР.

Использованные методы:.

Прямой, непрямой и конкурентный иммуноферментный анализ.

Электрофорез белков в ПААГ.

Дот-блот анализ.

Получение функционально активных Fab'-фрагментов из МА.

Выделение мРНК, обратная траскрипция и амплификация вариабельных и константных доменов МА.

Получение генноинженерых конструкций.

Анализ нуклеотидных и аминокислотных последовательностей.

Сборка вектора для экспрессии рекомбинантных антител в виде Fab 'фрагментов.!.

Экспрессия и очистка рекомбинантных Fab'-фрагментов к AflaT.

Масс-спектрометрический анализ МА. и рекомбинантных Fab'- фрагментов.

Расчет констант диссоциации антител к АфлаТ.

IV. РЕЗУЛЬТАТЫ И ОБСУЖДЕНИЕ

Анализ моноклональных антител против афлатоксинов Вь В2, G2.

Определение подтипов легких и тяжелых цепей антител.

Амплификация кДНК вариабельных и константных доменов моноклональных антител.

Получение плазмидных конструкций для экспрессии рекомбинантных антител.

Биосинтез и выделение рекомбинантных Fabфрагментов МА.

Определение аффинности и кросс-реактивности Fab'-фрагментов.

ВЫВОДЫ

БЛАГОДАРНОСТИ

Заключение диссертации по теме «Биохимия», Калиниченко, Артем Андреевич

выводы

1. Впервые получены гены панели из 10 моноклональных антител к афлатоксинам Вь B2, G2, определена их нуклеотидная последовательность, картированы гипервариабельные участки вариабельных доменов легких и тяжелых цепей антител.

2. Обнаружено необычное для мышей соотношение подклассов легких цепей - половина антител содержат легкие цепи лямбда подкласса.

3. Показано ограниченное разнообразие легких цепей антител, как каппа, так и лямбда подклассов. Это позволяет предположить, что основную роль в формирование аффинности по отношению к гаптену вносят тяжелые цепи полученных антител.

4. Разработан метод экспрессии и очистки функционально активных рекомбинантных антител к афлатоксинам в виде Fab'-фрагментов

5. Показано, что полученные рекомбинантные Fab'-фрагменты обладают аффинностью и кросс-реактивностью, не уступающими по значениям исходным моноклональным антителам.

БЛАГОДАРНОСТИ

Благодарю людей, с помощью которых состоялась эта работа. В первую очередь своего научного руководителя Долгих Дмитрия Александровича за ценные советы и направление в нужное русло, Алиева Теймура Кантамировича за помощь в интерпретации результатов и написании работы, Кирпичникова Михаила Петровича за интересную тему. Большое спасибо Топоровой Виктории Александровне за обучение хитростям молекулярной биологии, генной инженерии и пример целеустремленности. Большую признательность выражаю Паниной Анне Алексеевне и Литвинову Ивану Сергеевичу за многочисленные обсуждения, и редактирование печатных работ. Благодарю Болдыреву Елену Филипповну и Крюкову Елену Александровну за регулярную помощь в работе. Спасибо Шулепко Михаилу Анатольевичу, Копейной Гелине Сергеевне, Балабашину Дмитрию Сергеевичу и Хабибуллиной Нелле Фамзуловне за дружескую атмосферу и поддержку в сложных ситуациях. Выражаю глубокую признательность Петровской Ладе Евгеньевне и Шингаровой Людмиле Николаевне за ценные советы. Отдельная благодарность Шемчуковой Ольге Борисовне, Солоповой Ольге Николаевне и Поздняковой Любовь Петровне за анализ рекомбинантных белков и МА. Большая благодарность Свешникову Петру Георгиевичу за сотрудничество, гибридомы и разъяснение тонкостей иммунологического анализа.

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