Abstract
Rotavirus infection is the leading cause of acute gastroenteritis among infants. The development of new vaccines against rotavirus A is urgent because the virus has many genotypes, some of which have regional prevalence. Virus-like particles (VLP) is a promising way to create effective and safe vaccine preparations.The purpose of the study is to develop the technology for the production of VLP, containing VP2, VP4, VP6 and VP7 of viral genotypes prevalent on the territory of the Russian Federation, and to give its molecular genetic and virological characteristics. The virulent strain Wa G1P[8] of human RV A adapted to MARC-145 cell culture has been used. It was cultured and purified according to the method described by the authors earlier. Standard molecular genetic and cytological methods were used: gene synthesis; cloning into transfer plasmids; recombinant baculoviruses production in Bac-to-Bac expression system; VLP production in the insect cells; centrifugation in sucrose solution; enzyme-linked immunosorbent assay (ELISA); electron microscopy (EM); polyacrylamide gel electrophoresis (SDS-PAGE) and western blot analysis. VP4 and VP7 of the six most represented in Russia genotypes: G1, G2, G4, G9, P4, P8, as well as VP2 and VP6 were selected for VLP production. Recombinant baculoviruses were obtained with codon frequencies optimized for insect cells. Cabbage loopper (Trichoplusia ni) cell culture was coinfected with different combinations of baculoviruses, and VLP consisting of 2-4 proteins were produced. VLP were purified by centrifugation. The size and morphology of the particles matched the rotavirus A virion (by EM). The presence of rotavirus A proteins in VLP was confirmed by the ELISA, SDS-PAGE and western blot analysis. The technology for the synthesis of three-layer VLP consisting of VP2, VP4, VP6 and VP7 has been developed and optimized. The resulting VLP composition represents 6 serotypes of VP4 and VP7, which are most represented on the territory of Russia, and can be used for vaccine development.
Highlights
Rotavirus infection is the leading cause of acute gastroenteritis among infants
The purpose of the study is to develop the technology for the production of Virus-like particles (VLP), containing VP2, VP4, VP6 and VP7 of viral genotypes prevalent on the territory of the Russian Federation, and to give its molecular genetic and virological characteristics
VP4 and VP7 of the six most represented in Russia genotypes: G1, G2, G4, G9, P4, P8, as well as VP2 and VP6 were selected for VLP production
Summary
Синтез и характеристика вирусоподобных частиц ротавируса А (Reoviridae: Sedoreovirinae: Rotavirus: Rotavirus A) человека. Черепушкин С.А.1, Цибезов В.В.1, Южаков А.Г.1, Латышев О.Е.1, Алексеев К.П.1, Алтаева Э.Г.2, Хаметова К.М.1, Воркунова Г.К.1, Южакова К.А.1, Гребенникова Т.В.1. Применяли стандартные молекулярно-генетические и цитологические методы: синтез генов, клонирование в трансферные плазмиды, получение рекомбинантных бакуловирусов; производство ВпЧ с помощью бакуловирусной системы экспрессии Bac-to-Bac в клетках насекомых; центрифугирование в растворе сахарозы; иммуноферментный анализ (ИФА); электронная микроскопия (ЭМ); электрофорез (ЭФ) в полиакриламидном геле с додецилсульфатом (лаурилсульфатом) натрия (ПААГ-ДСН); иммуноблоттинг. Полученные частицы содержат белки VP4 и VP7 6 генотипов, наиболее представленных на территории России, и могут быть использованы для создания вакцинных препаратов против РВ А. Для цитирования: Черепушкин С.А., Цибезов В.В., Южаков А.Г., Латышев О.Е., Алексеев К.П., Алтаева Э.Г., Хаметова К.М., Воркунова Г.К., Южакова К.А., Гребенникова Т.В. – проведение экспериментальных исследований, написание текста; Южаков А.Г., Латышев О.Е., Алексеев К.П., Алтаева Э.Г.
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