цель-экспериментально обосновать возможность использования перевиваемой линии клеток сно-к1 для определения специфической активности холерного токсина и компонента вакцины холерогена-анатоксина в процессе производства холерной химической вакцины. материалы и методы. в исследованиях использовали перевиваемую линию клеток сно-к. учет результатов метода биоиндикации на перевиваемой клеточной линии проводили визуально с помощью инвертированного микроскопа и фотометрически в колориметрическом тесте для оценки метаболической активности клеток при длине волны 595 нм. результаты и обсуждение. предлагаемый метод позволяет определить активность продукции токсина у штамма Vibrio cholerae 569B при глубинном культивировании в биореакторе и специфическую активность холерогена-анатоксина по анатоксиносвязыванию с использованием клеточных культур. результаты коррелируют с данными, полученными при использовании методов внутрикожной пробы по крейгу, GM1-иФа и рпиг. введение в практику метода клеточных культур может обеспечить существенное сокращение использования животных на этапах производства вакцины холерной бивалентной химической. Ключевые слова: холерная химическая вакцина, культура клеток сно-к1, холерный токсин, холерогенанатоксин.
Pigmentation was examined in 214 plague microbe strains and 68 pseudotuberculosis agent strains. Plague microbe strains of different subspecies and pseudotuberculosis microbe strains demonstrated different ability of hemin and acidic dyes sorption. Genes of hms operon in typical Y. pestis strains of five subspecies and in talassica group strains, as well as in Y. pseu do tu ber cu losis O1 serotype strains, were sequenced and analyzed using PCR. Single nucleotide substitutions were detected in all genes of the operon in plague microbe strains as compared with the operon of pseudotuberculosis agent strains. Determined were single nucleotide substitutions promising for intraspecific differentiation of plague microbe strains.
Cellular morphologic variations of Yersinia pseudotuberculosis strain 164/84 were assessed depending on the cultivation temperature and the plague agent's fra-operon carriage by these cells. Notably slower population growth of the two isogenic variants of strain 164/84 (fra+ and fra-) was observed at the cultivation temperatures of 4 to 6 °C as compared with those at 26-28 °C and 36-38 °C. Only late growth stages at the temperatures of 36 to 38 °C brought about significant differences between the fra+ and fra-cell variants expressed in the occurrence of relatively bigger cells in the population of the capsule forming Y. pseudotuberculosis variant.
Developed is the method of identification and intraspecific typing of plague microbe strains along with their potential virulence determination. Intraspecific differentiation and focal attribution of the examined plague microbe strains can be determined by monolocus VNTR-PCR, and main virulence determinants (chromosomal pigmentation region and calcium-dependence plasmid genes) - by multiplex PCR.
Experimentally substantiated is the possibility to apply tangential ultrafiltration for desalting antigen components of the tableted bivalent chemical cholera vaccine. Specified are the technological parameters of the process. It is demonstrated that the properties of choleragen-anatoxin (produced by Vibrio cholerae strain 569B Inaba) and O-antigens (produced from V. cholerae 569B Inaba and M-41 Ogawa strains) obtained using the designed methodology are as efficient as the ones manufactured using certified procedure and satisfy regulatory requirements. Experimentally substantiated technology for the desalting of antigen components of chemical cholera vaccine provides for the reduction of the time elapsed up to 5-6 hours from the original 3 to 4 days. It also allows for the manufacturing under controlled conditions. This hardware controlled method of desalting has been implemented into the vaccine production practice.
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