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Master's Dissertation
DOI
10.11606/D.87.2016.tde-11032016-104211
Document
Author
Full name
Rafaela Tais Zanardo
Institute/School/College
Knowledge Area
Date of Defense
Published
São Paulo, 2015
Supervisor
Committee
Gonçalves, Viviane Maimoni (President)
Cheng, Elisabeth
Lucarini, Adriana Celia
Title in Portuguese
Purificação do polissacarídeo capsular de Streptococcus pneumoniae de sorotipo 14.
Keywords in Portuguese
Ácido tricloroacético
Cromatografia
Pneumococo
Polissacarídeo capsular
Purificação
SDS
Abstract in Portuguese
Streptococcus pneumoniae (pneumococo) é um importante patógeno humano, responsável por graves infecções das vias respiratórias. O principal fator de virulência desse microrganismo é a cápsula polissacarídica (PS), antígeno das vacinas atuais, que são elaboradas com os PS purificados de cepas de pneumococo prevalentes na população. O objetivo desse trabalho foi desenvolver um processo de purificação do PS do sorotipo 14, responsável por 39% das doenças em crianças de 0-6 anos no Brasil. A metodologia de purificação envolveu separação celular por microfiltração tangencial e concentração do microfiltrado com membrana de ultrafiltração tangencial de 50 kDa. O produto dessa etapa foi diafiltrado com dodecil sulfato de sódio em membrana de ultrafiltração tangencial de 30 kDa, seguido de precipitação com ácido tricloroacético a 5%, precipitação por etanol (20% e 60%) e cromatografia de troca aniônica. A pureza do PS foi avaliada pelo conteúdo de proteínas e ácidos nucleicos remanescentes e o tamanho por cromatografia de exclusão molecular. O PS foi obtido com pureza e tamanho requeridos pelos órgãos regulatórios e o rendimento final do processo foi de 65%.
Title in English
Purification of capsular polysaccharide of Streptococcus pneumoniae serotype 14.
Keywords in English
Capsular polysaccharide
Chromatography
Pneumococcus
Purification
SDS
Trichloroacetic acid
Abstract in English
Streptococcus pneumoniae (pneumococcus) is an important human pathogen responsible for severe respiratory tract infections. The main virulence factor of this microorganism is the capsular polysaccharide (PS), which is the antigen of all current vaccines, which are prepared with purified PS of pneumococcal strains prevalent in the population. The objective of this work was to develop a new purification process for PS of serotype 14, responsible for 39% of diseases in children of 0-6 years old in Brazil. The purification method involved cell separation by tangential microfiltration and concentration of cell-free culture broth containing PS by tangential ultrafiltration (50 kDa). The product of this step was diafiltrated with sodium dodecyl sulfate by tangential ultrafiltration (30 kDa), following by 5% trichloroacetic acid precipitation, 20% and 60% ethanol precipitation and anion exchange chromatography. The PS purity was evaluated by the content of residual proteins and nucleic acids, and the molecular mass by size exclusion chromatography. The purity and molecular mass requirements were achieved and the process global yield was 65%.
 
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Publishing Date
2016-03-11
 
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