RESUMO
The gene corresponding to mature PsaA from Streptococcus pneumoniae serotype 14 was cloned into a plasmid with kanamycin resistance and without a purification tag in Escherichia coli to express high levels of the recombinant protein for large-scale production as a potential vaccine candidate or as a carrier for polysaccharide conjugation at Bio-Manguinhos/Fiocruz. The evaluation of induction conditions (IPTG concentration, temperature and time) in E. coli was accomplished by experimental design techniques to enhance the expression level of mature recombinant PsaA (rPsaA). The optimization of induction process conditions led us to perform the recombinant protein induction at 25°C for 16 h, with 0.1mM IPTG in Terrific Broth medium. At these conditions, the level of mature rPsaA expression obtained in E. coli BL21 (DE3) Star by pET28a induction with IPTG was in the range of 0.8 g/L of culture medium, with a 10-fold lower concentration of inducer than usually employed, which contributes to a less expensive process. Mature rPsaA expressed in E. coli BL21 (DE3) Star accounted for approximately 30-35% of the total protein. rPsaA purification by ion exchange allowed the production of high-purity recombinant protein without fusion tags. The results presented in this work confirm that the purified recombinant protein maintains its stability and integrity for long periods of time in various storage conditions (temperatures of 4 or -70°C using different cryoprotectors) and for at least 3 years at 4 or -70°C in PBS. The conformation of the stored protein was confirmed using circular dichroism. Mature rPsaA antigenicity was proven by anti-rPsaA mouse serum recognition through western blot analysis, and no protein degradation was detected after long periods of storage.
Assuntos
Adesinas Bacterianas/biossíntese , Clonagem Molecular/métodos , Lipoproteínas/biossíntese , Proteínas Recombinantes/biossíntese , Adesinas Bacterianas/química , Adesinas Bacterianas/genética , Western Blotting , Cromatografia por Troca Iônica , Armazenamento de Medicamentos , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Escherichia coli/metabolismo , Glucose , Isopropiltiogalactosídeo , Lipoproteínas/química , Lipoproteínas/genética , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Reprodutibilidade dos Testes , TemperaturaRESUMO
Apresenta-se neste trabalho a situaçäo da vacinaçäo, produçäo e desenvolvimento tecnológico de vacinas no mundo e no Brasil; também säo feitas algumas reflexöes sobre a complexidade da inovaçäo tecnológica de vacinas e as diversas etapas do processo de desenvolvimento tecnológico requeridas para esse completo desenvolvimento. Descrevem-se várias etapas envolvidas, com análise dos parâmetros e fatores integrantes em cada etapa, os requisitos técnicos de instalaçöes e equipamentos, as normas de boas práticas de fabricaçäo(BPF_), a necessidade organizacional, de infra-estrutura e de gestäo, o longo período e o alto custo demandados para essa atividade.