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Expression of a human single-chain Fv antibody against HBsAg in Pichia pastoris / 生物工程学报
Article em Zh | WPRIM | ID: wpr-270045
Biblioteca responsável: WPRO
ABSTRACT
To express and secrete native HBscFv (anti-HBsAg single-chain Fv) in P. pastoris, HBscFv was amplified from plasmid pGEM-HBscFv, and then sub-cloned into expression vector pPICZalphaA. The resulting plasmid pPIC-HBscFv was linearized and transformed into P. pastoris GS115. The recombinant Pichia strains, identified by direct PCR and Zeocin-resistant screening of Pichia transformants, were cultured and induced with methanol. It was found that recombinant HBscFv, lead by alpha-factor, could be secreted into the culture supernatant to a level of 80mg/L. The bioactivity of Pichia produced HBscFv was confirmed by indirect ELISA, which also suggested that the bioactivity of HBscFv in the culture supernatant reached its peak in 72h and decreased in the late-stage of the induction. PAS staining suggests that HBscFv produced by yeast is poorly glycosylated or none-glycosylated protein.
Assuntos
Texto completo: 1 Índice: WPRIM Assunto principal: Pichia / Western Blotting / Reação em Cadeia da Polimerase / Eletroforese em Gel de Poliacrilamida / Alergia e Imunologia / Anticorpos de Cadeia Única / Genética / Antígenos de Superfície da Hepatite B / Metabolismo Tipo de estudo: Prognostic_studies Limite: Humans Idioma: Zh Revista: Chinese Journal of Biotechnology Ano de publicação: 2003 Tipo de documento: Article
Texto completo: 1 Índice: WPRIM Assunto principal: Pichia / Western Blotting / Reação em Cadeia da Polimerase / Eletroforese em Gel de Poliacrilamida / Alergia e Imunologia / Anticorpos de Cadeia Única / Genética / Antígenos de Superfície da Hepatite B / Metabolismo Tipo de estudo: Prognostic_studies Limite: Humans Idioma: Zh Revista: Chinese Journal of Biotechnology Ano de publicação: 2003 Tipo de documento: Article