Expression of hepatitis B virus core gene in Pichia pastoris / 中国病理生理杂志
Chinese Journal of Pathophysiology
;
(12)2000.
Artigo
em Chinês
| WPRIM
| ID: wpr-523801
ABSTRACT
AIM:
To study the expression of hepatitis B virus core gene in Pichia pastoris and to obtain high-level expressed recombinant HBcAg with good immunoreactivity and high specificity.METHODS:
HBV core gene was amplified by PCR from plasmid pHBV1 which contained HBV whole DNA sequence. The PCR product was cloned into pGEM-T vector by TA cloning strategy. After confirmed by DNA sequence analysis, the gene of interest was inserted into the yeast expression vector pPIC9. The recombinant plasmid pPIC9-cAg was constructed and transformed into GS115 by electroporation. The recombinant yeast GS115 was induced by 0.5% methanol. The expressed product was analysed by SDS-PAGE,Western blot and ELISA.RESULTS:
The restriction analysis and DNA sequence analysis proved that HBV core gene had already been cloned to yeast expression plasmid pPIC9. The expressed HBcAg existed in SDS-PAGE. Good immunoreactivity and high specificity of the recombinant HBcAg have been proved by ELISA and Western blot. The titre of the recombinant HBcAg in the cell lysate was 1∶ 12 800 .CONCLUSION:
The recombinant plasmid pPIC9-cAg was successfully constructed. The recombinant HBcAg with good immunoreactivity and high specificity was successfully expressed in Pichia pastoris expression system and can be applied to further developing HBcAb immunoassay. [
Texto completo:
DisponíveL
Índice:
WPRIM (Pacífico Ocidental)
Idioma:
Chinês
Revista:
Chinese Journal of Pathophysiology
Ano de publicação:
2000
Tipo de documento:
Artigo
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