Expression of hepatitis B virus core gene in Pichia pastoris
- VernacularTitle:乙型肝炎病毒C基因在毕赤酵母中的表达
- Author:
Zhaoxia LI
;
Minjian LIANG
;
Lin LI
;
Bo HU
;
Zhenyu ZHU
- Publication Type:Journal Article
- Keywords:
Hepatitis B core antigens;
Immunoreactivity;
Gene expression;
Pichia
- From:
Chinese Journal of Pathophysiology
2000;0(12):-
- CountryChina
- Language:Chinese
-
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. [
