2001•Pharmaceutical biotechnologyRequires access
Secreted Expression of Hepatitis E Virus ORF2 Gene in Pichia Pastoris
Chunjiang Zhang, Shen Xin-liang, Lei Qing, Qimin Li
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Abstract
We have used the methytotrophic yeast Pichia Pastoris to express the open reading frame2(ORF2) 1.3 kb gene of Hepatitis E virus(HEV). The recombinant secreted expression vector pPIC9HEV and pHUS1HEV were linearized by SaiI and BglII,and then transformed into Pichia Pastoris GS115 by electroporation. The expression vector carrying interested gene was integrated into GS115 chromone. MD and MM plates were used to selected Mut phenotype, and then positive clones were analyzed by PCR to confirm the integration of HEVORF2 gene. We test expression of both HIS+ MUTs and HIS+ MUT+ in BMGY/BMMY media. A high level of secreted protein could be detected by ELISA and SDS-PAGE. Westems-blot showed it could be recognized by the McAb of HEV ORF2.We get the strains that can express HEV ORF2 protein and we tried to find the best expression condition.