Establishment of homologous selectable system and development of hepatitis B virus oral vaccine in Flammulina velutipes
Date Issued
2012
Date
2012
Author(s)
Lin, Hao-Ye
Abstract
The mushroom molecular pharming refers to the use of mushrooms as bioreactor to produce valuable and pharmaceutical proteins, especially oral vaccines. Previously, a reliable Agrobacterium-mediated transformation for Flammulina velutipes has been established. However, the application was limited due to bacterial antibiotic selectable marker and low yield in heterologous proteins. Until now, the selectable markers using antibiotic resistance genes raise public concerns of biosafety. In this study, we developed a homologous selectable strategy via a single point mutation of succinate dehygrogenase to get transformants against harmless agent, carboxin. Besides, the heterologous gene expression was greatly enhanced by partial deletion of the glyceraldehyde-3-phosphodehydrogenase (gpd) promoters. We also successfully expressed the hepatitis B virus surface antigen (HBsAg) by partial deletion of gpd promoter with a maximum expression of 329.08 ± 35.37 ng/g total soluble protein. We demonstrated that the homologous selectable marker and the enhancement of protein expression using partial deletion of gpd promoter provide useful strategies in development of oral vaccines in F. velutipes.
Subjects
Mushroom molecular pharming
Flammulina velutipes
glyceraldehyde-3-phosphate dehydrogenase
homologous selectable marker
a single point mutation of succinate dehygrogenase
hepatitis B virus surface antigen oral vaccines
SDGs
Type
thesis
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