Establishment of poly-heterologous gene expression system in Flammunila velutipes and its application on enterovirus 71 virus-like particles production
Date Issued
2014
Date
2014
Author(s)
Lin, Yu-Ju
Abstract
A reliable Agrobacterium tumefaciens-mediated transformation (ATMT) for the basidiomycete Flammulina velutipes, or commonly knows as enoki mushroom, has been established in our lab previously. However, the enhancement of protein production and expression of multi-target genes remain a big challenge. The 2A peptides mediated cleavage is emerging as a highly effective and novel tool for co-expression of polyproteins. In this study, a feasible polycistronic expression system was developed in F. velutipes. The several objectives have been achieved including: (1) Establishment of polycistronic expression system in enoki mushroom F. velutipes using selectable marker, hph, and reporter gene, egfp, linked by 2A peptides from porcine teschovirus-1. After ATMT, the F. velutipes tramsformants carrying antibiotic resistant capability expressed EGFP as evidenced by fluorescent microscopy and western blotting. Position effect of transgene expression was also investigated by locating egfp at upstream or downstream of 2A peptides. The EGFP amount of F. velutipes transformants assayed by ELISA showed that transgene at upstream of 2A peptides, which was directly driven by the promoter, was expressed more than that at downstream. (2) Enhancement of transgene expression using tandem repeated egfp linked by 2A peptides. The results of western blotting, ELISA and fluorescent microscopy showed that heterologous protein expression in the transgenic F. velutipes was notably enhanced via 2A peptide-mediated cleavage to co-express up to three copies of single gene. (3) Establishment of an EV71 virus-like particles (VLPs) expression system. The genes of EV71 P1 structural protein and 3C protease were constructed in the same transcript linked by 2A peptides. After P1 and 3C were separated via 2A peptides mediated cleavage in co-translational level, 3C protease can proteolytically cleavage P1 into four subunits, VP1, VP2, VP3, and VP4. These four subunits automatically assemble to form VLPs within transgenic mycelia. To increase the efficiency of subunits production, a site-directed mutation K52R of 3C protease was made to prevent sumoylation from host cell. After ATMT and F. velutipes transformants selection, the crude extraction of EV71 VLPs from transgenic mycelia were purified and fixed by GraFix method. The subunits VP1 and VP2 were detected using specific monoclonal antibodies. The partial peptide sequences of VP1 to VP4 were assigned by LC-MS/MS and mascot. These results demonstrated that both of 2A peptides and 3C protease are functional and successfully produce the EV71 VLPs in transgenic F. velutipes via poly-heterologous gene expression system. Moreover, the 2D images of EV71 VLPs can be observed under TEM, and their 3D structure was reconstructed de novo from the TEM-images of class III. This study is the first report regarding the establishment of mushroom polycistronic system. The polycistronic strategy using 2A peptide-mediated cleavage developed in this study can not only be used to express single gene in multiple copies, but also to express multiple genes in a single reading frame. It is a promising strategy for the application of mushroom molecular pharming.
Subjects
菇類分子農場
金針菇
農桿菌媒介轉形
多基因表現系統
2A短鏈胜肽
2A胜肽媒介截切
腸病毒71型類病毒顆粒
Type
thesis
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