Expression of enterovirus 71 virus-like particles in transgenic enoki (Flammulina velutipes)

Abstract

No commercial vaccines are currently available for enterovirus 71 (EV71) infection. Oral virus-like particle (VLP) vaccines are regarded as a better choice for prevention from food-borne diseases compared with injected whole virus vaccines. Unfortunately, the application of oral VLP vaccines produced from transgenic plants was limited due to the concerns of gene contamination. Alternatively, using transgenic mushrooms retains the advantages of transgenic plants and tremendously reduce risks of gene contamination. Polycistronic expression vectors harboring the glyceraldehyde-3-phospho-dehydrogenase promoter to codrive EV71 structural protein P1 and protease 3C using the 2A peptide of porcine teschovirus-1 were constructed and introduced into Flammulina velutipes via Agrobacterium tumefaciens-mediated transformation. The analyses of the genomic PCR, Southern blotting, and RT-PCR showed that the genes of P1 and 3C were integrated into the chromosomal DNA through a single insertion, and their resulting mRNAs were transcribed. The Western blotting analysis combined with LC-MS/MS demonstrated that EV71 VLPs were composed of the four subunit proteins digested from P1 polyprotein by 3C protease. Through the use of a single particle electron microscope, images of 1705 particles with diameter similar to the EV71 viron were used for 3D reconstruction. Protrusions were observed on the surface in the 2D class averages, and a 3D reconstruction of the VLPs was obtained. In conclusion, EV71 VLPs were successfully produced in transgenic F. velutipes using a polycistronic expression strategy, which indicates that this approach is promising for the development of oral vaccines produced in mushrooms.

Development of a Transgenic Flammulina velutipes Oral Vaccine for Hepatitis B

“…Previously, we demonstrated that heterologous protein expression in the enoki mushroom Flammulina velutipes is notably enhanced by using 2A peptide-mediated cleavage to co-express multiple copies of a single gene ( 16 ). Using this polycistronic expression strategy, enterovirus 71 virus-like particles were successfully produced in transgenic F. velutipes ( 17 ).…”

“…Although single mating types have been known to produce fruiting bodies under severe stress, most fruiting requires the plasmogamy of two genetically different monokaryotic mycelia, AxBx/AyBy or AxBy/AyBx, to form the dikaryotic mycelia that consist of two nuclei in a cell and the structure of clamp connection ( 19 ). The generation of transgenic mushrooms usually involves Agrobacterium -mediated transformation using modified mycelial pellets, followed by the selection of transformants under appropriate pressures ( 17 ). Transformants are selected if the foreign gene is inserted into the chromosomal DNA in at least one nucleus of the mycelia.…”