caa a22 4500 605498458 CHVBK 20210128100546.0 cr unu---uuuuu 210128e20150801xx s 000 0 eng 10.1007/s00253-015-6588-z doi (NATIONALLICENCE)springer-10.1007/s00253-015-6588-z Expression of enterovirus 71 virus-like particles in transgenic enoki ( Flammulina velutipes ) [Elektronische Daten] [Yu-Ju Lin, Wen-Ti Liu, Holger Stark, Ching-Tsan Huang] 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. Springer-Verlag Berlin Heidelberg, 2015 Flammulina velutipes nationallicence Agrobacterium tumefaciens -mediated transformation nationallicence 2A peptides nationallicence Polycistron nationallicence Enterovirus 71 nationallicence Lin Yu-Ju Department of Biochemical Science and Technology, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, 10617, Taipei, Taipei, Taiwan aut Liu Wen-Ti Max Planck Institute for Biophysical Chemistry, Am Faßberg 11, 37077, Goettingen, Germany aut Stark Holger Max Planck Institute for Biophysical Chemistry, Am Faßberg 11, 37077, Goettingen, Germany aut Huang Ching-Tsan Department of Biochemical Science and Technology, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, 10617, Taipei, Taipei, Taiwan aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/16(2015-08-01), 6765-6774 0175-7598 99:16<6765 2015 99 253 https://doi.org/10.1007/s00253-015-6588-z text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-015-6588-z text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Lin Yu-Ju Department of Biochemical Science and Technology, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, 10617, Taipei, Taipei, Taiwan aut NATIONALLICENCE 700 1- Liu Wen-Ti Max Planck Institute for Biophysical Chemistry, Am Faßberg 11, 37077, Goettingen, Germany aut NATIONALLICENCE 700 1- Stark Holger Max Planck Institute for Biophysical Chemistry, Am Faßberg 11, 37077, Goettingen, Germany aut NATIONALLICENCE 700 1- Huang Ching-Tsan Department of Biochemical Science and Technology, National Taiwan University, No. 1, Sec. 4, Roosevelt Road, 10617, Taipei, Taipei, Taiwan aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/16(2015-08-01), 6765-6774 0175-7598 99:16<6765 2015 99 253