Expression of enterovirus 71 virus-like particles in transgenic enoki (Flammulina velutipes)
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.
KeywordsFlammulina velutipes Agrobacterium tumefaciens-mediated transformation 2A peptides Polycistron Enterovirus 71
We are grateful to TechComm (College of Life Science, National Taiwan University) for LC-MS/MS technical assistance. The financial support of the National Science Council of the ROC (Grant No. NSC102-2313-B-002-059) is greatly appreciated.
- Berends E, Ohm RA, de Jong JF, Rouwendal G, Wosten HA, Lugones LG, Bosch D (2009) Genomic and biochemical analysis of N glycosylation in the mushroom-forming basidiomycete Schizophyllum commune. Appl Environ Microbiol 75(13):4648–4652. doi: 10.1128/AEM.00352-09 PubMedCentralPubMedCrossRefGoogle Scholar
- Chen HL, Huang JY, Chu TW, Tsai TC, Hung CM, Lin CC, Liu FC, Wang LC, Chen YJ, Lin MF, Chen CM (2008) Expression of VP1 protein in the milk of transgenic mice: a potential oral vaccine protects against enterovirus 71 infection. Vaccine 26(23):2882–2889. doi: 10.1016/j.vaccine.2008.03.041 PubMedCrossRefGoogle Scholar
- Kastner B, Fischer N, Golas MM, Sander B, Dube P, Boehringer D, Hartmuth K, Deckert J, Hauer F, Wolf E, Uchtenhagen H, Urlaub H, Herzog F, Peters JM, Poerschke D, Luhrmann R, Stark H (2008) GraFix: sample preparation for single-particle electron cryomicroscopy. Nat Methods 5(1):53–55. doi: 10.1038/nmeth1139 PubMedCrossRefGoogle Scholar
- Palucha A, Loniewska A, Satheshkumar S, Boguszewska-Chachulska AM, Umashankar M, Milner M, Haenni AL, Savithri HS (2005) Virus-like particles: models for assembly studies and foreign epitope carriers. Prog Nucleic Acid Res Mol Biol 80:135–168. doi: 10.1016/S0079-6603(05)80004-2 PubMedCrossRefGoogle Scholar
- Souza AC, Vasques RM, Inoue-Nagata AK, Lacorte C, Maldaner FR, Noronha EF, Nagata T (2013) Expression and assembly of Norwalk virus-like particles in plants using a viral RNA silencing suppressor gene. Appl Microbiol Biotechnol 97(20):9021–9027. doi: 10.1007/s00253-013-5077-5 PubMedCrossRefGoogle Scholar
- Specht EA, Mayfield SP (2014) Algae-based oral recombinant vaccines. Front Microbiol 5. doi: 10.3389/fmicb.2014.00060
- Taghavian O, Spiegel H, Hauck R, Hafez HM, Fischer R, Schillberg S (2013) Protective oral vaccination against infectious bursal disease virus using the major viral antigenic protein VP2 produced in Pichia pastoris. PLoS One 8(12), e83210. doi: 10.1371/journal.pone.0083210 PubMedCentralPubMedCrossRefGoogle Scholar