Abstract
To obtain the P8 protein of Rice gall dwarf virus (RGDV) with biological activity, its outer coat protein gene S8 was expressed in Spodoptera frugiperda (Sf9) insect cells using the baculovirus expression system. The S8 gene was subcloned into the pFastBac™1 vector, to produce the recombinant baculovirus transfer vector pFB-S8. After transformation, pFB-S8 was introduced into the competent cells (E. coli DH10Bac) containing a shuttle vector, Bacmid, generating the recombinant bacmid rbpFB-S8. After being infected by recombinant baculovirus rvpFB-S8 at different multiplicities of infection, Sf9 cells were collected at different times and analyzed by SDS-PAGE, Western blotting and immunofluorescence microscopy. The expression level of the P8 protein was highest between 48–72 h after transfection of Sf9 cells. Immunofluorescence microscopy showed that P8 protein of RGDV formed punctate structures in the cytoplasm of Sf9 cells.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Fan G C, Wu Z J, Huang M H,et al. 2005. Molecular characterization of the genome segment S9 of Rice gall dwarf virus (in Chinese). Virol Sin, 20(5): 539–542. (in Chinese)
Fan G C, Wu Z J, Lin Q Y,et al. 2005. Complete sequence determination and structure analysis of genome segment S8 of rice gall dwarf virus. J Agric Biotechnol, 13(5): 679–683. (in Chinese)
Fan H Z, Zhang S G, He X Z,et al. 1983. Rice gall dwarf-a new virus disease epidemic in the west of Guangdong Province of South China. Acta Phytopathol Sin, 13(4): 1–6. (in Chinese)
Gao F L, Fan G C, Shen J G,et al. 2009. Structure analysis of P8 protein of Rice gall dwarf virus and it’s expression in E.coli. China Biotechnol, 29(8): 51–56. (in Chinese)
Hagiwara K, Higashi T, Namba K,et a1. 2003. Assembly of single-shelled cores and double-shelled virus-like particles after baculovirus expression of major structural proteins P3, P7 and P8 of Rice dwarf virus. J GenVirol, 84: 981–984.
Hibi T, Omura T, Saito Y. 1984. Double-stranded RNA of Rice gall dwarf virus. J Gen Virol, 65: 1585–1590.
Hibino H. 1996. Biology and epidemiology of rice viruses. Annu Rev Phytopathol, 34: 249–274.
Ichimi K, Kikuchi A, Moriyasu Y,et al. 2002. Sequence analysis and GTP-binding ability of the minor core protein P5 of Rice gall dwarf virus. Japan Agric Res Q, 36(2): 83–87.
Kar A K, Ghosh M, Roy P. 2004. Mapping the assembly pathway of Bluetongue virus scaffolding protein VP3. Virology, 324: 387–399.
Kim Y, Chang K O, Kim W Y,et al. 2002. Production of hybrid double- or triple-Layered virus-like particles of group A and C Rotaviruses using a baculovirus expression system. Virology, 302: 1–8.
Liu F X, Zhao Q, Ruan Xiao L,et al. 2008. Suppressor of RNA silencing encoded by Rice gall dwarf virus genome segment 11. Chin Sci Bull, 53(3): 362–369.
Miyazaki N, Hagiwara K, Naitow H,et al. 2005. Transcapsidation and the conserved interactions of two major structural proteins of a pair of Phytoreoviruses confirm the mechanism of assembly of the outer capsid layer. J Mol Biol, 345, 229–237.
Moriyasu Y, Maruyama-Funatsuki W, Kikuchi A,et al. 2007. Molecular analysis of the genome segments S1, S4, S6, S7 and S12 of a Rice gall dwarf virus isolate from Thailand; completion of the genomic sequence. Arch Virol, 152(7): 1315–1322.
Omura T, Yan J. 1999. Role of outer capsid proteins in transmission of Phytoreovirus by insect vectors. Adv Virus Res, 54:15–43.
Omura, T, Inoue H, Morinaka T,et al. 1980. Rice gall dwarf, a new virus disease. Plant Dis, 64: 795–797.
Ong C A, Omura T. 1982. Rice gall dwarf virus occurrence in peninsular Malaysia. Int Rice Res Newsletter, 7(2): 7.
Polayes D A, Harris R, Anderson D,et al. 1996. New baculovirus expression vectors for the purification of recombinant proteins from insect cells. Focus, 18: 10–13.
Putta M, Chettanachit D, Morinaka T,et al. 1980. Gall dwarf—a new virus disease in Thailand. Int Rice Res Newsletter, 5(3): 10–13.
Sambrook J, Russell D W. 1998. In: Molecular cloning: a laboratory manual, 2nd ed. New York: Cold Spring Harbor Laboratory, p888–897.
Spector D L, Goldman R D, Leinwand L A. 1998. In: Cells: A Laboratory Manual, New York: Cold Spring Harbor Laboratory Press, p556–575.
Takahashi Y, Tomiyama M, Hibino H,et al. 1994. Conserved primary structures in core capsid proteins and reassembly of core particles and outer capsids between Rice gall dwarf and Rice dwarf Phytoreoviruses. J Virol, 75: 269–275.
Vieira H L A, Estevao C, Roldao A,et al. 2005. Triple layered rotavirus VLP production: Kinetics of vector replication, mRNA stability and recombinant protein production. J Biotechnol, 120: 72–82.
Zhang H M, Xin X, Yang J,et al. 2008. Completion of the sequence of Rice gall dwarf virus from Guangxi, China. Arch Virol, 153: 1737–1741.
Zhang H M, Yang J, Xin X,et al. 2007. Molecular characterization of the largest and smallest genome segments, S1 and S12, of Rice gall dwarf virus. Virus Genes, 35(3): 815–823.
Zheng H, Yu L, Wei C,et al. 2000. Assembly of double-shelled, virus-like particles in transgenic rice plants expressing two major structural proteins of Rice dwarf virus. J Virol, 74(20): 9808–9810.
Author information
Authors and Affiliations
Corresponding author
Additional information
Foundation items: This project was supported by the National Science Foundation of China (30970135), The Key Project of Genetically Modified Organisms Breeding (2009ZX08009-044B), the Natural Science Foundation of Fujian Province of China (No.2006J0065) and the Public-interest Scientific Institution Basal Research Fund of Fujian Province (2009R10029-3)
Rights and permissions
About this article
Cite this article
Fan, Gc., Gao, Fl., Wei, Ty. et al. Expression of rice gall dwarf virus outer coat protein gene (S8) in insect cells. Virol. Sin. 25, 401–408 (2010). https://doi.org/10.1007/s12250-010-3152-y
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12250-010-3152-y