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The rice gall dwarf disease, caused by Rice gall dwarf virus (RGDV), is a severe virus disease of rice in Southeast Asia and China [24]. This disease was first reported in central Thailand in 1980 [15, 18], and then occurred successively in Malaysia [16] and the rice-producing areas of China [3]. In recent years, it has been occurring severely and frequently in parts of Guangdong and Guangxi provinces of China, and is distributed sporadically in such areas as Yunxiao, Zhaoan and Zhangpu of Fujian province, and is exhibiting a tendency of spreading further [1]. RGDV is a member of the genus Phytoreovirus of the family Reoviridae, and is mainly transmitted in a persistent manner by Nephotettix cincticeps and Recilia dorsalis [6, 7]. Its virions (particles) exhibit a spherical icosahedron shape with a diameter of 65~70 nm, and have double shells. The viral genome is composed of 12 double-stranded RNA segments, designated S1 to S12 in ascending order of their mobility during SDS-PAGE gel electrophoresis [6]. So far, the complete sequences of RGDV isolates from Thailand [13] and Guangxi of China have been determined [23], laying the foundation for the research of the function of various segments.
RGDV virions have a total of six structural proteins. The minor core protein P1 encoded by S1 is an RNA polymerase, S2 encodes the minor outer capsid protein P2, P3 protein encoded by S3 is a major inner capsid protein, the minor core protein P5 encoded by S5 has guanylyltransferase activity, the minor core protein P6 encoded by S6 is a nucleic acid binding protein, and the S8-encoded P8 protein is a major outer capsid protein [13]. The structural protein of RGDV, especially the outer capsid, not only protects the viral nucleic acid from being impacted and damaged by external factors, acting as the primary antigen component, but also has such important biological functions as adsorbing to, penetrating and entering the host cell during virus infection [14]. Obtaining the recombinant protein by in vitro expression can facilitate the research of related functions of these proteins. The authors have tried to express the RGDV S8 gene in E.coli using the prokaryotic expression system, but the recombinant proteins existed in the form of inclusion body [4], and lacked the biologic activity they should have. Accordingly, this research, using the Bac-to-Bac baculovirus-insect expression system, constructs the RGDV outer coat protein gene S8 that has been cloned and identified in the baculovirus shuttle vector pFastBacTM1, and explores its expression characteristics in the Spodoptera frugiperda insect cell line (Sf9).
Expression of Rice Gall Dwarf Virus Outer Coat Protein Gene (S8) in Insect Cells
- Received Date: 04 June 2010
- Accepted Date: 09 October 2010
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 pFastBacTM1 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.