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http://dx.doi.org/10.5423/RPD.2016.22.1.32

In Vitro Expression and Antibody Preparation of Rice black-streaked dwarf virus Coat Protein Gene  

Lee, Bong Choon (Crop Foundation Division, National Institute of Crop Science, Rural Development Administration)
Cho, Sang-Yun (Seed Testing & Research Center, Korea Seed & Variety Service)
Bae, Ju Young (Crop Foundation Division, National Institute of Crop Science, Rural Development Administration)
Kim, Sang Min (Crop Foundation Division, National Institute of Crop Science, Rural Development Administration)
Shin, Dong Bum (Crop Cultivation & Environment Research Division, Rural Development Administration)
Kim, Sun Lim (Crop Foundation Division, National Institute of Crop Science, Rural Development Administration)
Publication Information
Research in Plant Disease / v.22, no.1, 2016 , pp. 32-37 More about this Journal
Abstract
In this work, major outer capsid protein (P10) encoded by genome segment S10 of Rice black-streaked dwarf virus (RBSDV) was expressed in Escherichia coli. Genomic dsRNA was extracted from RBSDV-miryang isolate infected rice plants. Based on the sequence of S10 (RBSDV-miryang, GenBank JX994211), a pair of S10 specific primers were designed and used to amplify the fragment encoding the N-part of P10. We amplified the partial gene (S10 1-834 nt) of RBSDV P10 (1-278 aa) by RT-PCR. Amplified RBSDV S10 (1-834 nt) was cloned into the expression vector pET32a (+). Recombinant RBSDV S10 (1-834 nt) was expressed in E. coli BL21(DE3) and purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity column. We successfully obtained P10 partial protein of RBSDV and the purified protein was used to immunize rabbits. The resulting polyclonal antiserum specifically recognized RBSDV from infected plant in both Western blotting and enzyme-linked immunosorbent assay. In this study, we provide purified RBSDV P10 (1-278 aa), which would be good material for the serological study of RBSDV-miryang isolates.
Keywords
P10 partial protein; Polyclonal antiserum; Rice black-streaked dwarf virus;
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