Research in Plant Disease (식물병연구)

The Korean Society of Plant Pathology (한국식물병리학회)
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In Vitro Expression and Antibody Preparation of Rice black-streaked dwarf virus Coat Protein Gene

벼검은줄오갈병바이러스 외피단백질 유전자 단백질 발현과 항혈청 제작

  • 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)
  • 이봉춘 (농촌진흥청 국립식량과학원 작물기초기반과) ;
  • 조상윤 (국립종자원 종자검정 연구센터) ;
  • 배주영 (농촌진흥청 국립식량과학원 작물기초기반과) ;
  • 김상민 (농촌진흥청 국립식량과학원 작물기초기반과) ;
  • 신동범 (농촌진흥청 국립식량과학원 재배환경과) ;
  • 김선림 (농촌진흥청 국립식량과학원 작물기초기반과)
  • Received : 2015.10.12
  • Accepted : 2016.03.18
  • Published : 2016.03.31
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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.

본 연구에서는 RBSDV의 외피단백질 P10을 코드하는 S10을 E. coli에서 발현시켰다. RBSDV-miryang isolate (GenBank JX994211)로부터 추출한 게놈 dsRNA을 주형으로 S10의 특이적인 primer를 사용하여 P10의 N-말단영역(1-834 nt, 1-278 aa)을 RT-PCR에 의해 증폭하였다. 증폭된 RBSDV S10-N (1-834 nt)을 발현 벡터 pET32a(+)에 클로닝하여 E. coli BL21(DE3)에서 발현시킨 후 Ni-NTA affinity column으로 발현된 단백질을 정제하였다. 정제된 단백질을 면역 동물에 주사하여 항혈청을 제작하였다. 제작된 항혈청은 Western blot 및 ELISA 분석으로 RBSDV와의 특이성을 확인하였다. 본 연구에서 RBSDV 한국 isolate의 항혈청이 제작되었으며 금후 혈청학적 연구의 좋은 재료로 활용될 수 있을 것으로 기대한다.

Keywords

References

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