Establishment of a Bacterial Expression System and Immunoassay Platform for the Major Capsid Protein of HcRNAV, a Dinoflagellate-Infecting RNA Virus

• Wada Kei

  Department of Biological Sciences, Graduate School of Science, Osaka University

• Kimura Kei

  National Research Institute of Fisheries and Environment of Inland Sea, Fisheries Research Agency

• Hasegawa Akifumi

  Department of Biological Sciences, Graduate School of Science, Osaka University

• Fukuyama Keiichi

  Department of Biological Sciences, Graduate School of Science, Osaka University

• Nagasaki Keizo

  National Research Institute of Fisheries and Environment of Inland Sea, Fisheries Research Agency

抄録

HcRNAV is a small icosahedral virus that infects the shellfish-killing marine dinoflagellate Heterocapsa circularisquama, which harbors a dicistronic linear single-stranded RNA (ssRNA) genome ca. 4.4 kb in length. Its major capsid protein (MCP) gene sequence is not expressed by various strains of Escherichia coli, possibly because of a codon usage problem. To solve this problem, a chemically modified (i.e., de novo synthesized) gene was designed and cloned into the pCold-GST expression vector, and transformed into E. coli strain C41 (DE3), in which codon usage was universally optimized to efficiently express the polypeptide having the viral MCP amino acid sequence. The bacterially expressed protein, which was purified after a procedure involving denaturation and refolding, successfully formed virus-like particles that significantly resembled native HcRNAV particles. The purified, denatured protein was used as an antigen to immunize rabbits, and the resulting antiserum was shown to be strongly reactive to not only the bacterially expressed recombinant protein, but also to native HcRNAV MCP by Western blotting and dot immunoassays, respectively. These results indicate that an antiserum recognizing native HcRNAV MCP was successfully obtained using bacterially expressed HcRNAV MCP as the antigen.<br>

収録刊行物

• Ｍｉｃｒｏｂｅｓ　ａｎｄ　ｅｎｖｉｒｏｎｍｅｎｔｓ

  Ｍｉｃｒｏｂｅｓ　ａｎｄ　ｅｎｖｉｒｏｎｍｅｎｔｓ 27 (4), 483-489, 2012

  日本微生物生態学会 / 日本土壌微生物学会 / Taiwan Society of Microbial Ecology / 植物微生物研究会 / 極限環境微生物学会

キーワード

• bacterial expression
• <i>Heterocapsa circularisquama</i> RNA virus
• major capsid protein
• polyclonal antibody

詳細情報

• CRID: 1390282679323619200
• NII論文ID: 10031125948
• NII書誌ID: AA11173196
• DOI: 10.1264/jsme2.me12046
• COI: 1:STN:280:DC%2BC3s%2FjsFKjsg%3D%3D
• ISSN: 13474405
• ISSN: 13426311
• PubMed: 23047150
• Web Site: https://www.jstage.jst.go.jp/article/jsme2/27/4/27_ME12046/_pdf
• 本文言語コード: en
• データソース種別:
  • JaLC
  • Crossref
  • PubMed
  • CiNii Articles
  • KAKEN
• 抄録ライセンスフラグ: 使用不可