Separation of Structural Protein of Japanese Encephalitis Virus by Column Chromatography

精製した日本脳炎(JE)ウイルス粒子を溶解した試料,或はウイルス感染ヒトスジシマカ培養細胞クローンC6/36の乳剤からJEウイルスの構造蛋白質を分離することを試みた.試料の分離にはガラスウール,DEAE-Sephacel, Sepharose 4B又は6B,Sephadex G-150,及びHydroxylapatiteカラムを種々の緩衝液と組み合わせて使用した.カラムから溶出され280nmの吸光度でピークを示した分画をドデシル硫酸ソーダ存在下のポリアクリルアミドゲル電気泳動(SDS-PAGE)で分離し,Coomassie Brilliant Blueで染色した.いくつかの分画にはJEウイルス外被膜糖蛋白質V3 (E)と同じ分子量(54K)を示すバンドがSDS-PAGEで検出された.これらの分画は,破壊した精製ウイルス粒子からSepharose 6B又はSephadex G-150カラムを用いて,或はウイルス感染細胞乳剤からSepharose 6B又はHydroxylapatiteカラムを用いて分離され,ELISAによってウイルス抗原性を有することが示された.ウイルス粒子をSDSと2メルカプトエタノール(2ME)で溶解後,Sepharose 6Bカラムを用いた場合V3 (E)は最もよく分離された.ウイルス粒子を溶解してカラムで分離する前の試料にはSDS-PAGEで分子量54KのバンドとV1 (M)蛋白質に相当する分子量8Kの二つのバンドが染色された.Attempts were made to separate structural proteins of Japanese encephalitis (JE) virus from purified and disrupted virion or virus-infected Aedes albopictus, clone C6/36, cell homogenate. The specimens were applied on the columns containing various materials, such as glass wool, DEAE-Sephacel, Sepharose 4B and 6B, Sephadex-G 150, and Hydroxylapatite with various elution buffers. The eluted peak fractions of OD_280 were further analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE) followed by Coomassie Brilliant Blue (CBB) staining. In several fractions, the presence of a protein band was identified by SDS-PAGE with estimated molecular weight (Mw) of 54K, which corresponds to the envelope glycoprotein V3 (E) of JE virus. These fractions were obtained from disrupted virion by Sepharose 6B or Sephadex G-150, and also from infected cell homogenate by Sepharose 6B or Hydroxyl-apatite columns. These fractions were associated with virus antigenicities measured by the ELISA. The best separation of V3 (E) was observed in Sepharose 6B column fractions obtained from the virion disrupted by SDS and 2-mercaptoethanol (2ME). While, disrupted but unfractionated virion showed 2 stained bands with Mw of 54K and 8K, the latter corresponded to virion membrane protein V1 (M)