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Western blotting法によるRocioウイルス感染細胞および培養液内のウイルス特異的ポリペプチド抗原の検出


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Title: Western blotting法によるRocioウイルス感染細胞および培養液内のウイルス特異的ポリペプチド抗原の検出
Other Titles: Detection of Rocio Virus-specific Antigenic Polypeptides in Virus-infected Cells and Culture Fluid by Western Blotting Method
Authors: SAKURAI, Tiyo / 分藤, 桂子 / 五十嵐, 章
Authors (alternative): Sakurai, Tiyo / Bundo, Keiko / Igarashi, Akira
Issue Date: 30-Sep-1985
Publisher: 長崎大学熱帯医学研究所 / Institute of Tropical Medicine, Nagasaki University
Citation: 熱帯医学 Tropical medicine 27(3). p129-140, 1985
Abstract: フラビウイルスの一つであるRocioウイルスを28℃のヒトスジシマカ培養細胞クローンC6/36細胞,および37℃のBHK21とVero細胞で増殖させた. C6/36細胞では感染後36時間で10^8PFU/ml以上のウイルス産生が見られたが,Vero細胞とBHK21細胞でのウイルス産生は各々10^8PFU/mlと10^7PFU/mlであった.C6/36細胞ではウイルス感染価とELISA抗原価は共に感染細胞乳剤よりも感染培養液中で高かったが,BHK21とVero細胞では細胞乳剤中のELISA抗原価の方が感染培養液中の値よりも高かった.ウイルス感染C6/36細胞培養液と細胞乳剤とをSDS-ポリアクリルアミドゲル電気泳動後,Western blottingによってウイルス特異的な抗原性を有するポリペプチドを検出した.還元剤を加えない条件下で試料を作成した場合,感染培養液中の主なポリペプチドは分子量55Kダルトンであり,他に分子量170K, 140K, 115K, 100K, 90K, 52K,および23Kダルトンの7種のポリペプチドが少量ずっ検出さた.一方,感染細胞内には,分子量105K, 83K, 60K, 56Kダルトンの4つのペプチドが多量に検出された他,分子量170K, 140K, 115K, 24K, 23Kダルトンの5つのポリペプチドが少量づつ検出された.これらのポリペプチドのうち105Kと60Kのものは感染後12時間目より出現しはじめ,他のポリペプチドも感染後24~36時間では充分に検出されるようになった.還元状態で試料を作成すると感染細胞内の分子量170K, 60K, 56K, 24K, 23Kのポリペプチドおよび感染培養液中のポリペプチドは検出されなくなるか量的に検出されにくくなる.それに対して,感染細胞内の分子量140Kと83Kのポリペプチドは易熱性であるらしい.感染培養液中の分子量55Kと52KのポリペプチドはWestern Blotting法で他のフラビウイルスと交叉反応を示し,恐らくウイルス外被膜の糖タンパクであろうと思われる. / Rocio virus, a member of flaviviruses, was grown in cultured Aedes albopictus, clone C6/36, cells at 28℃, or BHK21, and Vero cells at 37℃. Over 10^8 PFU/ml of virus production was obtained in C6/36 cells after 36 hours of infection, while the virus titers in Vero and BHK21 cells were 10^8 and 10^7 PFU/ml, respectively. Virus infectivity and ELISA antigen titers in the infected C6/36 cell homogenate did no exceed that in the infected fluid, however, the ELISA titers were higher in the infected cell homogenates than in the fluids in the case of BHK21 and Vero cells. Virus infected C6/36 cells and fluids were analysed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by the Western blotting to reveal antigenic and virus-specific polypeptides. Under nonreducing conditions, major antigenic component in the infected fluid was 55K dalton polypeptide with 7 minor components of 170K, 140K, 115K, 100K, 90K, 52K, and 23K daltons. While, there were 4 major (105K, 83K, 60K, and 56K daltons) and 5 minor (170K, 140K, 115K, 24K, and 23K daltons) polypeptides in the infected C6/36 cells. Some of these polypeptides (105K and 60K) began to appear at 12 hours and others were fully observed at 24-36 hours after infection in C6/36 cells at 28℃. The 170K, 60K, 56K, 24K, and 23K polypeptides in the infected cells disappeared or markedly reduced when the specimens were treated under reducing condition, so were the polypeptides in the infected fluid. On the other hand, 140K and 83K polypeptides in the infected cells appeared to be heat sensitive. The 55K and 52K polypeptides in the infected fluid were cross-reactive to several flaviviruses by the Western blotting method, and could probably be the envelope glycoproteins of the virus.
URI: http://hdl.handle.net/10069/4412
ISSN: 03855643
Type: Departmental Bulletin Paper
Appears in Collections:Volume 27, No. 3

Citable URI : http://hdl.handle.net/10069/4412

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