| Project/Area Number |
62480154
|
|---|
| Research Category |
Grant-in-Aid for General Scientific Research (B)
|
| Allocation Type | Single-year Grants |
|---|
| Research Field |
細菌学
|
|---|
| Research Institution | The University of Tokushima |
|---|
Principal Investigator |
OHNISHI Yoshinari The University of Tokushima, Dept.of Bacteriol., Professor, 医学部, 教授 (10037400)
|
|---|
| Co-Investigator(Kenkyū-buntansha) |
KINOUCHI Takemi The University of Tokushima, Dept.of Bacteriol., Lecturer, 医学部, 講師 (80136217)
ONO Tsuneko The University of Tokushima, Dept.of Bacteriol., Assistant Professor, 医学部, 助手 (40035514)
AKIMOTO Shigeru The University of Tokushima, Dept.of Bacteriol., Lecturer, 医学部, 講師 (10159337)
|
|---|
| Project Period (FY) |
1987 – 1989
|
| Project Status |
Completed (Fiscal Year 1989)
|
| Budget Amount *help |
¥6,300,000 (Direct Cost: ¥6,300,000)
Fiscal Year 1989: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 1988: ¥1,500,000 (Direct Cost: ¥1,500,000)
Fiscal Year 1987: ¥3,800,000 (Direct Cost: ¥3,800,000)
|
|---|
| Keywords | Bacteroides / pathogenicity / gene cloning / neuraminidase / DNA sequence / mobilization / electroporation / positive-selection vector / 偏性嫌気性菌 / バクテロイデス / 遺伝子発現 / 遺伝子クローニング / neruaminidase / エレクトロポレーション / 組換え体選択ベクター / Bacteroides gingivalis / Bacteroides fragilis / トリプシン様プロテアーゼ / neuraminidase / DNase / shultle vector |
|---|
| Research Abstract |
1. A gene encoding neuraminidase of Bacteroides fragilis strain YCH46 was cloned into the cosmid vector pHC79. The neuraminidase gene nanH was subcloned from the cosmid and was located within a 1.8-kilobase-pair XhoI-KpnI restriction endbnuclease fragment. DNA sequencing of the insert revealed an open reading frame which encodes 296 amino acids. The predicted protein sequence had no marked similarity to known bacterial neuraminidase sequences. A 5.4-kb BamHI-SaII fragment containing the nanH gene was cloned into the Escherichia coli-Bacteroides shuttle vector pVAL-1. The resulting plasmid, pKK105, was mobilized from E. coli strain HB101 to Bacteroides uniformis strain BU1001 by the conjugative plasmid R751.
The neuraminidase activity in crude cell extracts from strain BU1001 carrying pKK105 was found to be 1600-fold greater than that found in strain HB101 carrying the lame plasmid.
2. High voltage electroporation was applied to transformation of B. thetaiotaomicron strain 5482 using the shuttle plasmid pVAL-1. With the optimized procedure, B. thetaiotaomicorn was transformed with an efficiency up to 3xlO^5 transformants per mug pVAL-1 plasmid DNA.
3. A plasmid vector pUCST5 carrying a lethal gene pnd was constructed for the use of positive-selection of recombinant plasmids.
|
