碩士 / 國立海洋大學 / 水產生物技術研究所 / 88 / The sodA genes coding fore manganese superoxide dismutase from Vibrio cholerae O1 Inaba(-), Inaba(+) and O139 were cloned by PCR techniques using the degenerate primers designed from Mn-SOD conserved amino acid regions and random primed gene walking PCR techniques. Vibrio cholerae O1 Inaba(-) sodA gene was over-expressed in the heterologous host E. coli. BL21(DE3)pLysS using the pET20b(+) expression vector. The full-length gene was 615 base pairs long and encoded 205 amino acid residues. The recombinant protein was efficiently purified from the host cell lysate by His-tag affinity chromatography. The recombinant VCI(-)Mn-SOD protein was active and insensitive to inhibitors such as NaN3, H2O2, KCN and DDC.
Several gene specific primer pairs designed from nucleotide sequences of V. cholerae O1 Inaba(-) sodA were used to analyze the sodA gene probe specificity for Vibrio cholerae strain identification by cross-species PCR. Results indicated the specificity of the gene probe based on the upstream and downstream nucleotide sequences of V. cholerae sodA was very high. Vibrio cholerae O1 Inaba(+), Inaba(-), Ogawa(+), Ogawa(-), Non-O1 and O139 strains could be further distinguished from each other by randomly amplified polymorphic DNA analysis (RAPD).
The VCI(-)sodA -Eagh gene fusion was constructed by Vibrio cholerae O1 Inaba(-) sodA and growth hormone gene from Epinephelus awoara. The fusion gene was over-expressed in E. coli. host BL21(DE3)pLysS using the pET20b(+) expression vector. The over-expressed recombinant fusion protein was insoluble, but could be purified from the inclusion bodies by His-tag affinity chromatography under the denatured conditions.
| Identifer | oai:union.ndltd.org:TW/088NTOU0613009 |
| Date | January 2000 |
| Creators | Keng-Hao Chang, 張耿豪 |
| Contributors | Fu-Pang Lin, 林富邦 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 145 |
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