碩士 / 國立海洋大學 / 水產生物技術研究所 / 88 / The partial ORF of superoxide dismutase gene from Vibrio cholerae O1 Ogawa(+), Ogawa(-), and Non-O1 were obtained successfully by PCR techniques using the degenerate Mn-SOD No and Mn-SOD Co primers. The start codon and stop codon of the sod gene from these three Vibrio strains were found using the gene specific primers and random primed gene walking PCR techniques. The full-length sod gene from Vibrio cholerae O1 Ogawa(+), Ogawa(-), and Non-O1 were all composed of 615 base pairs and encoded 205 amino acid residues. There were only two amino acid residues difference among them. Vibrio cholerae Non-O1 sod gene was chosen and cloned into the expression vector pET20b(+) and was expressed in E. coli BL21(DE3)pLysS. The sod gene was induced by 0.4 mM IPTG at 25℃ and the recombinant protein was efficiently purified from the host cell lysate by His-Bind Resin affinity chromatography. The recombinant VCNO1Mn-SOD protein was active and insensitive to inhibitors such as NaN3, H2O2, KCN and DDC.
The Eagh-VCI(-)sodA fusion gene was constructed from Vibrio cholerae O1 Inaba(-) sodA and growth hormone gene from Epinephelus awoara. The full-length Eagh-VCI(-)sodA gene were composed of 1173 base pairs and encoded 391 amino acid residues. 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-Bind Resin affinity chromatography under the denatured conditions.
| Identifer | oai:union.ndltd.org:TW/088NTOU0613006 |
| Date | January 2000 |
| Creators | Tin-Hao Wang, 王廷豪 |
| 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 | 115 |
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