碩士 / 國立彰化師範大學 / 生物技術研究所 / 100 / Peroxiredoxin (Prx), a new family of antioxidant enzymes, which can be induced by cellular oxidative stress and associated primarily with the removal of H2O2. Prx1 as a pivotal regulator in controlling neuronal differentiation in the spinal cord, but their distributions and roles have not been resolved clearly in the embryonic development. In RAW 264.7 mouse macrophage cells Prx1 was up-regulated on the mRNA and protein level by LPS. The present study was undertaken to determine the role of Prx1 in zebrafish embryonic development and to investigate the gene expression and oxidative damage of Prx1 in the zebrafish in response to LPS. We have expressed and purified recombinant zebrafish Prx1 from E. coli BL21 (DE3). The recombinant Prx1 was obtained by affinity chromatography of His-tag and the yield was about 6 mg in 1 L culture. The gene contains an open reading frame which encodes a protein of 199 amino acid residues with calculated molecular mass of 24 kDa. The recombinant Prx1 retained about 42% activity at 70℃ for 10 minutes, but lost its activity at 80℃. It was stable under a broad pH range from 2.2 to 11 and pH 4 was the highest activity. The enzyme samples were incubated with imidazole of different concentrations and 0.4 M retained about 63% activity. RT-PCR and western blot were used to analysis Prx1 gene and protein expression during embryonic development. The results showed Prx1 mRNA and protein level with constant expression from 0 to 48 hpf during embryonic development. The embryos were treated with LPS (10μg/ml) for 48 hours to induce oxidative stress. The results showed Prx1 mRNA and protein have no significant difference when compared with WT. The Prx1 gene expression knock down by injecting morpholino into 1-cell stage embryos to confirm its physiological function. We also try to increase Prx1 to protect embryos away from oxidative stress by microinjection, and results showed Prx1 can protect embryos. Our data indicated that Prx1 may be important for the embryonic development of zebrafish.
| Identifer | oai:union.ndltd.org:TW/100NCUE5108001 |
| Date | January 2011 |
| Creators | Han-Chang Chou, 周漢璋 |
| Contributors | Chuian-Fu Ken, 耿全福 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 114 |
Page generated in 0.0085 seconds