碩士 / 國立高雄海洋科技大學 / 水產養殖研究所 / 104 / In recent years, fish has evolved miniaturization caused by inbreeding species and poor quality of brood fish that prolonged the culture period and increased the cost and risk of aquaculture farming. In order to enhance fish growth, previously studies had produced IGF-1 by E. coli, but it extremely forms inclusion body when overexpressed in E. coli. To prevent the formation of inclusion body in E. coli, the most effective method is to increase the solubility of IGF-1. In this study, Gram-negative bacteria secreted protein osmotically inducible protein Y (osmY) was used to improve the solubility of IGF-1. We fused IGF-1 to N- or C terminal of osmY to form IGF-osmY and osmY-IGF, respectively, and fused to pelB signal peptide as control (pelB-IGF). To confirm these proteins expression by western blot and verify the solubility of IGF-1 by SDS-PAGE. Our results showed that the solubility of IGF-OsmY (95 ±3.1%) was significant higher than osmY-IGF (66 ±0.8%) and pelB-IGF (3.97 ± 0.18%) ,pelB-IGF was almost found in inclusion body. We had purified osmY-IGF and IGF-osmY and rIGF-1 which was removed osmY from IGF-osmY with thrombin and then examined the effect of these proteins on grouper fin (GF-1) cells growth. We found that rIGF-1 could significantly enhance grouper fin cells proliferation, but IGF-1 fused with osmY affect the function of IGF-1 on GF-1 cells proliferation, the optimal concentration to enhance GF-1 cell proliferation was 40 nM in rIGF-1、300 n M in IGF-osmY and 300 nM in osmY-IGF. Then we had intraperitoneal injected 2μg/ g purified IGF-osmY and rIGF-1 into juvenile tilapia once a week, there were significant increases (p < 0.05) at 5 weeks after last IP injection in body weight ,body length and percent weight gain. The percent weight gain was 357.57 ± 32.16% for IGF-osmY, 375.59 ± 20.61 for rIGF-1, and 236.38 ± 56.32% for PBS. These results suggest that IGF-1 fused to osmY could enhance the solubility of IGF-1, but the steric hindrance between IGF-1 and osmY might affect the function of IGF-1 Moreover, the osmY fusion protein might overcome the problems that the inclusion bodies formation of proteins applied in aquaculture, such as vaccin and improve the availability of proteins applied in aquaculture in future.
| Identifer | oai:union.ndltd.org:TW/104NKIM0086002 |
| Date | January 2016 |
| Creators | SYU,JIA-NING, 許家寧 |
| Contributors | Cheng,Chiu -Min, 鄭秋敏 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 82 |
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