| Summary: | 碩士 === 大葉大學 === 分子生物科技學系碩士班 === 93 === ABSTRACT
Antifreeze proteins (AFPs) were first found in the blood and tissues of fish in the polar region and were characterized by lowering freezing temperature and preventing ice crystal formation, so which help fish to survive under low temperature in the polar region. The aims of this research were to investigate (1) the production capacity and bioactivity of type I antifreeze protein. Type I antifreeze protein sequence of winter flounder was constructed on the pET-28b() vector, after transformed into E. Coli, followed by induction by IPTG and purification, its production capacity and effect on the cryopreservation of zebrafish embryos were analyzed; and (2) the cryotolerance of zebrafish with type I AFP on pEGFP-N1 and pAAV-CMV-1RES- hrGFP-Neo vectors. The production of AFP was higher at 2 hours after induction with 0.4 mM IPTG than that at 1 hour ( 33.1 mg vs. 7.4 mg ). In general, the amount of AFP production was increased with culture peroids, however, the production at 3～24 hours showed no significant differences ( 39.9～65 mg, p＞0.05 ). The average concentration of AFP was 65 mg when AFP was induced in one liter of bacterial suspension for 6 hours. Six groups of various concentrations ( 0, 1, 5, 10 and 15 mg/mL ) of AFP and 5% glycerol were applied as cryoprotectants to freeze the zebrafish embryos at -10C. The survival rate was determined by using fluorescent dye. The results showed the survival rates of 0, 1, 5, 10, 15 mg groups and 5% glycerol group were 70.2%, 82.4%, 81.5%, 83.4%, 84.3% and 90.2%, respectively. Though higer surviral rate was observed in 5% glycerol group, no significant differences existed between groups ( p＞0.05 ). The success of cryopreservation of embryos depends on the optimal concention of AFP and cooling rate. The survival rates of zebrafish embryos injected with different constructions of pEGFP-N1 (control), pEGFP-N1-AFP, pAAV-CMV-IRES-hrGFP-Neo (control) and pAAV-CMV-IRES-hrGFP- Neo-AFP were 58.1%, 60.6%, 43.8% and 34.3%, respectively; and the fluorescence expression rates of pEGFP-N1 and pEGFP-N1-AFP groups were significant higher than those of pAAV-CMV-IRES- hrGFP-Neo and pAAV-CMV-IRES-hrGFP-Neo-AFP groups ( 40.6% and 52.9% vs. 9.6% and 6.1%, p＜0.01 ). Two hundred and seventy six bp were cloned from transgenic zebrafish, from which 120 bp were identified, and had a similarity of 43.4% when compared to AFP sequence. Hopefully, a AFP-transgene zebrafish model can be established through this study, and the technique can be applied to other fishes for preventing them from chilling injury in the fishery in Taiwan.
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