| Summary: | 碩士 === 國立宜蘭大學 === 生物技術與動物科學系動物科學碩士班 === 106 === Mycotoxins are secondary metabolites produced by fungi. Deoxynivalenol (DON) produced by Fusarium graminearum and Fusarium colmorum is a common mycotoxin in economic animal feed. Its chemical structure is thermally stable and cannot be effectively removed during feed processing. On animal, DON inhibit protein synthesis, health status of the gastrointestinal tract and the brain, induce feed refusal, organ damage, increased disease incidence, and malabsorption of nutrients. It seriously affects the health state of animals and humans. The toxins are rapidly absorbed into the small intestine and then distributed through the blood to tissues, such as liver, kidneys and brain, leading to affect cell membrane structure and oxidative stress. The purpose of this experiment is to clone the gene for DON degrading enzyme and express recombinant protein through Pichia pastoris system, the DON degrading enzyme gene fragment are isolate from the soil. In this study the gene is obtained by chemical synthesis, and constructed into the yeast expression vector. This expression system has the characteristics of being easy to culture and capable of expressing recombinant proteins in high amounts. This gene has been successfully constructed into yeast expression vectors, and the correctness of the gene is confirmed by sequencing. Further, the vector was introduced into the yeast genome by electroporation. After electrophoresis analysis and antibiotic screening, the gene was inserted in the yeast. In this experiment, the transformed yeast has been successfully confirmed the recombinant protein (65 kDa) by c-Myc antibody, the molecular weight between tri101 enzyme and recombinant protein are identity. The total length of Tri101 gene is 1356 bp and the yeast gene is 351 bp. The converted amino acid size is 65 kDa. The results show that the recombinant protein has ability to degrade deoxinivalenol. In the future, this yeast expression system can be used to secrete recombinant protein of DON degrading enzyme, and further test its enzyme activity and animal experiments for verification.
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