Production of recombinant Human Serum Albumin in rice and its application
| Autor: | Yu-Ning Chang, 張佑寧 |
|---|---|
| Rok vydání: | 2016 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 104 Recombinant proteins are important in pharmaceutical market. Recombinant protein produced in plant cells has advantage in bioactivity and low cost, and was not affected by animal pathogen contamination. Therefore, a well developed rice suspension cell expreaaion system was chosen to produce recombinant proteins for medical usage. This protein expression system features for its secretion of recombinant protein to cell culture, and can eliminate the beak down of cells for an easy downstraem protein purification. In addition, rice cells do not have negative feedback inhibition of human proteins to maintain the recombinant protein yield. Human Serum Albumin (HSA) is a 66.5 kDa of non-glycosylated protein, and is accounts for about 60% of the total plasma protein. HSA maintains blood osmolarity and increases half-life of it bound drugs. Previous research has shown that a variety of short half-life growth factors can increase their protein stability after fused with HSA protein, including the unstable epidermal growth factor (EGF) that can stimulate the proliferation of epidermal cells for wound healing and cosmetics. Our previous study indicated that after fused with HSA, the recombinant HAS-EGF (H - EGF) protein can be stored at 4℃ after secreted from transgenic rice suspension cell for more than 4 months. In this study, a rice recombinant protein production system was applied to produce transgenic cell lines and regenerated plants with high yield of HAS. Six transgenic rice plants were sucesfully regenerated via agrobacterium-mediated gene transformation, and the cell line HSA a10 produced 1.2 times more HSA than previous cell lines in the lab. To benefit commercial application, both HSA and H-EGF transgenic suspension cell lines were selected for cryopreservation. After comparsion osmotic pretreatment by either SGP or SGD, SGD showed a better protection for rice suspension cells. By selection of small to big cell mass of suspension cell lines, the average cell mass of H –EGF-43 was less than 1 mm diameter, the average diameter of cell mass for HSA -a1 and -a3 were from 1 to 1.5 mm, and HSA - a9 and -a10 were the biggest in cell mass, 2-3 mm, were compared for their re-growth ability after one day of liquid nitrogen storage.The survival rate of small size H-EGF-43 was 90%, medium size of HSA - a1 and HSA - a3 was 80%, and the big size of HSA - a9 and HSA - a10 was 60%, suggesting that cell mass about 1 mm is good for cryopreservation of rice suspension cells in a cell bank. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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