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標題: | 應用基因重組生長激素促進魚類生長 Growth Enhancement of Fish by Recombinant Fish Growth Hormone |
作者: | Bing-Chang Lee 李秉璋 |
出版年 : | 1999 |
學位: | 碩士 |
摘要: | 天然的生長激素( growth hormone , GH ) ,是由腦下垂體萃取而得,來源相當不易;若以基因工程的技術,選殖出 GH 的 cDNA ,即可經由原核系統 Escherichia coli及真核系統 Pichia pastoris大量生產 recombinant GH ( rGH )。 培養 100 ml 含有黃鰭鯛( Acanthopagrus latus ) GH cDNA 表現載體的 E . coli 菌液,在菌體打破後,以離心法沈降,得到約 88 . 57 士 0 . 42 mg 的內涵體,再以 6M Guanidine HCl 溶解後,經過透析純化出 33 . 66 士 0 . 16 mg rGH 。以質譜儀( mass spectrometer )及 Densito-pattern analyzer 偵測出分子量為 2 , 1397 kDa ,純度約 80 %。純化後的 rGH 以浸泡方式促進石斑魚( EPinephelus malabaricus )魚苗成長,實驗進行 17 天後,結果顯示浸泡 30 分鐘、 500 μg / liter ,效果最好,增重率比對照組高出 170 % ,體重高出約 69 %。 將帶有 ypGH cDNA 的質體( pSJ04 )轉形至 P pastoris 酵母中,經營春缺陷培養基( RDB plate )及抗生素培養基( YPD-G418 , 4 mg / ml )篩選出酵母轉形株( BC01 )。以 100 %甲醇誘發轉形酵母菌株表現 rGH ,經 SDS-PAGE 發現在分子量約 22kDa 處,有一對照組所無之蛋白質條紋:進一步以 rabbit anti-ypGH polyclonal antibody進行 western blot 分析,結果為免疫正反應; 800 ml 菌體培養液,在誘發 72 小時復,經 ELISA 測定轉形酵母菌其萃取液中 rGH 含量為 0 . 425 士 0 . 014μg/ml 。大量培養後的轉形酵母菌經冷凍乾燥,依比例添加於飼料中,餵食吳郭魚( Oreochromis niloticus ×O. aureus )魚苗,實驗進行 28 天後,結果顯示添加 0 . 16 %含 rGH 之酵母菌對促進魚苗生長最為明顯( P < 0 . 01 ) ,增重率比對照組高出 257 % ,體重高出約 101 % 。 本實驗證實了利用 E . coli及 P. pastoris 表現系統可以大量生產具生物活性的 rGH ,並能定量出轉形酵母所內含之 rGH 。此外,也提供了利用浸泡與餵食 rGH 的方式於水產養殖之可行性。 It is difficult to gain pure growth hormone (GH) via extraction of animal pituitary glands. However, with genetic engineering technology, large amount of recombinant GH (rGH) can be obtained through either prokaryotic or eukaryotic expression system. In prokaryotic system, 33.66 ± 0.16 mg of rGH was obtained out of 100 ml E. coli culture containing yellowfin porgy GH (ypGH) cDNA. Mass spectrometer and densito-pattern analyzer analysis show that the molecular weight of the E. coli-expressed rGH is 2,1397 kilo Dalton (kDa) and constitutes 80% of the purified protein. Biological activity of the rGH was assayed via immersing fries of grouper (Epinephelus malabaricus) in seawater containing rGH. After 17 days of experiment, results showed that 30 min immersion with 500 mg rGH/liter promoted fries growth most prominently. Compared with control group, the weight gain rate and average body weight were 170% and 69% higher, respectively. In eukaryotic system, plasmid (pSJO4) containing ypGH cDNA was introduced into yeast (P. pastoris). The transformed yeast was selected by nutrient limited medium (RDB) and antibiotic-containing (YPD-G418, 4 mg/ml) culture plates. One unique protein band of 22 kDa was identified exclusively in experimental group in SDS-PAGE analysis, and the 22- kDa protein reacted positively against rabbit anti-ypGH polyclonal antibody in western blotting analysis. After being induced for 72 hr, 800 milliliters of the transformed yeast were subjected to ELISA. The rGH concentraction was 0.425 ± 0.0 14 μg/ml. The transformed yeast harvested from large-scale preparation was frozen-dried, added with various proportion to the feed powder as a supplements. After 29 days of experiment, result showed that feeding with 0.16% transformed yeast containing rGH enhanced the growth of tilapia fries (Oreochromis niloticus × 0. aures) most prominently (P<0.0l). Compared with control group. the weight gain rate and average body weight were 257% and 101% higher, respectively. The experiments showed above indicate that using both E. coli and P.pastoris expressing systems, large amount of biologically active rGH could be obtained, and the quantity of rGH in the transformed yeast could he measured. Furthermore, it is feasible to apply rGH to aquaculture by either immersing or feeding. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/75076 |
全文授權: | 未授權 |
顯示於系所單位: | 漁業科學研究所 |
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