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Title: | 具活性重組魚類生長激素的生? Production of Biologically Active Recombinant Fish Growth Hormone |
Authors: | Ming-Hui Hish 謝銘煇 |
Publication Year : | 1995 |
Degree: | 碩士 |
Abstract: | Growth hormone(GH)具有刺激動物生長之作用,可惜天然的GH產量有限;若以基因工程的方法便可由Escherichia coli得到大量rGH (recombinant GH)。但是得到的rGH並不具有生物活性,它們以inclusion bodies(I. B.)的形式存在,必須經過適當地恢復原性(renaturation)處理才能具有活性。因此,本實驗嘗試了各種不同因數來探討恢復rGH活性,溶解性及其產量的效果。結果發現最好的條件分別是:pH10, 0℃, I.B。溶解濃度為0.1mg/ml,添加2mM oxidized glutathione(GSSG), 2mM reduced glutathione(GSH)和0.1ML-arginine的混合(2G+0.1L)及採用24小時之內將 guanidine hydrocholide (Gdn.Cl)濃度由6M降到70mM的直接透析方式最好。然而當個別的最佳條件結合在一起時,最好的條件則為:pH10,0~4℃, I.B。溶解濃度為0.5mg/ml,添加2G+0.1L及採用直接透析的方式。如此條件得到的具folding rGH的比率約佔I.B.量的20%左右。另外,若回收透析過程中產生的不溶解物質,經再溶解透析後,可以再得到具有folding的rGH;如此重復回收處理至少可得到佔原來I.B.量17.7%的folding rGH。並且由實驗中得知,第一次透析時folding rGH的產生量很低(佔I.B.量的1.83%),但從第二次透析後,其產量便急劇地提高(4.43~8.86%)。在菌體製造rGH方面,培養100ml含生長激素表現載體的E. coli BL21(DE3)(Tsai et al., 1995)在經過1mM IPTG誘發後20個小時,其產生rGH的量最高;I.B.乾重為73mg。然而,若用0.1mM β-lactose誘發,I.B.重量則顯著增多(P<0.05)到95mg,並且經過檢驗得知兩者等量I.B.中所含有的rGH比率是相同的,細菌密度也一樣。根據此條件,每培養100ml的E. coli BL21(DE3)約可得到19mg的folding rGH。生產出來的folding rGH以對烏魚(Mugil cephalus)稚魚施行腹腔注射的方式進行生物活性測試。注射期為12週(每2週注射一次),後續觀察期4週。注射後第4週(第2次注射)後,低劑量(0.1ug rGH/g)組的平均體重和控制組(不處理)相比已開始有顯著性差異(P<0.01),而平均體長在第6週(第3次注射)後才開始有差異;低劑量組比中(1ug rGH/g)或高劑量組(2ug rGH/g)更能有效地促進烏魚稚魚的成長,該組的淨增重比控制組增加了138%,飼料效率增加了65%,在停止注射(第12週)後其促進成長的效力仍持續維持至少到第16週。因此,本實驗的結果證實folding rGH的確具有生物活性,並且只要極少量便能促進魚類顯著的增長,將來運用在水產養殖上必定極具經濟效益。 Growth hormone (GH) enhances the growth rate of aquacultured fish. However, it is hardly to extract native GH from pituitary glands. Recombinant GH (rGH) produced by Escherichia coil may solve this difficulty. But E. coli-produced rGH exists in inclusion bodies (TB.) without folding structure. In order to obtain biologically active rGH, we found that rGH had to be renatured respectively at the following parameters for refolding (1) dialysis buffer was under the condictions of pH 10, 0℃ adding the mixture of 2mM oxidized glutathione, 2mM reduced glutathione and 0.1M L-arginine (2G+0.1L); (2) the concentration of I.B. was 0.1mg/ml; and (3) dialysis starting at 6 M guanidine hydrocholide (Gdn.Cl) but ending at 0.07M Gdn.Cl within 24h. When these parameters were combined, the optimal conditions was: pH 10; 0~4°C; the concentration of I.B. was 0.5mg/ml; adding 2G+0.1L and taking the method of direct dialysis. About 20% of folding rGH were obtained from I.B. In addition, at least 17.7% of folding rGH were got from the remainder I.B. after reprocessing. We found that the yield of folding rGH was very low (1.83%) in the first dialysis but the yield increased rapidly (4.43~8.86%) after the second dialysis. We cultured 100 ml of E. coli BL21 (DE3) containing GH-expression plasmid (Tsai et al., 1995), about 73.43±5.47mg I.B. (dry weight, n=3) were produced after 20h induction by 1mM IPTG. However, if the bacteria is induced by 0.1mM β-lactose, 95.3±3.43mg of I.B. was obtained (significant differences at P<0.05 level), and resulted in 19.06±0.69mg folding rGH. Same amount of I.B. from two kinds of inducer were analyzed by SDS-polyacrylamide gel electrophoresis, we found the percentage of rGH in I.B. was identical. The processed rGH was administrated biweekly by intraperitoneal injections to juvenile striped mullet (Mugil cephalus) for 12 weeks, and the succeed observed periods were 4 weeks. The average body weight of low dosage group (0.lug rGH/g) has significant differences (P<0.05) from that of control group (without treatment) since the fourth week after injection (the second injection), but the significant differences in average body length didn’t happen till the sixth week (the third injection). Low dosage group has a greater degree of growth enhancement than that of middle (lug rGH/g) and high dosage groups (2ug rGH/g). The net weight gain and feed efficiency of low dosage group resulted in increases in 138% and 65%, respectively, relative to the control group at week 12 onwards. After cease of injection, the effect of low dosage rGH on growth enhancement continued at least till the 16th week. Results prove that folding rGH has a biological activity even in the extremely low quantity. Therefore, rGH surely will achieve the better economic benefit in the aquaculture. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/76183 |
Fulltext Rights: | 未授權 |
Appears in Collections: | 漁業科學研究所 |
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