臺灣博碩士論文加值系統

　　利用聚合酉每鏈反應，複製出含石斑魚及香魚生長激素成熟胜　密碼的DNA，將此DNA接合到pET-20b(+)的表現載體上，在表現載體中，石斑魚及香魚生長激素受到T7啟動子的調控，經由IPTG的誘導，可以得到大量的重組生長激素。由這兩段PCR複製的DNA推測，可得分別是194個胺基酸的石斑魚重組生長激素和195個胺基酸的香魚重組生長激素，並且這個種重組生長激素都在碳端具有6個Histidine 胺基酸，以便於利用His-tag binding 樹脂管柱來進行純化工作。由於這兩種重組生長激素在大腸菌中大量表現的結果形成了內涵體，於是，利用0.4%的Sarkosyl將內涵體的重組生長激素溶解於萃取液中，以透析將Sarkosyl濃度降低後，再以His-tag binding 樹脂管柱純化得到每升約為10mg大於99%純度的重組生長激素。
　　為測試重組生長激素的活性，本實驗將不同劑量的重組生長激素，分別注射入莫三比克吳郭魚的腹腔中，發現每公克體重注射10μg的重組石斑魚生長激素提昇了吳郭魚肝臟中的IGF-I表現有十倍之多。為了更進一步測定重組生長激素的活性，分別以每隔兩週和每隔一週，注射不同劑量的重組生長激素於石斑魚苗，經連續測量魚體重量及魚體長度。結果發現在每隔一週注射一次的實驗組中，重組生長激素，對魚體的生長速率並沒有顯著的促進作用，但是在隔兩週注射實驗中，10μg劑量之重組石斑魚生長激素則具有促進生長之效益。

　　CDNAs encode mature grouper and ayu growth hormones were amplified by PCR from the grouper and ayu cDNA clones. The obtained PCR products were ligated into pET-20B(+) expression vector and expressed in E. coli, BL21(DE3) pLysS, under the T7 promoter regulation. Through IPTG induction, large amount of recombinant GH were produced and the size of the recombinant grouper and ayu GH are 194 and 195 amino acids, respectively. Since the expressed GH were packed in inclusion bodies, 0.4% Sarkosyl was used to dissolve the inclusion bodies and followed by dialysis to remove Sarkosyl. The recombinant peptides consist of 6 Histidines on the C-terminal for binding to His-tag binding resin. With this His-tag binding resin purification method, 10 mg/l recombinant GH peptides in more than 99% purity was obtained. In order to analyze the activity of recombinant GH, we injected tilapia intraperitoneally with 1μg 10μg per gram body weight of recombinant GH. We found that 10μg grouper recombinant GH induced higher level expression of IGF-I than 1μg grouper recombinant GH. However, injection of ayu recombinant GH into tilapia seems to have no promoting effect on the IGF-I expression level. Besides, we injected juveniles of grouper every week or every two weeks at concentration of 1μg and 10μg of these recombinant GH to each gram body weight of fish. We found that these two recombinant GH seem to have no growth promotion with weekly injection. However, with two weeks injection, 10 μg recombinant GH hormone promote fish growth very well.