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題名 | 利用基因工程量產斑馬魚腦型芳香酶=Overexpression and Purification of Recombinant Zebra Fish Aromatase (Brain Form) with Genetic Engineering |
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作者 | 林念儀; 郭應誠; Lin, Nien-yi; Guo, Ing-cherng; |
期刊 | 動物園學報 |
出版日期 | 20011200 |
卷期 | 13 2001.12[民90.12] |
頁次 | 頁11-21 |
分類號 | 387.15 |
語文 | chi |
關鍵詞 | 斑馬魚; 腦型芳香酶; 重組蛋白質; Zebra fish; Brain from aromatase; Recombinant protein; |
中文摘要 | 芳香?在細胞內催化雄性素為動情素,在動物的生殖生理上扮演重要的角色。近來在斑馬魚發現有別於卵巢型芳香?的腦型芳香?,可能對於腦的性別分化、動物性行為的表現以及體內性內泌素的回饋機制等有重要意義。本研究即利用基因工程量產斑馬魚腦型芳香?,將藉以研究其功能。首先利用具有EcoRI切位的特異性引子對與斑馬魚腦型芳香?之cDNA進行聚合?連鎖反應,得到407 bp的DNA片段,將此產物與蛋白質表現載體pET29a進行接合反應,後經轉形作用移入大腸桿菌JM109內生產表現質體。所獲質體經由限制?切割、核酸定序、並確認胺基酸讀碼後,確定得到腦型芳香?表現質體。將此質體轉入大腸桿菌BL21的三個品系:BL21(DE3)、BL21(DE3)pLysS、BL21(DE3)pLysE,並利用三種濃度的IPTG(0.1mM、1mM、10mM)於七個時間點(0、1、2、3、4、5、6小時)嘗試誘導重組蛋白質表現。結果以BL21(DE3)pLysE品系的大腸桿菌在0.1mM IPTG 六小時的誘導下,能最有效率地表現腦型芳香?的重組蛋白質,接著再以NTA-親和管柱進行重組蛋白質的純化。本研究成功地利用大腸桿菌量產斑馬魚腦型芳香?的重組蛋白質,並加以純化。 |
英文摘要 | Cytochrome P450 aromatase converts androgens into estrogens. The newly identified brain form aromatase are proposed to play an important role in brain sexual differentiation, control of sexual behavior and steroids feedback mechanism. To further study its functions, we produced recombinant brain form aromatase with genetic engineering in this paper. The polymerase chain reaction was performed specific EcoRI-tailed primers and brain form aromatase cDNA of zebra fish. After EcoRI digestion, the PCR product (407bp) was ligated with a protein expression vector, pET29a, linearized with EcoRI and dephosphorylated with CIAP. The transformants were screened with EcoRI for insertion, with KpnI for orientation, and sequencing. The overexpression of 24kDa recombinant brain form aromatase, packed in inclusion body, reached to the highest level in BL21(DE3)pLysE 6 hours after 0.1mM IPTG induction. Two denature buffers, containing 4.5M urea pH7.0 and 6M urea pH 7.0 respectively, efficiently dissolved this overexpressed protein. Furthermore, the dissolved recombinant brain form aromatase was purified by affinity column. Our study successfully overexpressed and purified recombinant brain form aromatase of zebra fish with genetic engineering. |
本系統之摘要資訊系依該期刊論文摘要之資訊為主。