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標題: 果蠅前胸腺刺激激素基因之表現與功能分析
Expression and Functional Analysis of Drosophila Prothoracicotropic Hormone(PTTH) Gene
作者: 杜泳穎
Du, Yung- Ying
關鍵字: Drosophila melanogaster;果蠅;Prothoracicotropic Hormone (PTTH);baculovirus expression system;ring gland;ecdysteroidogenesis;前胸腺刺激激素;昆蟲細胞-桿狀病毒表現系統;環狀腺;蛻皮固醇合成
出版社: 生物科技學研究所
昆蟲前胸腺刺激激素 (Prothoracicotropic Hormone, PTTH) 在昆蟲發育過程中扮演刺激前胸腺分泌蛻皮固醇 (Ecdysteroids) 的重要角色。因由大腸桿菌表現系統表現之果蠅 PTTH 蛋白無活性,故改用昆蟲細胞-桿狀病毒表現系統 (baculovirus expression system) 生產具活性之重組蛋白。先由實驗中選殖出重組桿狀病毒vABhRpPTTH、vABhRhcmPTTH、vABhRpX並感染昆蟲細胞,經由比較不同啟動子對蛋白表現之差異、調整病毒感染濃度、及觀察不同天數重組蛋白表現量,發現於感染第四天培養液中可得到較佳重組蛋白,並以西方點墨法得知表現之重組PTTH分子量為52 kDa。為了確定重組PTTH蛋白具有生物活性,進一步完成果蠅環狀腺放射性免疫測定,確認其具有刺激果蠅環狀腺組織分泌蛻皮固醇之能力。重組蛋白在稀釋濃度10-5有最高之活性比例約2.57倍,並呈現劑量反應。由實驗結果得知此52 kDa重組PTTH蛋白和果蠅腦部萃取PTTH具有相似之生理活性,皆能刺激蛻皮激素之產生。

Prothoracicotropic Hormone (PTTH) is an insect neuropeptide which stimulates the secretion of molting hormone, ecdysone. Since the Drosophila PTTH from E. coli expression system without detectable activity, we tried to use baculovirus expression system to obtain functional PTTH. We have screened and isolated the recombinant baculovirus: vABhRpPTTH, vABhRhcmPTTH, and vABhRpX with different constructs derived from pABhRpX and pABhRhcmX vector. The molecular weight of expressed PTTH from Western blotting experiments shows 52 kDa, and negative control without any detectible PTTH. From the comparison of expression by different promoters, recombinant virus infection conditions, and time course studies, we found vABhRpPTTH 1 clone secreted rPTTH into culture medium at high-level on 4th day after infection and gave the best production. Subsequently, the biological function of recombinant PTTH was determined by in vitro ring gland assay/radioactive immunoassay. Dose response curve indicated that the rPTTH in 10-5 diluted medium significant induces ecdysone release 2.57 fold, under assay conditions. These results showed that recombinant PTTH could stimulate Drosophila ring gland ecdysteroidogenesis, which is comparable to the activity of native Drosophila PTTH.
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