Expression and Functional Analysis of Drosophila Prothoracicotropic Hormone(PTTH) Gene
| Autor: | Yung- Ying Du, 杜泳穎 |
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| Rok vydání: | 2004 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 92 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. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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