碩士 / 國立彰化師範大學 / 生物學系 / 100 / CHH (crustacean hyperglycemic hormone) was originally identified in a crustacean neurosecretory complex, the X-organ/sinus gland. In addition, we purified the native CHH1 and CHH1-L (CHH1-like) from neurosecretory tissues. Tissue extracts (sinus gland: SG; thoracic ganglia: TG) were chromatographically separated. CHH1 and CHH1-L, two structural variants of the crustacean hyperglycemic hormone family identified in the crayfish (Procambarus clarkii), and assays of biological activity showed CHH1-L are functionally different from CHH1, with respect to hyperglycemic activity. In this study, recombinant proteins (rCHH1 and rCHH1-L) were produced by an E. coil expression system, with attempts to increase rCHH1 and rCHH1-L yields by decreasing amount of rare codons, change either stability of mRNA secondary structure or tag fusion protein in the N-terminal. In results, expression with pelB-leader protein or decreases amount of rare codons, that can increase the rCHH1 and rCHH1-L yields, respectively. And then refolded, purified, and confirmed by western blotting and mass spectrometric analyses. In addition, Circular dichromatic spectra of rCHH1 and rCHH1-L indicate they are rich in α-helixes (30.5 % and 41 %, respectively). To purified rCHH1 was C-terminally amidated (rCHH1amide) in accordance with native counterpart. Functionally, rCHH1amide at 10 pmole/animal elicited significant hyperglycemic responses, that likes native CHH1 response. In the other hand, present study examined the effects of white spot syndrome virus (WSSV) on the CHH and CHH-like peptide system, that P. clarkii challenged by the white spot syndrome virus (WSSV), WSSV genomic DNA was first detected at 12 hr. At 48 and 72 hours of post challenge, the viral load increased substantially. The CHH1 protein levels in the eyestalk ganglia were significantly decreased after challenge at 24 and 48 hr, but CHH1-L protein levels in the thoracic ganglia did not change significantly after challenge. The WSSV-activated peptide systems might be involved in mediating adaptive processes (e.g., hyperglycemic response) in response to pathogen-related stresses or in the processes of immune defense against invading pathogens.
| Identifer | oai:union.ndltd.org:TW/100NCUE5112015 |
| Date | January 2012 |
| Creators | 陳彥州 |
| Contributors | 李奇英 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 122 |
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