碩士 / 國立臺灣大學 / 動物學研究所 / 96 / Hemocytes play an important role in crustacean immunity. Generation of new hemocytes (hematopoiesis) is thus necessary to maintain homeostasis which is vital to crustaceans. In vertebrates, certain cytokines from various types of cells have been demonstrated to regulate hematopoiesis and immune responses. But in invertebrates, little is known about cytokines related to hematopoiesis. In the present study, we cloned an astakine gene from hemocytes cDNA of tiger shrimp (Penaeus monodon). It is 1,509 bp in length with a 5’-UTR of 143 bp, a coding region of 375 bp and a 3’-UTR of 991 bp. The deduced protein has 124 amino acid residues, including a signal peptide of 21 amino acid residues, one prokineticin domain, one epidermal growth factor-like subdomain and five pairs of disulfide bonds. The calculated molecular weight of the mature peptide is 11,295 Da and isoelectric point is 5.2. Based on Bayesian estimation, this molecule is phylogenetically most similar to astakine-related molecules of arthropod and the distant was found from vertebrate prokineticin and dickkopf molecules, the common among these sequences was that all contained cysteine-rich domain in C-terminal region. Nested RT-PCR showed that this molecule is expressed in eyestalk, subcuticular epithelium, gill, heart, hepatopancreas, lymphoid organ, intestine, muscle, nerve and hemocytes. Real-time PCR revealed that mRNA of the present molecule is expressed most in tissues with the highest hemocyte abundance. The recombinant protein was expressed by insect cell-baculovirus expression system. The authenticity of recombinant protein was examined by MALDI-MS/MS spectrometry. The molecular weight of C-terminally histidine-tagged recombinant protein is determined by ESI-MS as 12,107 Da, which is 10 Da less than the expected recombinant protein, suggesting formation of five pairs of disulfide bonds. Injection of recombinant protein into tiger shrimp promotes proliferation of hematopoietic cells and this phenomenon occurred early than control group. The present study may shed light on shrimp hemocyte culture and interpretation of hematopoiesis.
| Identifer | oai:union.ndltd.org:TW/096NTU05312003 |
| Date | January 2008 |
| Creators | Chun-Yi Hsiao, 蕭君儀 |
| Contributors | Yen-Ling Song, 宋延齡 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 56 |
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