碩士 / 國立成功大學 / 生物科技研究所碩博士班 / 94 / In the PCR-selected subtractive hybridization experiment of grouper, with NNV(grouper nervous necrosis virus) being infected or without, we discovered that sparc gene is one of the targeted genes expressed differently in these two population. By using the RACE method, we cloned grouper’s SPARC cDNA which consists of an open reading frame of 912 nucleotides encoding a 303 amino-acid protein. Alignment of grouper SPARC amino acid sequence with the various species, we found that it is highly conserved in every functional domain. The protein consists of an acidic domain in the N-terminus of the polypeptide as well as a signal sequence; a follistatin-like domain which is a cysteine-rich region contains a copper binding sequence-(K)GHK and a EC domain which contains two EF-hand motif located in the C-terminus of SPARC. Overexpression of recombinant grouper SPARC in E.coli BL21(DE3) and inoculating the rabbit with this protein are for the preparation of anti-grouper SPARC antibody. Also, after incubating GF-1 cells with E.coli expressing SPARC, we found that this protein has induced a change in cell morphology and elicited classic anti-adhesion and anti-proliferation characteristics. By expressing grouper’s sparc gene in GF-1 cells via transfection, we found that the recombinant protein was secreted and cleavaged in condition medium. We have found that the expression level of sparc mRNA in the grouper have decreased when infected with NNV which was detected by RT-PCR.
| Identifer | oai:union.ndltd.org:TW/094NCKU5111008 |
| Date | January 2006 |
| Creators | Jhong-Jian Liao, 廖中健 |
| Contributors | Tzong-Yueh Chen, 陳宗嶽 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 70 |
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