從點帶石斑魚感染神經壞死病毒(grouper nervous necrosis virus)與否之魚隻進行PCR-selected subtractive hybridization的比較試驗，其中發現點帶石斑魚sparc (secreted protein acidic and rich in cysteine/osteonectin )基因為具有差異性的標的基因之一，並輔以RACE (rapid amplification of cDNA ends)的方法將點帶石斑魚SPARC cDNA全長序列選殖完成，其開放性讀架(ORF)為912 bp，可轉譯出含303個氨基酸的蛋白，此蛋白含有(1) N端之acidic domain，具有一段訊息胜肽；(2) follistatin-like domain，具有一銅離子結合序列-(K)GHK；(3)C端之EC domain，具有兩個會與鈣離子結合之EF-hand motif，並由不同物種間的SPARC氨基酸序列比對可發現該蛋白中各功能區域具有高度保留的特性。從所獲得之點帶石斑魚sparc基因建構表現質體以大量表現點帶石斑魚SPARC蛋白，再以此重組蛋白免疫兔隻以取得抗石斑魚SPARC蛋白之抗體。以含不同濃度重組蛋白之細胞培養液進行細胞培養發現SPARC具有影響細胞型態的特性，同時也具有抑制細胞貼附以及抑制細胞生長的功能。此外，也以細胞轉染(transfection)方式將點帶石斑魚sparc基因送入GF-1細胞株，發現所表現之SPARC蛋白可被分泌至細胞外並可於細胞外被切割，推測點帶石斑魚SPARC蛋白如哺乳動物之SPARC蛋白具有被細胞外蛋白酶作用以增強生物功能之特性。此外以RT-PCR方法偵測，發現當神經壞死病毒感染點帶石斑魚後會導致魚體內sparc mRNA表現量有減少的現象發生，這也將值得繼續探討SPARC與魚病及魚生理的相關性。

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.

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