本實驗以幾丁質酶高保留區域的退化性引子，透過RT-PCR篩得點帶石斑魚兩種幾丁質酶gchi1與gchi2片段基因，經由5’與3’ RACE獲得基因全長，推演出的胺基酸序列，根據資料庫序列比對後，顯示兩者都是屬於醣類水解酵素18家族，其序列亦具有典型的幾丁質酶結構，包括：signal peptide、hinge region、chitin binding domain。以此引子作標幟，發現只在胃部偵測到gchi1與gchi2，再藉由免疫組織化學染色法分析石斑魚胃的組織切片，則進一步指認Gchi與Gchi2表現在胃腺細胞。接著在點帶石斑魚胃之粗萃取蛋白測出具水解幾丁質的活性，進一步以陰離子交換層析管柱及膠體過濾管柱純化出具高活性的幾丁質酶，藉著質譜蛋白質鑑定，以及幾丁質酶抗體免疫原性的辨認，確認純化的幾丁質酶即為所選殖的gchi1基因之蛋白質產物。將Gchi1進行N端定序後，並與上述轉殖得到之基因核酸序列比對，發現其signal peptide已被切除。另外，分析幾丁質酶之表現調控，則是以蝦子餵食點帶石斑魚時發現，gchi1與gchi2 mRNA以及Gchi1表現量都有增加。由於Gchi1與Gchi2在胃腺細胞表現，並且在餵食蝦子時增加表現，故推測Gchi1與Gchi2是作為幫助消化分解幾丁質的幾丁質酶。

Two chitinase genes (gchi1 and gchi2) were identified from orange-spotted grouper, Epinephelus coioides by RT-PCR. Their full-length cDNAs were obtained by 5’ and 3’ RACE. The deduced amino-acid sequences of grouper chitinase revealed a typical chitinase structure containing glycol-18 domain, signal peptide, a hinge region and chitin binding domain. The gchi1 and gchi2 mRNA were only expressed in the stomach. The localization of Gchi1 and Gchi2 in grouper, according to immunohisochemistry analysis of it’s stomach tissue section proved that Gchi1 and Gchi2 were expressed in the cell of gastric glands. Chitinolytic activities was measured in stomach from grouper. High activity of chitinase was purified from stomach by anion exchange chromatography and gel filtration. By mass protein identification and recognition by chitinase antibody, indicates that the purified chitinase is Gchi1. After sequencing the N-terminal of Gchi1, the result demonstrated that the signal peptide was cleaved. The mRNA and protein expression level of gchi1 and gchi2 can be enhanced by feeding the shrimps, and those result suggest that Gchi1 and Gchi2 is the chitinase for digesting chitin.

黃貴民 (1999)，實用石斑魚養殖，水產出版社出版
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