罗非鱼胰蛋白酶ⅡcDNA克隆与序列分析

Cloning and sequencing of trypsin Ⅱ cDNAs in tilapia

应用 3′ RACE方法对尼罗罗非鱼 (Oreochromisniloticus) 和奥利亚罗非鱼 (Oreochromisaureus) 胰蛋白酶ⅡcDNA进行了克隆并进行序列测定和分析。结果表明, 胰蛋白酶Ⅱ序列中均具有催化活性必需的高度保守的催化三联体氨基酸残基 (His、Asp、Ser) 和构成二硫键的 10个半胱氨酸残基, 以及决定底物特异性的保守性氨基酸残基———天冬氨酸残基和S1结合袋。奥利亚罗非鱼和尼罗罗非鱼胰蛋白酶ⅡmRNA序列以及氨基酸序列同源性分别为 96 4%和 97 5%。

Trypsin Ⅱ cDNAs in two species of tilapia(Oreochromis aureus and Oreochromis niloticus)were cloned and sequenced by means of 3′-RACE.The results indicated that trypsin Ⅱ of Oreochromis niloticus and Oreochromis aureus had highly conserved amino acid residues essential for the catalytic activity and conformational maintenance,being His,Asp,Ser of catalytic triad and ten cysteines forming disulfide bridges,together with the residues determining substrate specificity and generating the S1 substrate-binding pocket of a typical trypsin nature.Trypsin Ⅱ and its mRNA of Oreochromis niloticus showed identity of 96.4% and 97.5% respectively to those of Oreochromis aureus.