斑马鱼3种胰蛋白酶原基因的克隆、序列分析及组织表达

 胰蛋白酶是丝氨酸蛋白酶类超家族成员之一，在动物蛋白消化中起着重要作用。为深入研究胰蛋白酶在鱼类中的蛋白结构和生理功能，利用RT-PCR和RACE方法，成功获得了斑马鱼3种胰蛋白酶原cDNA序列(zftry1a、zftry1b和zftry2)。结果表明，zftry1a和zftry1b均有242个氨基酸残基组成，其中包括15个氨基酸的信号肽和5个氨基酸(LDDDK)的激活肽。zftry2由247个氨基酸残基组成，其中包括15个氨基酸的信号肽和9个氨基酸(APLGDDDDK)的激活肽。氨基酸序列比对结果显示，三者具备胰蛋白酶原的保守结构特征，如含有催化三联体氨基酸(His-57、Asp-102和Ser-195)，12个半胱氨酸，位于底物结合口袋底部Asp-189和口袋开口处的Gly-216、Gly-226等。进化树结果显示，斑马鱼zftry1a和zftry1b属于group I，为阴离子胰蛋白酶原；斑马鱼zftry2属于group II，为阳离子型胰蛋白酶原。RT-PCR结果显示，三者组织分布模式类似，且在肠中有最高表达量。这些结果为研究鱼类胰蛋白酶原的基因进化和功能以及进一步探讨鱼类消化生理的分子机制奠定了基础。

Molecular Cloning,Sequence Analysis and Tissue Expression of Three Forms Trypsinogens in Zebrafish,Danio rerio

Trypsin, one member of the serine protease family, plays important roles in animal protein digestion. It is synthesized and secreted as proenzyme trypsinogen, and removed the N-terminal activation peptide by enterokinase converting to active form in the intestine. The active trypsin can specifically cleavage at the peptide bond on the carboxyl side of basic L-amino acids such as arginine or lysine residuse. For studying the protein structure and physiological funtions of trypsinogen in fish, we successfully got three trypsinogen cDNAs (zftry1a、zftry1b and zftry2) from zebrafish by RT-PCR and RACE. The results showed zftry1a and zftry1b consisted of 242 amino acids which contained a signal peptide(SP) of 15 amino acids and an activation peptide(AP) of five amino acids, LDDDK. zftry2 consisted of 247 amino acids which contained a SP of 15 amino acids and an AP of nine amino acids, APLGDDDDK. The alignment based on the amino acid sequences revealed that they had the conversed structural characteristics, such as the catalytic triad (His-57, Asp-102, and Ser-195), 12 cysteines forming 6 disulfide bonds, Asp-189 at the bottom of the substrate-binding pocket and Gly-216 and Gly-226 lining the sides of the binding pocket, and so on. The results from isoelectric and phylogenetic analyses suggested that zftry1a and zftry1b were group with teleost anionic trypsinogen group I, while zftry2 was phylogenetically related to teleost cationic group II. Tissue expression pattern was similar to each other, and all trypsinogens were mainly expressed in the intestine. These results from zebrafish trypsinogens can provide the foundations for further study of its expression profiles, the molecular characteristics and evolution of fish trypsinogen.