水牛NAAA基因克隆分析及其真核表达载体构建

为了阐明水牛N-酰基乙醇胺酸酰胺酶（N-acylethanolamine acid amidase，NAAA）基因在水牛卵泡发生和胚胎发生过程中的作用及分子机制，本试验采用了3'Race、生物信息学分析、载体构建和细胞转染等技术对NAAA进行了研究。结果表明：水牛NAAA基因的编码区全长1 071 bp，共编码356个氨基酸；多重序列比较分析显示：水牛NAAA核苷酸序列与牛、山羊、绵羊、猪、马、猫和人相应序列的相似性分别为98%、96%、95%、87%、87%、85%和84%，结合系统进化树分析结果，NAAA基因在不同物种以及进化的过程中具有高度保守性。对NAAA蛋白质的分析表明：该蛋白呈弱酸性，第26~27位氨基酸为其信号肽，细胞亚定位于溶酶体，存在NAAA-beta和Ntn_AC_NAAA结构域。成功构建水牛NAAA基因真核表达载体，转染293 T后，能够正确形成NAAA-EGFP融合蛋白，产生绿色荧光信号。研究结果为阐明NAAA基因在水牛卵泡发生和胚胎发生过程中的作用及分子机制奠定了理论基础。

Cloning and Construction of Eukaryotic Expression Vector of Buffalo NAAA Gene

In order to elucidate the function and molecular mechanisms of buffalo NAAA gene during folliculogenesis and embryogenesis, buffalo NAAA gene was studied with 3'-full Race, eukaryotic vector construction and cell transfection technology in this study. The results show that the coding region of buffalo NAAA is 1 071 bp in length, encoded 356 amino acids. The buffalo NAAA gene shares 98%, 96%, 95%, 87%, 87%, 85% and 84% similarity of nucleotide sequences with that of Bos taurus, Capra hircus, Ovis aries, Sus scrofa, Equus caballus, Felis catus and Homo sapiens, respectively. NAAA protein is weakly acidic, 26th~27th region was its signal peptide, located in the lysosome, and existed NAAA-beta and Ntn_AC_NAAA structural domains. The buffalo NAAA eukaryotic expression vector was successfully constructed. After transformed into 293T cell lines, NAAA-EGFP fusion protein was detectable. This study provided an important reference for surveying the regulation mechanism of NAAA gene during buffalo folliculogenesis and embryogenesis.