鲤鱼肥胖基因的分子克隆及在大肠杆菌中的表达  

Molecular cloning of the obese gene from the carp Cyprinus carpio and its expression in E.coli

作　　者：戴汉川[1] 龙良启[1] 丁光[2] 

机构地区：[1]华中农业大学水产学院淡水增养殖生物学重点实验室,武汉430070 [2]云南农业大学动物科技学院,昆明520001

出　　处：《动物学报》2005年第1期95-100,共6页ACTA ZOOLOGICA SINICA

基　　金：湖北省自然科学基金资助 (NO 2 0 0 0J10 4)~~

摘　　要：为了研究鲤鱼肥胖基因的结构特点和体外表达产物的生物学活性 ,利用RT PCR技术从鲤鱼肠系膜脂肪组织中扩增出鲤鱼肥胖基因的cDNA编码序列 ,分析表明该cDNA序列由 4 38个核苷酸组成 ,编码 14 6个氨基酸组成的多肽 ,鲤鱼肥胖基因与人、猪、鼠的相比 ,核苷酸同源性分别为 :84 %、 86 %、 95 % ;氨基酸的同源性分别为 84 %、 82 %、 96 %。构建了原核表达载体 pET 2 8a li,利用IPTG在大肠杆菌中进行了诱导表达 ,并对表达产物进行了初步纯化和生物活性检测 ,结果表明 ,鲤鱼肥胖基因在大肠杆菌中进行了高效特异性融合表达 ,融合蛋白质分子量约为 2 0kD ,经薄层扫描分析 ,目的蛋白占菌体总蛋白的 2 0 3%。表达产物经过纯化和复性能够明显抑制小鼠的摄食和生长 。With the aim of analyzing the characteristic of the carp obese gene structure and the biological activity of the expression product, the obese gene of the carp was amplified by RT-PCR from the carp mesentery adipose tissue RNA.Sequence analysis revealed that it has a length of 438 nucleotides which encoded a 146-amino peptide.When Sequence nucleotide and deduced amino acid sequences were compared with homologous sequences from humans, pigs and rats, it displayed a fairly high degree of conservation.The homologue of the nucleotide sequences are respectively 84%, 86% and 95%; the homologue of the amino acid sequence are respectively 84%, 82% and 96%.The cDNA fragment was inserted into expression vector pET-28a and the resulted plasmid was expressed in E.coli BL21(DE3) by IPTG induction.The results of SDS-PAGE analysis indicated that fusion protein was specifically expressed in the E.coli BL21(DE3).The weight of the fusion protein was about 20 kD and 16 kD protein was expressed from the carp obese gene.Through gel thin layer scanning analysis, the amount of target protein is about 20.3%.The purified product was found to be biologically active, reducing the food intake and body weight gain upon testing in mice

关 键 词：肥胖基因 生物学活性 表达产物 大肠杆菌 RT—PCR技术 肠系膜 核苷酸 鲤鱼 同源性 利用 

分 类 号：Q785[生物学—分子生物学]