鲤鱼肥胖基因的分子克隆及在大肠杆茵中的表达

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438个核苷酸组成,编码146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-28a-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20 kD,经薄层扫描分析,目的蛋白占菌体总蛋白的20.3%.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,说明表达产物Leptin具有明显的生物学活性动物学报 51(1)95-100,2005].

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

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