鸭肥胖基因的分子克隆、序列分析及原核表达

Molecular Cloning of the Obese Gene from Duck and Its Expression in E.coli

根据人、小鼠、猪等动物的肥胖基因编码区序列的保守性设计1对引物,利用RTPCR技术扩增出鸭肥胖基因的cDNA编码序列,将PCR产物插入pGEMT载体,经PCR和双酶切鉴定正确后进行序列测定,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,鸭与人、猪、小鼠、大鼠的核苷酸相似性分别为83%、84%、98%、94%;氨基酸的相似性分别为86%、83%、99%、96%。为了研究鸭肥胖基因体外表达的特点,构建了原核表达载体pET28aYa,在大肠杆菌中进行了诱导表达。结果表明,鸭肥胖基因在大肠杆菌中进行了高效特异性融合表达,融合蛋白分子量约为20ku,其中16ku为鸭肥胖基因表达的蛋白质,经薄层扫描分析,目的蛋白约占菌体总蛋白的30%。鸭肥胖基因的克隆和表达研究,为进一步研究鸭leptin的功能与应用奠定了基础。

According to the conservative sequences of announced obese gene in human ,mouse and pig, a pair of primers were designed. The obese gene of the duck was amplified by RT-PCR. The product was cloned into pGEM-T vector. Sequence analysis revealed that it has a length of 438 nucleotides which encods a 146-amino peptide. When nucleotid sequence and deduced amino acid sequence were compared with homologous sequence of human, pig and rat, it displayed a fairly high degree of conservation. The homologue of the nucleotide sequence are 83%,84%,98% and 94% respectively; the homologue of the amino acid sequence are 86%,83%,99% and 96% respectively. With the aim of analyzing the characteristic of the expression, the cDNA fragment was inserted into expression vector pET-28a and the resulted plasmids were expressed in E.coli BL21(DE3) by IPTG induction. The results of SDS-PAGE analysis indicated that protein was specifically expressed in the E.coli BL21(DE3).The weight of the fusion protein was about 20ku,which included the 16ku protein expressed from duck obese gene. The cloning and expression of the duck obese gene established the foundation of the further research about the function and application of the duck leptin.