【作者】 杨银凤； 朱雪敏； 都格尔斯仁；

【Author】 YANG Yin-feng~1 ZHU Xue-min~1 DU Ge-er si ren~1 (1.Laboratory of Animal Embryo and developmental biology,Inner Mongolia Agriculture University,Huhhot 010018)

【机构】 内蒙古农业大学动物科学与医学学院动物胚胎与发育生物学研究室；

【摘要】 Ghrelin主要是由胃产生的一种含有26～28个氨基酸残基的多肽。本研究根据本课题组已获得的驯鹿Ghrelin cDNA的部分片段设计一条序列特异性引物作为上游引物,3′接合器引物作为下游引物,克隆驯鹿Ghrelin cDNA的3′末端序列。另外,采用反向嵌套PCR RACE法,根据驯鹿Ghrelin cDNA的已知序列,设计一条5′末端磷酸化的特异性反转录引物和两对特异性反向嵌套PCR引物,首先进行反转录（RT）,然后将反转录成的cDNA进行环化,最后进行反向嵌套巢式PCR,克隆驯鹿Ghrelin cDNA的5′末端序列。结果成功地克隆出了驯鹿Ghrelin cDNA的3′和5′末端序列,从而得到604 bp的驯鹿Ghrelin cDNA全序列,其中包含57bp 5′非翻译区（UTR）、405bp的开放读码框（ORF）、128bp的3′UTR和poly（A）₁₄。序列同源性比较显示驯鹿Ghrelin cDNA与山羊、绵羊和牛的同源性分别是92.0%、90.8 %和89.5%:与猪和人的同源性分别是69.5%和65.2%;与鸡的同源性仅为33.8%。表明Ghrelin的结构具有明显的种属特异性。驯鹿Ghrelin cDNA全序列的获得为进一步研究其生理和病理生理作用奠定了基础。更多还原

【Abstract】 Ghrelin is produced by stomach and is a kind of polypeptide composed of 26～28 amino acid.In this study,we cloned the 3′cDNA end of reindeer ghrelin using a sequence specific primer designed basing on the known partial reindeer ghrelin cDNA sequence as upper primer and 3′sites adaptor primer as down primer.In addition,the 5′cDNA end was amplified using inverse nestd PCR with a specific RT primer whose 5’end is phosphated and two pairs specific inverse nested PCR primers,which are designed basing on the known reindeer ghrelin cDNA sequence.The reindeer ghrelin cDNA which come from reindeer ghrelin mRNA by RT was encircled,then inverse nestd PCR is performed.The results indicate 3’and 5’cDNA end were amplified successfully.The full-length reindeer ghrelin cDNA is 604bp and includes an 5’untranslated region （UTR） of 57 bp,a open reading frame （ORF） of 405bp,a stop codon of TAA,3’ UTR of 128bp and ploy（A）₁₄.The sequence alignment shows that the cDNA of reindeer ghrelin has homology of 92.0%,90.8 % and 89.5% with goat,sheep and cattle,69.5% and 65.2% with pig and human,only 65.2% with chicken.The results indicate that the structure of ghrelin has a obvious varietal speciality.The obtaining of full-length reindeer ghrelin establishes bases for further studying the function of physiology and pathological physiology.更多还原