蒙古绵羊Ghrelin基因的克隆和原核表达  

The cDNA Cloning and Prokaryotic Expression of Mongolia Sheep Ghrelin Gene

作　　者：梁建荣[1] 曹贵方[1] 赵鹏伟[1] 温世勇[1] 程兰玲[1] 凃勇[1] 

机构地区：[1]内蒙古农业大学动物胚胎与发育生物学研究室,呼和浩特010018

出　　处：《畜牧兽医学报》2011年第12期1782-1786,共5页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：内蒙古自然科学基金项目(200711020506);国家自然科学基金(30860201)

摘　　要：为了克隆蒙古绵羊Ghrelin基因,构建其原核表达载体,并检测其在大肠杆菌中的表达情况。本研究用RT-PCR方法从蒙古绵羊胃组织mRNA扩增获得Ghrelin基因的cDNA序列,克隆到pMD-19T载体后进行序列分析。将测序正确的cDNA序列与原核表达载体pET-32a(+)连接,并转化BL21(DE3)大肠杆菌。经IPTG诱导后进行SDS-PAGE和Western-blot分析。结果表明克隆的蒙古绵羊Ghrelin基因序列与已发表基因序列有2个碱基的差异,该碱基的变化并不影响氨基酸序列,目的蛋白主要以可溶性形式存在,Western-blot初步证实了所获得的融合蛋白是特异的。本研究成功构建了蒙古绵羊Ghrelin基因的原核表达载体,并在大肠杆菌中获得高效表达,为进一步研究蒙古绵羊Ghrelin基因的功能提供参考。To construct Mongolia sheep Ghrelin prokaryotic expression vector,and observe its expression in host Escherichia coli strain BL2l（DE3）,the cDNA of Ghrelin gene was amplified from abomasum fundic gland mRNA of Mongolia sheep by RT-PCR.PCR product was cloned into the T vector pMD-19T to construct pMD-19T-Ghrelin for sequencing.Then the cDNA was subcloned into the prokaryotic expressing plasmid vector pET-32a（＋） and transformed into host Escherichia coli strain BL2l（DE3）for expression.The clone was induced by IPTG and was identified by SDS-PAGE and Western-blot.Comparing with the Ghrelin sequence of sheep reported in GenBank,there were two nucleotide differences,but it did not affect the amino acid sequence;the expression product was observed with soluble protein;The result of Western-blot showed that the recombinant protein was recognized by His-antibody specifically.The nucleotide sequence isolated from the recombinant plasmid pET-32a-Ghrelin was the same as expected;the Ghrelin protein was expressed successfully in Escherichia coli,which provided a basis for further investigation of Ghrelin function.

关 键 词：GHRELIN 基因克隆 原核表达 蒙古绵羊 

分 类 号：S826.82[农业科学—畜牧学] S813.3[农业科学—畜牧兽医]