蒙古绵羊ghrlin cdna的克隆和原核表达.pdfVIP

摘 要 Ghrelin 是Kojima 等 1999 年发现的生长激素促分泌素受体(GHSR) 的第一个内 源性配体，Ghrelin 除调节生长激素分泌外，还有增加食欲，调节能量平衡，促进胃 酸分泌等作用。根据Genbank 中检索羊prepro Ghrelin mRNA 序列，设计了蒙古绵 羊Ghrelin 原核表达引物。通过RT-PCR 技术扩增 Ghrelin cDNA，将其与pMD-19T Vector 连接，转入大肠杆菌经鉴定正确后，回收目的片段与原核表达载体pET-32a(+) 相连接，并转化BL21(DE3)大肠杆菌。经IPTG 诱导后进行SDS和Western-blot 分析。结果显示克隆的蒙古绵羊 Ghrelin cDNA 序列与已发表序列有两个碱基的差 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 初步证实了所获的融合蛋白是特异的，结果表明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。 关键词：Ghrelin ；基因克隆；原核表达；蒙古绵羊 The cDNA Cloning and Prokaryotic Expression of Mongolia Sheep Ghrelin Abstract Ghrelin is the first endogenous ligand of growth hormone secretogogue (GHSR), it is found in rat stomach in 1999 by Kojima. Ghrelin can not only regulate the secretion of GH, but also stimulate appetite ，regulate energy balance and stimulate gastric acid secretion. According to the sequence of preproghrelin mRNA，a pair of primer of the expression system of prokaryotic was designed. Ghrelin gene was amplified by RT-PCR and the PCR product were cloned into the pMD-19T Vector, subcloned into the expression vector pET-32a(+) and transformed into host Escherichia coli strain BL2l(DE3)for expression. Comparing with the sequences of sheep Ghrelin 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. 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 expect