淇河鲫生长激素基因cDNA的克隆和原核高效表达  

Cloning of cDNA for Carassius auratus gibelio var GH and Its Highly Efficient Expression in Prokaryocyte

作　　者：王松涛[1] 陈丽丽[2] 杜启艳[3] 常重杰[3] 

机构地区：[1]新乡医学院解剖学教研室,河南新乡453003 [2]新乡学院生命科学与技术系,河南新乡453003 [3]河南师范大学生命科学学院,河南新乡453007

出　　处：《贵州农业科学》2009年第7期110-113,共4页Guizhou Agricultural Sciences

基　　金：国家自然科学基金(30771666)

摘　　要：从脑垂体中提取总RNA,用RT-PCR方法扩增并克隆到淇河鲫(Carassius auratus gibelio var)的生长激素(GH)基因cDNA,其GenBank注册号为DQ 350437。分析其核苷酸序列和推测的氨基酸序列,结果显示:克隆到的淇河鲫生长激素基因的开放阅读框(ORF)包括633个核苷酸,编码210个氨基酸,其中,包括22个氨基酸的信号肽和188个氨基酸的成熟肽。把GH成熟肽的cDNA克隆入表达载体pET-28 a,在大肠杆菌BL21(DE3)表达N端含6个组氨酸的融合多肽。SDS-PAGE结果表明,0.1 mmol/LIPTG诱导表达的蛋白约为23.5 kD,其表达量超过蛋白总量的50%,主要为不溶性的包涵体。细菌裂解液沉淀溶于8 mol/L尿素后,用固定化金属配体亲和层析纯化,获得了分子量约为23.5 kD的单一蛋白带。GH cDNA was amplified and cloned from total RNA isolated from pituitary gland of Carassius auratus gibelio vat by RT-PCR. The accession number in GenBank is DQ350437. The sequence includes an ORF of 633 bp which encodes a precursor of 210 aa comprising 22 aa signal peptide and an 188 aa mature protein. The eDNA fragment encoding the mature peptide of GH was amplified and subcloned to expression vector pET-28 a and expressed in E. coli BL21 （DE3） as fusion polypeptide containing a His 6 at the N-terminus. The addition of 0.1 mmol/L IPTG induced expression of a protein band with molecular weight of about 23.5 kD. The recombinant protein was composed of more than 50% of the total bacterial proteins and accumulated as inclusion bodies. The inclusion bodies were solubilized in 8 mol/L urea and further purified by immobilized metal affinity chromatography （IMAC） under denaturing condition due to the fused N-terminal his · tag. The purified GH fusion polypeptide migrated as a single band of 23.5 kD on SDS-PAGE.

关 键 词：淇河鲫 生长激素 基因克隆 原核表达 

分 类 号：Q786[生物学—分子生物学]