作　　者： ; ; ; ; ;

机构地区： 中山大学生命科学学院水生经济动物繁殖营养和病害控制国家重点学科专业实验室

出　　处： 《水产学报》 2003年第5期391-397,共7页

摘　　要： 通过构建斜带石斑鱼垂体cDNA文库,克隆了其生长激素(GH)全长cDNA。斜带石斑鱼GH全长cDNA为955bp,编码的多肽为204aa。应用PCR方法把编码GH成熟肽的cDNA片段克隆到表达载体pET-15b,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,0.4mmol·L-1IPTG诱导表达的蛋白约为24kDa,主要为不溶性的包含体。细菌裂解液沉淀溶于6mol·L-1盐酸胍后,用Ni2+-NTA树脂进行亲和分离纯化,纯化产物在SDS-PAGE上表现为一条24kDa的蛋白带。在黑鲷GH放射免疫分析系统中,纯化产物能与黑鲷GH竞争结合GH抗体,表明大肠杆菌表达的斜带石斑鱼GH融合多肽具有GH免疫活性。 Epinephelus coioides GH cDNA was cloned from pituitary cDNA library by random sequencing. The sequence obtained spanned 955bp, with an open reading frame encoding a protein of 204 amino acids, which is composed of a putative signal peptide of 17 residues and a mature polypeptide of 187 amino acids. The cDNA fragment encoding the mature polypeptide of GH was PCR amplified and subcloned to expression vector pET-15b (Novagen), and expressed in E.coli BL21(DE3) as fusion polypeptide containing a His6 at the N-terminus. The addition of 0.4 mmol·L^(-1) IPTG induced expression of a protein band with molecular weight of about 24 kDa. The expressed protein accumulated as inclusion bodies, which were solubilized in 6 mol·L^(-1) guanidine HCl, and further purified and renatured on Ni^(2+)-NTA resin. The purified Epinephelus coioides GH fusion polypeptide migrated as a single band of 24 kDa on SDS-PAGE and exhibited GH immunoreactivity in sea bream GH RIA system.

关 键 词： 斜带石斑鱼 生长激素 克隆 大肠杆菌 文库 表达 海水养殖

领　　域： [农业科学] [农业科学] [生物学]