斜带石斑鱼生长激素cDNA克隆及其在大肠杆菌中的融合表达

Epinephelus coioides growth hormone cDNA cloning and fusion expression in Escherichia coli

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.