论文部分内容阅读
从脑垂体中提取总RNA,用RT-PCR方法扩增并克隆到了胭脂鱼的生长激素(GH)基因cDNA。胭脂鱼生长激素基因的开放阅读框(ORF)均包括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,用IPTG诱导重组蛋白的表达,其表达量超过细胞蛋白总量的50%,主要为不溶性的包含体。细菌裂解液沉淀溶于8M尿素后,用固定化金属配体亲和层析纯化,获得了相对分子质量为24kDa的单一蛋白带。
Total RNA was extracted from the pituitary gland, and cDNA of growth hormone (GH) gene was amplified and cloned by RT-PCR. The open reading frame (ORF) of the gilts growth hormone gene consists of 633 nucleotides and encodes 210 amino acids, including a signal peptide of 22 amino acids and a mature peptide of 188 amino acids. The cDNA of GH mature peptide was cloned into the expression vector pET-28a. The recombinant protein was induced by IPTG and expressed more than 50% of the total cellular protein, mainly consisting of insoluble inclusion bodies. The bacterial lysate was dissolved in 8M urea and purified with immobilized metal ligand affinity chromatography to obtain a single protein band with a relative molecular mass of 24 kDa.