鲶生长激素基因cDNA的克隆和原核表达

Molecular Cloning and Prokaryotic Expression of Growth Hormone in Silurus asotus

从脑垂体中提取总RNA,用RT-PCR扩增并克隆到鲶鱼的生长激素(GH)基因cDNA。分析其核苷酸序列和推测的氨基酸序列,结果显示:鲶鱼生长激素基因的开放阅读框(ORF)包括603个核苷酸;编码200个氨基酸;其中包括22个氨基酸的信号肽和178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8 mol/L尿素后,用固定化金属配体亲和层析纯化,获得分子量为22.5 kD的单一蛋白带。

GH eDNA was amplified and cloned from total RNA isolated from pituitary gland of Silurus asotus by RT-PCR. With sequence analyses of DNA and deduced amino acids, the open reading frame（ORF） of growth hormone gene is of 633 bp which encoded a precursor of 200 AA comprising a 22 AA signal peptide and a 178 AA mature protein. The eDNA encoding the mature peptide of GH was subcloned into expression vector pET-28a and expressed in Escherichia. coli BL21 （DE3） as fusion polypeptide containing a His 6 at the N-terminus. By induction of IPTG, the recombinant protein was expressed and composed of more than 50% of the total bacterial proteins and accumulated as inclusion bodies. The inclusion bodies were solubilized in 8mol/L urea and further purified by immobilized metal affinity chromatography （IMAC） under denaturing condition. The purified GH fusion polypeptide migrated as a single band of 22.5 kD on SDS-PAGE.