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

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.