黄河鲤生长激素基因cDNA的克隆和原核高效表达

[目的]研究黄河鲤生长激素基因cDNA的克隆和原核高效表达。[方法] 从黄河鲤脑垂体中提取总RNA，用RT-PCR方法扩增并克隆黄河鲤的生长激素（GH）基因cDNA，分析其核苷酸序列和推测的氨基酸序列。[结果] 经克隆得到的黄河鲤生长激素基因的开放阅读框包括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。将GH成熟肽的cDNA扩增并克隆入表达载体pET-28a，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/L IPTG 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。包涵体经尿素溶解和亲和层析，获得了单一蛋白条带。[结论]该研究克隆到黄河鲤的生长激素基因，构建其原核表达质粒，得到了高效表达的基因工程菌。

Cloning of cDNA for Growth Hormone of Cyprinus carpio haematopterus and Its High Efficient Expression in Prokaryocyte

[Objective] The study aimed to research cloning of cDNA for growth hormone(GH) of Cyprinus carpio haematopterus and its high efficient expression in prokaryocyte.[Method] The total RNA was isolated from the pituitary gland of C.carpio haematopterus.The GH cDNA was amplified and cloned by RT-PCR method and its nucleotide sequence and the deduced amino acid(aa) sequence were analyzed.[Result] The open reading frame of GH gene of C.carpio haematopterus included 633 nucleotide which encoded a precursor of 210 aa comprising of a 22 aa signal peptide and a 188 aa mature peptide.The cDNA fragment encoding the mature peptide of GH was amplified and subcloned to the expression vector pET-28a and expressed in E.coli BL21(DE3) as fusion polypeptide containing a His6 at the N-terminus.SDS-PAGE result showed that the addition of 0.1 mmol/L IPTG induced the expression of a protein band with molecular weight of about 23.5 kD.The recombinant protein was more than 50% of the total bacterial proteins and accumulated as the insoluble inclusion bodies.When the inclusion bodies were solubilized in urea and further purified by affinity chromatography,the purified GH fusion polypeptide migrated as a single band.[Conclusion] This study could clone GH gene of C.carpio haematopterus,build its prokaryocyte expression plasmid and obtained a genetic engineering bacteria with high efficient expression.