摘要
[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。
[ Objective ] The study aimed to research cloning of eDNA 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 eDNA 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 subotoned 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.
出处
《安徽农业科学》
CAS
北大核心
2009年第14期6369-6371,6386,共4页
Journal of Anhui Agricultural Sciences
基金
国家自然科学基金(30771666)
关键词
黄河鲤
生长激素
基因克隆
原核表达
Cyprinus carpio haematopterus
Growth hormone（GH）
Gene cloning
Prokaryotic expression
