军曹鱼（Rachycentron canadum）生长激素基因cDNA的克隆和原核表达

摘要

运用同源克隆方法，从军曹鱼脑垂体中分离了生长激素基因（GH）的全长cDNA。结果表明，该基因cDNA全长862bp，含615bp的开放阅读框（ORF），编码204个氨基酸；其中包括22个氨基酸的信号肽和182个氨基酸的成熟肽。采用T-A克隆构建pMD18-T-CoGH重组质粒，测序正确后经Nde I和Xho I双酶切获得CoGH片段，插入表达载体pET-28a，获得pET-CoGH重组质粒并在表达宿主菌Escherichia coli BL21（DE3）中用IPTG诱导表达。SDS—PAGE电泳显示在22．4kDa处有特异性的蛋白条带出现，Western—blotting检测表明已经成功表达了融合蛋白，该蛋白主要以非可溶性的包涵体形式存在于菌体沉淀中。%The full length eDNA encoding growth hormone of Rachycentron canadum was amplified and cloned from total RNA isolated from pituitary gland by RT-PCR. The sequence was 862 nucleotides long, including.an ORF of 615 base pairs. The encoded protein contains 204 amino acids, comprising a 22 amino acids signal peptide and a 182 amino acids mature protein. The resulting cDNA sequence was undergone sequence analysis and subcloned into expression vector pET-28a between Nde land Xho I restriction sites and expressed in Escherichia coli BL21（DE3） cells. SDS-PAGE and Western-blotting analysis showed that the expressed protein accumulated as inclusion bodies and the molecular weight of expressed fusion protein was 22.4kDa.