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

LONING OF cDNA FOR RACHYCENTRON CANADUM GROWTH HORMONE AND ITS EXPRESSION IN PROKARYOCYTE

作　　者：郝羽[1,2] 邓思平[1,2] 刘丽[1,2] 刘楚吾[1,2] 

机构地区：[1]广东海洋大学水产学院,湛江524025 [2]南海水产经济动物增养殖广东普通高校重点实验室广东海洋大学,湛江524025

出　　处：《海洋与湖沼》2011年第3期409-413,共5页Oceanologia Et Limnologia Sinica

基　　金：国家"十一五"科技支撑计划项目;2007BAD29B03号;国家自然科学基金项目;30972253号;广东省自然科学基金项目;8452408801001272号

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pMD 18-T-CoGH重组质粒,测序正确后经NdeⅠ和XhoⅠ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.

关 键 词：军曹鱼 生长激素基因 CDNA克隆 原核表达 

分 类 号：Q789[生物学—分子生物学]