金钱鱼催乳素基因SaPRL的克隆及原核表达  

Cloning and Prokaryotic Expression of Prolactin Gene SaPRL from Spotted Scat(Scatophagus argus)

作　　者：梁雪梅[1] 余琳 张俊彬[1,2] Liang Xuemei;Yu Lin;Zhang Junbin(College of Fisheries and Life Science,Shanghai Ocean University,Shanghai,201306;College of Life Sciences and Oceanography,Shenzhen University,Shenzhen,518060)

机构地区：[1]上海海洋大学水产与生命学院,上海201306 [2]深圳大学生命与海洋科学学院,深圳518060

出　　处：《基因组学与应用生物学》2019年第11期4875-4884,共10页Genomics and Applied Biology

基　　金：中国国家科学技术部项目(项目编号:DF)资助

摘　　要：金钱鱼(Scatophagus argus)对水环境的盐度变化具有很强的适应能力。为了解该鱼的催乳素基因(SaPRL)在其渗透压调节过程中所起到的作用,本研究采用RACE技术,对SaPRL基因进行了全长cDNA序列的克隆及序列分析。结果表明,SaPRL的cDNA序列全长为1550 bp,可编码212个氨基酸,具有与其他物种共同的保守区域Growth hormone like superfamily;在氨基酸序列上,它与黑棘鲷(Acanthopagrus schlegelii)的催乳素基因(PRL)相似性较高,达82%,而与人(Homo sapiens)的PRL的相似性仅为39%。用半定量法(sq-PCR)进行组织分布分析显示,在盐度为2.5%的海水中,金钱鱼SaPRL基因主要于脑和垂体中有高丰度表达。此外,SaPRL主要分布在金钱鱼脑组织的脑膜中。最后,本研究成功构建了SaPRL-pET28a(+)原核表达质粒,当在含该质粒的宿主菌中加入1 mmol/L的IPTG并以32℃诱导4h后,能获得大量融合蛋白的表达;该诱导条件下的蛋白主要以包涵体形式存在,且于4 mol/L尿素中的溶解度最大。上述研究结果,为探讨SaPRL基因在金钱鱼盐度适应过程中的作用机理提供了重要的基础资料。Scatophagus argus has a strong ability to adapt to the salinity change of water environment.To study the role of Scatophagus argus prolactin(SaPRL)during osmoregulation,full-length cDNA sequence of SaPRL were cloned using RACE method.cDNA sequence of SaPRL consists of 1550 bp,encoding 212 amino acids,and SaPRL has the same’Growth hormone like superfamily’conserved domain with other species.In the amino acid sequence,the similarity of the prolactin gene(PRL)of Acanthopagrus schlegeli is as high as 82%.However,SaPRL shared only 39%amino acid identity with Homo sapiens PRL.sq-PCR resulted that SaPRL was predominantly expressed in brain and pituitary of seawater-reared S.argus(2.5%in salinity).Besides,SaPRL mainly located in the cerebral cortex of S.argus.We successfully constructed the prokaryotic expression plasmid,SaPRL-pET28a(+).In the host bacteria that contained SaPRL-pET28a(+),we added IPTG with 1 mmol/L final concentration.After 4 hour induction at 32℃,massive recombinant proteins expressed.Under this optimum induced expression condition,SaPRL recombinant protein existed mainly in the form of inclusion body,which had the maximum solubility in 4 mol/L urea.This paper provided major basis data for the follow-up study on the role of SaPRL in osmoregulation of S.argus response to environmental salinity change.

关 键 词：金钱鱼 催乳素 基因克隆 序列分析 原核表达 

分 类 号：S917.4[农业科学—水产科学]