淇河鲫生长激素基因的克隆与原核表达

Cloning and prokaryotic expression of the growth hormone in Carassius auratus gibelio var

【目的】克隆、分析淇河鲫生长激素(Growth hormone,GH)基因,并进行原核表达。【方法】采用RT-PCR方法对淇河鲫GH基因cDNA进行克隆,对其序列进行分析,并构建其原核表达载体PQE30-GH,进行原核表达。【结果】淇河鲫GH基因长633 bp,与鲫鱼、鲤鱼、团头鲂和斑马鱼具有较高的同源性,且处于同一进化分支上;与胡子鲇、鳗鲡、家鼠和人等的同源性较低。构建的表达载体PQE30-GH含有GH基因完整的阅读框架(ORF),转化到大肠杆菌DH5α中,经终浓度0.1 mol/L IPTG诱导后成功地表达出24 ku的融合蛋白。【结论】淇河鲫GH基因克隆成功。淇河鲫GH基因可以在原核中诱导表达,但表达量较低。

【Objective】 The study aims to clone and analyse the growth hormone（GH） gene in Carassius auratus gibelio var,then to study the prodaryotic expression of GH.【Method】 The cDNA encoding GH was amplified from Carassius auratus gibelio var by reverse transcription polymerase chain reaction（RT-PCR） method;the amplified cDNA sequence was analysed and its fragment was cloned into the prokaryotic expression vector,pQE30,to produce the expression vector pQE30-GH;and then the expression of GH in E.coli was studied.【Result】 The result showed that the coding region of the cDNA fragment was 633 bp in size.A high homology was found in the GH coding region within Carassius auratus,Cyprinius carpio,Megalobrama amblycephala and Danio rerio,but the homology was lower compared with that within Anguilla japonica,Mus musculus and Homo sapiens.The recombinant plasmid contained the entire ORF of GH gene;The recombinant plasmid was transformed into E.coli DH5α.The fusion protein cabout 24 ku was obtained after the addition of 0.1 mol/L IPTG into the growth media.【Conclusion】 So it can be concluded that the GH gene of Carassius auratus gibelio var has been sucessfully cloned and could be prodaryotic expression,but the expression quanitity was low.