以ECHO克隆系统构建人生长激素的哺乳动物表达载体  

Cloning human growth hormone gene into the mammalian expression vector with ECHO system

作　　者：汪宗桂[1] 郑文岭[2] 马文丽[3] 梁念慈[1] 

机构地区：[1]广东医学院生物化学教研室,广东省湛江市524023 [2]华南基因组研究中心,广东省广州市510800 [3]南方医科大学基因工程研究所,广东省广州市510515

出　　处：《中国临床康复》2006年第20期110-112,共3页Chinese Journal of Clinical Rehabilitation

基　　金：国家自然科学基金资助项目(39880044);广州市重点科技攻关项目(99-Z-022-01)~~

摘　　要：目的:构建ECHO克隆系统构建人生长激素的哺乳动物表达载体,以克服原核生物表达系统生产人生长激素存在的缺陷。方法:实验于2002-08在解放军广州军区广州总医院医学实验科进行。①先把人生长激素基因片段克隆到ECHO系统的供载体中,构建供载体pUni/Lox-HGH。②让该供载体与具Lox位点的受载体在Cre重组酶的作用下融合,构建出人生长激素的哺乳动物表达载体pcDNA4.1/pUni-HGH,使人生长激素基因处于CMV启动子的调控下,EcoRI单酶切及BamHI/EcoRI双酶切鉴定重组质粒。结果:由琼脂糖凝胶电泳和序列测定证实,用ECHO克隆系统构建的融合质粒中,确实含有人生长激素带内含子的基因序列。结论:用ECHO克隆系统可以构建人生长激素的哺乳动物表达载体,且方便、快捷,为取代原核生物表达系统生产人生长激素,提供了依据。AIM： To construct mammal expression vector of human growth hormone （HGH） by using ECHO system, which overcame the expression limitation of HGH gene in the recombinant prokaryotic expression system. METHODS： The experiment was conducted in Department of Medical Research, General Hospital of Guangzhou Area Command of Chinese PLA in August 2002. ①The HGH gene fragment was cloned into the donor carriers by using the ECHO system firstly. ② Fused with the acceptor vector having Lox site by Cre recombinase and successfully made the HGH cDNA into the pcDNA4.1/pUni-HGH, under the CMV promoter. Recombinant plasmid was identified with EeoRI and BamHI/EeoRI. RESULTS： Agarose gel electrophoresis and DNA sequencing had proved that the fusion plasmid by using the ECHO system contained the gene sequence of HGH with intron. CONCLUSION： Successful construction of mammal expression vector of HGH can be undergone by using the ECHO system conveniently and rapidly. It offers evidence for replacement with novel prokaryotic system to express HGH.

关 键 词：人生长激素 遗传载体 电泳 琼脂凝胶 

分 类 号：R394.8[医药卫生—医学遗传学]