牛nanog基因原核表达载体构建及其在大肠杆菌中的表达  

Construction of Prokaryotic Expression Vector of Bovine nanog Gene and Its Expression in Escherichia coli

作　　者：郑喜邦[1,2] 云彦[1] 胡勇策[1] 王华岩[1] 窦忠英[1] 

机构地区：[1]西北农林科技大学国家干细胞工程技术中心陕西分中心,杨凌712100 [2]广西大学动物科技学院,南宁530005

出　　处：《农业生物技术学报》2008年第5期770-774,共5页Journal of Agricultural Biotechnology

基　　金：国家高技术研究与发展计划(863)项目(No.2005AA219050);教育部重大项目(No.03160);国家自然科学基金(No.36071067);陕西省重大科技专项(No.2006kz05-G1)资助

摘　　要：从6周龄的胎牛(Bostaurus)原始生殖嵴中提取总RNA,通过RT-PCR扩增nanog基因,将其克隆到PMD-18T载体,然后再亚克隆到pGEX-KG表达载体上,获得原核表达质粒pGEX-KG-nanog,限制性内切酶分析和DNA测序证明所插入片段为牛nanog基因编码序列。重组质粒转化大肠杆菌JM109(Escherichia coli),在不同的培养温度(25、30和37℃)和不同浓度的IPTG(0.10、0.25、0.50、1.00和2.00mmol/L)诱导下均获得了表达,结果表明,培养温度和IPTG浓度对GST-Nanog融合蛋白在大肠杆菌中的表达影响甚微;经Western blot检测证实该蛋白约60kD,并具有GST抗原活性,证实目的蛋白为Nanog蛋白。cDNA of Bos taurus nanog gene from the primodial genital ridges of a fetus at age of six weeks was amplified by RT-PCR, inserted into PMD18-T vector, and subeloned into vector pGEX-KG. And a recombinant plasmid pGEX-KG-nanog was obtained. Restriction enzyme digestion and sequencing confirmed that the inserted fragment was the complete coding sequences of Bos taurus nanog. The recombinant plasmid pGEX-KG-nanog was then transformed into JM 109 （Eseherichia coil ） and induced to express GST-Nanog fusion protein by IPTG （0.10,0.25,0.50,1.00 and 2.00 mmol/L） at 25, 30 and 37 ℃, respectively. The results showed that （1） the GST-Nanog fusion protein expressed efficiently, IPTG concentration and culture temperature exerted little impact on the expression yield of GST- Nanog fusion protein in JM109; （2） Western blotting assay showed that the fusion protein was about 60 kD, and reactivated with GST antibody, indicating that the fusuion protein was obtained sueeessfully.

关 键 词：nanog分子克隆 原核表达 牛 

分 类 号：S188[农业科学—农业基础科学]