大鼠β神经生长因子前体基因的克隆及其真核表达载体pEGFP-NGF的构建  

Cloning the Gene of Rat Premature Nerve Growth Factor and Constructing Its Eukaryotic Expression Vector pEGFP-NGF

作　　者：丁鹏[1] 冯忠堂[1] 王廷华[1] 董坚[2] 杨志敏[3] 路华[1] 路钢[1] 

机构地区：[1]昆明医学院神经科学研究所,昆明650031 [2]昆明医学院第一附属医院中心实验室 [3]昆明医学院第一附属医院神经外科

出　　处：《四川大学学报（医学版）》2004年第6期761-763,共3页Journal of Sichuan University(Medical Sciences)

基　　金：国家自然科学基金 (批准号 3 0 2 60 112 )资助

摘　　要：目的　克隆大鼠脑组织中神经生长因子 (NGF) β亚基前体的全长序列 ,构建其真核表达载体 p EGFP-NGF。方法　采用 RT- PCR方法扩增 NGFβ亚基前体的全长序列 ,克隆入载体 p MD18- T,转化大肠杆菌 JM10 9,挑选白色菌落行酶切鉴定、PCR鉴定及序列分析 ;利用高保真 Taq酶自重组质粒 p MD18- T/ NGF中扩增目的基因NGF,将目的基因与真核表达载体 p EGFP- N1行双酶切 ,T4连接酶连接 ,转化大肠杆菌 DH- 5 α,挑选白色菌落行酶切鉴定。结果　 p MD18- T/ NGF测序结果与 Gen Bank的序列完全一致 ,p EGFP- NGF行限制性内切酶 H ind 、Bam H 酶切 ,可见酶切片断与插入基因长度相符。结论　成功从大鼠脑组织中克隆神经生长因子 (NGF) β亚基前体的全长序列 ,并构建其真核表达载体 p EGFP- NGF,为从分子水平开展 NGF转基因相关研究奠定了基础。Objective To clone the entire coding sequence of rat premature nerve growth factor (NGF) β subunit and construct its eukaryotic expression vector. Methods The gene of premature nerve growth factor (NGF) β subunit was amplified by RT-PCR from SD rat brain. RT-PCR product was ligated into pMD 18-T Vector, the recombinant plasmid was identified by the restriction enzymes, PCR and DNA sequence analysis. Then the gene of premature nerve growth factor (NGF) β subunit was cloned into the eukaryotic expression vector pEGFP-N1. The recombinant plasmid pEGFP-NGF was identified by the restriction enzymes analysis. Results The DNA sequence was identical to the published sequence encoding NGF gene, the restriction enzymes mapping product of the recombinant plasmid pEGFP-NGF was nearly 750 bp which matched the expected size. Conclusion The entire coding sequence of premature nerve growth factor (NGF) β subunit was successfully cloned and its eukaryotic expression vector pEGFP-NGF was constructed, which will provide the basis for studying the gene of NGF.

关 键 词：NGF 真核表达载体 神经生长因子 EGFP 大鼠 克隆 脑组织 酶切 全长序列 目的基因 

分 类 号：R346[医药卫生—基础医学]