hBDNF基因原核表达重组质粒的构建及其在大肠杆菌中的表达  

Expression of Human Brain Derived Neurotrophic Factor Gene in E.coli

作　　者：刘智敏[1] 陈俊杰[2] 林佳[2] 王若菡[2] 游乐然[2] 东云华[2] 

机构地区：[1]华西医科大学附一院外科实验室,成都610041 [2]华西医科大学生物化学与分子生物学研究所重组DNA研究室,成都610041

出　　处：《生物医学工程学杂志》2001年第1期68-71,共4页Journal of Biomedical Engineering

摘　　要：我们按照 h BDNF基因全长编码序列设计合成引物 ,从人基因组 DNA中扩增出 76 0 bp的片段 ,反向插入到 p GEM- 3Zf(+ )载体上 ,获得 p GEMBF18克隆 ,限制性酶分析和 DNA序列测定均证实该克隆插入片段为h BDNF基因全长编码序列。从 p GEMBF18克隆中获取 h BDNF全长编码片段 ,与原核表达载体 p GEX- 5 T连接 ,构建了 p5 TBF34原核表达重组质粒。重组质粒转化大肠杆菌 JM10 9,经 IPTG诱导表达 ,SDS- PAGE特异区带分子量为 43k Da,此重组蛋白占菌体可溶性蛋白总量的 7.5 3% ,Western杂交证实该特异区带具 hThe primers specific for the full-length BDNF coding sequence was designed and synthesized. The BDNF coding sequence was directly amplified from human genomic DNA by using PCR and inserted into vector pGEM-3Zf(+). The recombinant DNA was transformed into the host cells JM109 to obtain the positive clone pGEMBF18. The restriction enzyme analysis and DNA sequence detection confirmed that the inserted fragment of clone pGEMBF18 is the full-length BDNF coding sequence. The hBDNF DNA fragment was recovered from the clone pGEMBF18 and ligated with prokaryotic expression vector pGEX-5T to construct the recombinant expression plasmid p5TBF34. The E. coli JM109 transformed with p5TBF34 was induced with IPTG. A new protein band with apparent molecular weight 43 kDa was detected in the lysate of the transformed cell by using SDS-PAGE. The result of western hybridization showed that this fusion protein reacted specifically to the antibodies to human BDNF. The amount of the soluble fusion protein was about 503.04mg/L lysate, 7.53% of total bacterial soluble protein of transformed cells, estimated by absorbance scanning of SDS-PAGE and protein quantitation.

关 键 词：HBDNF 基因克隆 SDS-PAGE 原核表达 Western杂交 脑源性神经营养因子 质粒 

分 类 号：R346[医药卫生—基础医学]