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

我们按照hBDNF基因全长编码序列设计合成引物，从人基因组DNA中扩增出760bp的片段，反向插入到pGEM-3Zf( )载体上，获得pGEMBF18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hBDNF基因全长编码序列。从pGEMBF18克隆中获取hBDNF全长编码片段，与原核表达载体pGEX-5T连接，构建了p5TBF34原核表达重组质粒。重组质粒转化大肠杆菌JM109，经IPTG诱导表达，SDS-PAGE特异区带分子量为43kDa，此重组蛋白占菌体可溶性蛋白总量的7.53%，Western杂交证实该特异区带具hBDNF抗原活性。

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

The 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.