Expression of Human Brain Derived Neurotrophic Factor Gene in E. coli
10.3321/j.issn:1001-5515.2001.01.018
- VernacularTitle: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
- Author:
Zhimin LIU
1
;
Junjie CHEN
;
LinJia
;
Ruohan WANG
;
Leran YOU
;
Yunhua DONG
Author Information
1. 华西医科大学附属第一医院
- From:
Journal of Biomedical Engineering
2001;18(1):68-71
- CountryChina
- Language:Chinese
-
Abstract:
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 inser ted fragment of clone pGEMBF18 is the full-length BDNF coding sequence. The hBD NF DNA fragment was recovered from the clone pGEMBF18 and ligated with prokaryot ic expression vector pGEX-5T to construct the recombinant expression plasmid p5 TBF34. The E.coli JM109 transformed with p5TBF34 was induced with IPTG. A new pr otein band with apparent molecular weight 43 kDa was detected in the lysate of t he transformed cell by using SDS-PAGE. The result of western hybridization show ed 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 sc anning of SDS-PAGE and protein quantitation.
