title: [Expression of human brain derived neurotrophic factor gene in E. coli].
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 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 503.04 mg/L lysate, 7.53% of total bacterial soluble protein of transformed cells, estimated by absorbance scanning of SDS-PAGE and protein quantitation.
authors.affiliation: Laboratory of Surgical Department, First Affiliated Hospital, West China University of Medical Sciences, Chengdu 610041.
authors.forename: Z
authors.initials: Z
authors.lastname: Liu
authors.forename: J
authors.initials: J
authors.lastname: Chen
authors.forename: J
authors.initials: J
authors.lastname: Lin
authors.forename: R
authors.initials: R
authors.lastname: Wang
authors.forename: L
authors.initials: L
authors.lastname: You
authors.forename: Y
authors.initials: Y
authors.lastname: Dong
mesh_terms: D019208:Brain-Derived Neurotrophic Factor / Q000096:biosynthesis / Q000235:genetics*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D066298:In Vitro Techniques; D021141:Nucleic Acid Amplification Techniques; D010957:Plasmids
chemical_list: D019208:Brain-Derived Neurotrophic Factor