شبیه سازی و بیان فاکتور نوروتروفیک مشتق از مغز (BDNF) در اشریشیا کلی

Brain-derived neurotrophic factor, also known as BDNF, is a member of the neurotrophic family of growth factors. The BDNF protein is present in high concentrations in the hippocampus and cerebral cortex of the brian. The precursor protein contains 247 amino acids and in the later process the protein is cleaved to yield the 119 amino acid residue BDNF. This mature BDNF is secreted to extracellular sites and binds to TrkB receptor. Interaction of BDNF with TrkB can lead to a variety of intracellular signaling cascades such as Ras-MAP kinase cascade. These cascades help to keep the survival of existing nerve cells, and stimulate the growth and differentiation of new neurons and their synapses. The aim of this research was to clone the functional BDNF coding sequence in a prokaryotic vector to produce BDNF in E. coli (BL21). The functional part of the protein containing 357bp was synthesised into PUC57 plasmid. This plasmid transformed in E.coli (DH5) for amplifying purposes. Then, the plasmids were extracted and treated by BamH1 and Xho1 restriction endonucleases and the 372bp fragments were inserted into the BamH1-Xho1 site in pET26b vector. In the next step, the recombinant vector was transformed into TOP10 E.coli cells for amplifying purposes. The recombinant plasmid was extracted and transformed into BL21 E. coli cells for gene expression. The expression of the recombinant protein was confirmed by the SDS-PAGE technique.