Production Of Blood-brain-barrier-penetrating PTD-BDNF And Its Biological Effect

The brain-derived neurotrophic factor (BDNF) is one of the members in the neurotrophin family. Its gene locates at chromosome 11p in humans ,consisting of 744bp . The length of the cDNA consists 360bp. BDNF initially produces as a proneurotrophin(252 aa,30 kDa) ,then cleaved by prohormone convertases, such as furin to form the mature neurotrophin (14 kDa,isoelectric point 9.99).BDNF has survival and growth-promoting actions on a variety of neurons, including dorsal root ganglion cells , hippocampal and cortical neurons,and certain peripheral sensory neurons ,BDNF can rescue the death of cells and attenuate learning and memory deficits. However, protein therapeutics including the BDNF,are generally excluded transport from blood to brain, owing to the negligible permeability of these drugs to the brain capillary endothelial wall of the blood-brain barrier (BBB) in vivo. The efficacy of BDNF via peripheral (i.v.) administration is limited by the lack of transport of the neurotrophin through BBB .None of the neurotrophin family's member has been applied in CNS disease at present as a result of the absence of a rational way across BBB. Recently, an approach to deliver therapeutic peptides to the brain is the application of fusion proteins linked to so-called protein transduction domain (PTD), derived from the human immunodeficiency virus trans-activator transcription (TAT) protein, capable of passing through the blood-brain barrier and cell membranes. To deliver the therapeutic proteins, in this study the BDNF mature peptides were linked to the protein transduction domain (PTD) of the human immunodeficiency virus (HIV-1) TAT protein by genetic engineering, and the soluble PTD-BDNF has been expressed successffuly in E.Coli. On the other hand ,we constructed another bacterial expression vector by computer-assisted prediction of the RNA secondary structure, and calculated the codon adaptation to redesign the nucleotide coding sequence of the PTD-BDNF . The level of the PTD-BDNF protein expression was highly elevated. We obtained highly purified obtained highly purified inclusion by optimizeing the inclusion washing technology. The effective production of the soluble and biologically active BDNF is optimized using the dilution renaturation technique. The present study showed that the i.v administered recombinant human BDNF(rhBDNF) conjugated with a protein transduction domain(PTD ) was able to promote the survival and growth of hippocampal neurons impaired by Aβ25-35(10μM) in vitro , and transported through the BBB in vivo . The Morris water maze test indicated that the i.v PTD-rhBDNF improved the spatial learning and memory of mice impaired by the aggregated Aβ25-35. The peripherally administered PTD-rhBDNF exhibited neuroprotective effects in brain, and raise the possibility of delivery of the exogenous rhBDNF in treatment of the brain diseases. The result also showed that PTD-BDNF via i.v injection was also efficient in the impairedDA neuron recovery.