Molecular Clonging And Expression Of Human Neurotophin-3 And Biol-activity Detection

Neurotrophic factors (NTFs) are traditionally viewed as secretory proteins that regulate thesurvival, development and differentiation of neurons, including more than thirty proteins, such asneurotrophin family (NTs), glial cell line-derived neurotrophic factor (GDNF) family ligands(GFLs) and the other types of neurotrophic factors. The bioactivities of NTFs are exerted by thebinding between ligands and receptors and their signal transduction pathway. The various types ofneurons, including hippocampal cholinergic neurons, substantia nigra dopaminergic neurons, locusceruleus noradrenergic neurons, corpus striatum GABAergic neurons in the central nervoussystem (CNS) and motor neurons, sensory neurons and sympathetic neurons in the peripheralnervous system (PNS), are responded to neurotrophic factors, and NTFs also influence the otherorgans and tissues. Neurotrophic factors decrease the denaturation of neurons to prevent theneurodegeneration and promote the outgrowth and regeneration of axon. So neurotrophic factorsare considered to play the important roles to cure Parkinson's disease (PD), Alzheimer's disease(AD), amyotrophic lateral sclerosis (ALS), spinal cord injure, pathological changes of peripheralneurons and the other disease of nervous system in future.Neurotrophin-3 (NT-3) is a member of neurotrophin family (NFs), and mRNA of humanneurotrophin-3 is expressed in CNS and PNS extensively. There are two types receptors of humanneurotrophin-3, the low appetency receptor p75^NTR and the high appetency receptor TrkB. NT-3promotes the growth, development, differentiation and survival of neurons and acts on therestoration and regeneration of the injured neurons.Here, human neurotrophin-3 cDNA is amplified by RT-PCR with human fetal brain RNA asthe template and inserted into plasmid pGEM-T to construct the cloning vector pGEM-T-hNT-3.The positive recombinant is identified by restriction digest and PCR and sequenced. The resultsuggests that the nucleotide sequence of the cloned human neurotrophin-3 cDNA is identical withthe reported one (Genbank MW002527) and codes 119 amino acids with 357nt.With the restriction sites of EcoRâ… and Salâ… , human neurotrophin-3 cDNA is obtained fromthe cloning vector pGEM-T-hNT-3 and ligased with the lined plasmid pGEX-6p-1 to construct theprokaryotic expression vector pGEX-6p-1-hNT-3. The positive recombinant identified byrestriction digest and PCR is transformed into the E coll. BL21(DE₃)PlysS and the positivebacterium is induced by IPTG and the expressed fusion protein is identified by SDS- ployacarylamide gel electrophoresis.The experiment suggests human neurotrophin-3 fusion protein is expressed only as inclusionprotein at 28℃and 37℃, The result of the best induced expression indicates that the volumeyield of human neurotrophin-3 fusion protein is induced by 1.0m mol/L IPTG for 4hr at 37℃. Themolecular weight (Mr) of human neurotrophin-3 fusion protein is approximately 39.6kD, incldingMr of glutathione S-transferase (GST) tag protein is 26kD and Mr of human neurotrophin-3 is13.6kD. so inclusion bodies are dissolved and renatured by the oxidoreducuion system. Finally,The human neurotrophin-3 fusion protein is purified by Glutathione Sepharose 4B andconcentrated by PEG 8000.Neurons from the brain of the new born mice is cultured with Neurobasal and B27 in vitro toanalyze the bioactivity of the recombinant human neurotrophin-3. The result indicates therecombinant human neurotrophin-3 promote the outgrowth of neurites and the survival of neurons.Results got from the experiment, human neurotrophin-3 are expressed in the prokaryoticexpression system and the bioactivity of recombinant human neurotrophin-3 is analyzed, couldprovide the material for researching their bioactivities and the application of diseases in thenervous system.