| Alzheimer's disease and related neurodegenerative disorders are prevalent among the elderly and might be considered as the plague of the 21st century. It is thus imperative to find cures for these conditions. The use of nerve growth factor proteins as neuroprotective therapeutics is limited by their hindered mobility through the blood-brain barrier. Peptides provide an attractive alternative. ADNP, activity-dependent neuroprotective protein, one of the factors within the neurotrophic milier produce by VIP (vasoactive intestinal peptide) stimulated astroglia. ADNP provided neuroprotection at subfemotomolar concentrations against toxicity associated with tetrodotoxin (electrical blockade), the β-amyloid peptide (the Alzheimer's disease neurotoxin), N-methyl-D-aspartate (excitotoxicity), and the human immunodeficiency virus envelope protein. An 8-amino acid peptide derived from ADNP (NAP, NAPVSIPQ) has been reported that protects cultured neurons from multiple neurotoxins. NAP has greater potency and broader effective concentration range (10-15 to 10-13M) than ADNP in preventing neuronal death associated with tetrodotoxin treatment. NAP is a so small peptide (<1000) that it is able to cross the blood-brain barrier (BBB). But some results suggested that the halflife of NAP in the cortex is 15 min and the price of the peptides which synthesized with solid-phase technology and purified to homogeneity by HPLC is very high, all of these restrict its using in clinical therapy. This experiment focused on the molecular cloning and sequencing of NT4-NAP fusion gene, constructed the prokaryotic expression vector bearing fusion gene NT4-NAP and evaluation the bioactivity of NAP. That will continue the research on the gene therapy of Alzheimer's disease intensively.Methods: By means of asymmetrical primer/template, double stranded cDNA of NAP was gained, which included restriction enzymes sites on the two extremities. NAP cDNA was ligated to the signal and leader peptides of neurotrophin 4 (NT4), the fusion gene was named NT4-NAP. Through genetic recombination technique, the fusion gene NT4-NAP was inserted into polylinker site of prokaryotic expression vector pBV220, to generate a recombinant plasmid pBV220/NT4-NAP. The plasmid was induced expressing in E.coli at 42℃. Addition of 0.1ml expression NAP protein to dissociated spinal cord-dorsal root ganglion (SC-DRG). Compared the text and control groups to evaluation the bioactivity of NAP. Results: Evidences of DNA sequence analysis and restriction enzymes digestion showed that we recombined NAP cDNA to the 3'terminal of the signal and leader peptides of NT4, and the fusion gene was subcloned into pBV220 successfully and open reading frame is not exchange. Bioactivity of the products was proved that it could support the cell survival and neurite growth in the primary cultures of DRG of embryonic day 8 chickens neurons as compared the control (P<0.01). Conclusion: This study successfully cloned and sequenced the fusion gene of NT4-NAP. Prokaryotec expression vector pBV220/NT4-NAP can be constructed successfully and the NAP protein is a satisfactory bioactivity. It woule be convenient for us to study the expression of NAP in eukaryote and rAAV,and to continue the research on the gene therapy。To sum up, There are great protential theraphy function of the neurotrophic factors in the neurodegeneration disease. But they cann't go through the BBB because of their mulmolecule, so it's difficult to use in clinic for they must be injected in ventricle. NAP as an 8 peptides produce by VIP stimulated astroglia. It has been reported that protects cultured neurons from multiple neurotoxins. Examples are put forth to strengthen our opinion threat peptides are important candidates for future drug development for AD. This experiment used the means of asymmetrical primer/template, double stranded cDNA of NAP was gained. Because there are no secretion and expression elements in NAP, so it is difficult to secret NAP invo or intro by means of gene technology. This i...
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