The Construction Of PSSHG/NT4-GFP-Ant Recombinant AAV And The Delivery Of Expressive Macromolecular Proteins In Vivo To The Brain Via "Nose-brain" Pathway

Recently,Scientists have found a lot of macromoleculal biological proteinswhich had the function of neurocyte protection .Unfortunately,they can't beapplied in the clinic for the bellowing reasons:1.The macromoleculal proteinscan't go through the blood-brain barrier(BBB), so there're not suitableadministration routes in the clinic.Local cerebral ventricle administration wayhas the disadvantage of trauma,which the patientscan'taccept .Besides,scientists found macromoleculal proteins modified by cellpenetrating peptides(CPP)could go through the BBB by vein or abdominaladministration way.However,these administration ways could deliver thebiological drugs all over the body.which caused the encranial drugconcentration was low and the systemic organs had the severe side effect.2.Thehalf-life time of some biological proteins is very short.they should beadministrated dozens of times every day .which caused inconvenience in theclinic. So,how to choose a suitabale administration route to delivermacromoleculal biological proteins into the brain chronically is the capital issuein the study of CNS disease gene therapy. The research expored intranasal administration way .We use therecombinant AAV express proteins in the nasal mucosa cells and observe if theexpressive macromolecule protein can be delivered into the brain via"nose-brain" pathway. First, we cloned the GFP gene by using PCR and T-vector cloningmethod.The positive clone was identified by the restriction enzymes, and thenthe cloned amplified fragments were sequenced and analyzed. Second,theresulting gene of GFP and Ant,PBV220/NT4 were conjunct by DNAligase,and thus construct PBV220/NT4-GFP–Ant,then the NT4-GFP–Antfragments was gained and identified by the restriction enzyme.Third,Theresulting gene of NT4-GFP-Ant fragment was inserted into the EcoRⅠ-BamHⅠsite of vector plasmid pSSHG to construct the vector of NT4-GFP-Antrecombinant AAV. The recombinant AAV viral sock was packaged. Renalembryo 293 cell was co-transfected with the rAAV vector of plasmid pSSHG/NT4-GFP-Ant ,packaging plasmid pAAV/ Ad and helper adeno virus pasmidpFG140.The recombinant AAV viral stock was titrated by dot blot.Finally,wedesigned the anminal experiment.The kunming rats were divided into twogroups.One group were given pSSHG/ NT4-GFP-Ant AAV at a dose of 10ulper day of each nostril by intranasal administration.The other group were givenpSSHG/ GFP AAV in the same way. rats were killed after seven days and madein frozen slides ,and then were detected by laser confocal microscope.The cloned GFP gene was coincident with gene bank sequence.NT4-GFP-Ant fusional gene was identified correct by the restriction enzymes. The combinant viral stock vector of plasmid pSSHG/NT4-GFP-Ant was constructed successfully. The results of dot blot showed that we had obtained the rAAV stocks of high titre. The fluorescence can be observed at the membrana mucosa nasi ,olfactory bulb and hippocampus in the groupwhich was given pSSHG/NT4-GFP-Ant by intranasal administration.By contrast,the fluorescence can be observed only at the membrana mucosa nasi in the group which was given pSSHG/ GFP.From the above results, We can draw the conclutions that we cloned GFPgene and constructed NT4-GFP-Ant gene successfully.We constructed andobtained The viral stock of PSSHG/NT4-GFP-Ant AAV successfully. Theannimal experiment confirmed that macromolecular protein modified by cellpenetrating peptides can be deliverde into brain via "nose-brain" pathway.So,We delivered macromolecule proteins modified by CPP into the brainvia "nose-brain" pathway,which is a immediate administration way and can avoid thesystemic side effect. Besides,we use the recombinant AAV express proteins inthe nasal mucosa cells constantly,which solved the question of administrationinconvenience in the clinic.We can further replace the GFP gene by other therapy gene,such as nerve growth factor gene,neurotrophic factor gene,anti-apoptosis gene .Then construct recombinant adeno-associated virus,delivered the macromolecule therapyproteins into the brain via "nose-brain" pathway,which can bring a new method and route to deliver macromolecule protein into the brain to treatCNS disease.