Construction Of Recombinant Adeno-associated Virus Vectors Encoding Ciliary Neurotrophic Factor And Green Fluorescent Protein

ObjectiveTo construct bicistronic adeno-associated virus(AAV) vectors encoding ciliary neurotrophic factor(CNTF) and enhanced green fluorescent protein(GFP) in order to establish a novel AAV vector platform for the gene therapy of eye disease.MethodsTo design CNTF gene primers of the two extremes. High fidelity polymerase increases the whole CNTF array. The vector plasmid was constructed with the restriction endonuclease and T4 DNA ligase. To insert the target gene into pAAV-IRES- hrGFP to construct the pAAV—CNTF-IRES- hrGFP plasmid. The recombinant expression plasmid is co-transfected into the AAV-293 cells with pHelper and pAAV-RC.Recombinant AAV viral particles are prepared from infected AAV-293 cells and then be used to infect retinal pigment epitheliums(RPEs) in vitro. The infection efficiency of the RPEs could be ascertained by fluorescence microscopy.Result1. The successfully inserted target gene was proved by PCR and SalⅠ/BamHⅠenzyme identification.The result of sequencing showed that the cloned CNTF gene is consistent with the sequence reported in the GenBank.The pAAV-CNTF-IRES-hrGFP plasmid was indentified to be correct.2. After three plasmids of pAAV-IRES-hrGFP vector co-transfected into the AAV-293 cells,a great amount of green fluorescence in AAV-293 cells was observed with the fluorescence microscope.The inserted CNTF was confirmed by PCR.3. After the recombinant virus infected RPEs,the green fluorescence in RPEs were observed.ConclusionsRPEs are able to be infected by recombinant pAAV-CNTF-IRES-hrGFP virus in vitro.