Construction Of Adeno-associated Virus Encoding Growth Associated Protein 43 And Its Expression In Rat Retina

Objectives: To construct recombinant adeno-associated virus of Growth Associated Protein 43(GAP-43), determine its infective viral titer, and conduct intravitreal injection of rats in order to monitor its expression after retinal infection. It also tried to investigate the feasibility for the treatment of opticneuropathy and retinopathy via the introduction of GAP-43 gene to the adeno-associated virus.Methods: TO clone target gene segment of GAP-43,construct the recombinant plasmid of pAAV-IRES-hrGFP-GAP-43. pAAV-IRES-hrGFP- GAP-43 is co-transfected AAV-293 cells with pAAV-RC and pHelper plasmid in calcium-phosphate protocol to construct rAAV-GAP-43. The viruses, purified and condensed via chloroform-PEG8000/NaC1-chloroform method,infected HT1080 cells. The fluorescent cell counting was recorded under the fluorescent microscope and the virus titer was determined. The intravitreal injection of rAAV-GAP-43 into the right eyes of rats was performed; 4 weeks after the injection, the retina from rats were subjected to frozen sectioning to monitor green fluorescence under fluorescent microscope and to detect the GAP-43 expression with western blot analysis.Results: The recombinant plasmid of pAAV-IRES-hrGFP-GAP-43 was constructed;After the adeno-associated viruses (rAAV-GAP-43 and AAV-GFP) were constructed and purified and condensed, the titer of rAAV-GAP-43 was 3.0×109 /ml. Four weeks after the intravitreal injection in the right eye, the frozen sections showed the fluorescent expression under the fluorescent microscope and western blot analysis revealed high GAP-43 protein expression in the retinal tissues.Conclusion: The constructed rAAV-GAP-43 can infect retina and successfully express the GAP-43 protein and green fluorescent protein on the retina, which paves the way for the treatment of opticneuropathy and retinopathy.