Construction And Expression Of Fused Genes Of GAP-43 And Red Fluorescent Protein In Recombinant Adeno-associated Virus Vectors

ObjectiveTo construct a recombinant adeno-associated virus ( rAAV2) vector carrying the rat growth associated protein 43(GAP-43) and red fluorescent protein(RFP) fusion gene(pAAV-GAP43-RFP plasmid),and to examine the expression of GAP43-RFP fusion protein in 293 cell line, for further studying of effect of GAP-43 on the injured rat retinal ganglion cells(RGC).MethodsThe RFP primers were designed and RFP OFP DNA fragment were amplified by polymerase chain reaction (PCR) with pDsRed1-C1 plasmids template.The pAAV-GAP43-EGFP plasmid and RFP DNA fragment were digested with restriction enzymes Sal I and BglⅡ, and then the digested products were purified by using CASPure gel extraction Kit. The purified pAAV-GAP43 and RFP DNA fragment were ligated into pAAV-GAP43-RFP by T4DNA ligation enzyme. And transformed into E.coliDH5α.The pAAV-GAP43-RFP plasmid was identified by PCR , double digestion with restriction enzymes Sal I and BglⅡand DNA sequencing. pAAV-GAP43-RFP and the other two plasmids ( pHelper,pAAV-RC) were co-transfected into AAV-293 cells by Lipofectamine 2000 liposome. The expression of GAP43-RFP fusion protein was observed by fluorescent microscope.