Construction And Expression Of Adeno-associated Virus Vector Containing IRES Sequence

Objective: To construct the adeno-associated virus (AAV) vector of containing internal ribosome entry sites(IRES) sequence.Use to get the message of the expression of CNTF gene in the target cell conveniently and quickly.0Methods :1.To apply PCR technology to amplify the IRES sequence in the plasmid pIRES and utilize the gene recombination technology to insert the IRES sequence to the adeno-associated virus vector pAAV-CNTF-EGFP. 2.Co-transfect AAV-293 cells with three plasmids ( pAAV-CNTF-IRES-EGFP, pAAV-RC and pHelper ) by lipofectamine 2000. To use the inverted fluorescence microscope to detect the expression of the Enhanced Green Fluorescent Protein(EGFP). 3.To apply RT-PCR to detect the expression of CNTF gene.Results : 1. PCR,enzyme cutting and sequencing verificate that the plasmid pAAV-CNTF-IRES-EGFP is constructed successfully. 2. The expression of the Enhanced Green Fluorescent Protein(EGFP) is detected under the inverted fluorescence microscope.3.The expression of the CNTF is detected in the transfected AAV-293 cells by RT-PCR.Conclution: The IRES sequence control the expression of the CNTF and the EGFP in the AAV-293 cells at equal pace and independently.That will provide a foundation for investigating the effects of CNTF on the regeneration of the optic nerve.