Construction Of The Recombinant Plasmid PAAV-CNTF-EGFP And Expression In Eukaryotic Cells

Objective Amply CNTF cDNA fragment from the total RNA of rat sciatic nerve to construct a recombinant adeno-associated virus vector ( rAAV2) carrying CNTF cDNA and EGFP, to package the recombinat virus carrying CNTF.Methods 1) cloning rat' s CNTFcDNA : total RNA was extracted from the mature Sprague-Dawley rats. Amplify CNTF cDNA fragment from the total RNA through RT-PCR method using Trizol Kit and cloned into the plasmid pUcm-T vector ( 即 pUcm- CNTF) . 2) construction of the recombinant plasmid pAAV-EGFP : EGFP cDNA was amplified from the plasmid pEGFP-N₁ through polymerase chain reaction (PCR) and digested with Sal I and Bgl II. The digested EGFP cDNA was cloned into the plasmid pAAV-MCS. The recombinant plasmid pAAV-EGFP was constructed. 3) construction of the recombinant plasmid pAAV-CNTF — EGFP: CNTF cDNA was inserted into the plasmid pAAV-EGFP and constructed the recombinanat plasmid pAAV-CNTF — EGFP. 4)The recombinant plasmid was identified by digestion with Bam HI , Sal I and Bgl II sequenced by Sanger-dideoxy-mediated chain termination. 5)transfection:The recombinant plasmid ,pAAV-RC and pHelper were co-transfected into 293 cell using Lipofectamine. a viral production positive control (pAAV-EGFP) and negative control were performed. 6)Expression of the recombinant plasmid pAAV-CNTF—EGFP: After 48 hours of transfection , the expressions of CNTF-EGFP in AAV293 were demonstrated...