Construction of recombinant adeno-associated viral vectors in human neurenergen-3 gene

Abstract

Background Research of transgene brings hope for gene therapy of various diseases; in addition, some projects have been tested in clinic. Recently, the focus has been to find an ideal vehicle and a suitable therapeutic gene. Objective To explore an effective way to construct recombinant adeno-associated viral vectors expression in human neurnnergen-3 gene. Design Gene directed cloning. Setting Central Laboratory of Northern China Coal Medical College. Materials DH5a competent bacillus coli strain was provided by Capital Medical University; pCDNA3-NT-3 by professor Chen from Bengbu Medical College; pAAV-Laze, pAAV-Helper, pAAV-RC and pAAV-MCS plasmids by Capital Medical University; HEK293 cells by Cell Center of Basic Medical College of Tongji Medical University. Methods NT-3 genes which were selected from pCDNA3-NT-3 plasmids were cloned in pAAV-MCS to form a recombinant adeno-associated viral plasmid (pAAV-NT-3). pAAV-NT-3, pAAV-RC, pAAV-LacZ and pHelper plasmids were extracted, purified and subjected to enzyme-shearing evaluation. In addition, pAAV-NT-3 and pAAV-LacZ were cotransfected with pHelper and pAAV-RC, respectively into AVV-293 cells with DNA mediated by calcium superphosphate transfection gene; and then, AVV-293 cells were packed into recombinant adeno-associated viral rAAV-NT-3 and rAAV-LacZ. After collection of viral particles, rAAV-LacZ viral stock solution was diluted based on ratio of 10:1 and the mixture was used to infect HT1080 cells. X-gal stain was used to measure virus titer. Main Outcome Measures Size of targeted gene fragments, validity of vehicle construction and virus titer. Results Targeted gene NT-3 was successfully inserted into the relative vehicle pAAV and pAAV-NT-3 was correctly recongnized by enzyme-shearing evaluation. Enzyme-shearing electrophoresis demonstrated that pAAV-NT-3, pAAV-RC, pAAV-LacZ and pHelper plasmids were successfully extracted and purified. β-galactoside staining in situ indicated that LacZ genes were expressed in human fibrosarcoma cells (HT1080) and the recombinant virus titer was measured as 1×10 12 virus particles per milliliter. Conclusion Total-length cDNA fragment of NT-3 gene, which is obtained from pCDNA3-NT-3 plasmids, is closely matched to polyclone enzyme-shearing sites of adeno-associated viral vectors, while the combination can be used to construct recombinant adeno-associated viral vectors expression in hNT-3 gene.