Construction of recombinant adenoviral vector Ad-CMV-hTGFbeta1 for reversion of intervertebral disc degeneration by gene transfer / 中华创伤杂志(英文版)

ABSTRACT

<p><b>OBJECTIVE</b>To provide a highly efficient adenoviral vector Ad-CMV-hTGFbeta1 for the study of gene therapy for reversion of the intervertebral disc degeneration.</p><p><b>METHODS</b>A newly developed recombinant adenoviral vector construction system was used in the study. The cDNA of hTGFbeta1 was first subcloned into a shuttle plasmid pShuttle-CMV. The resultant plasmid was linearized by digesting with restriction endonuclease PmeI, and subsequently transformed into E.coli. BJ5183 cells with an adenoviral backbone plasmid pAdEasy-1. Recombinants were selected by kanamycin resistance and confirmed by restriction endonuclease analysis. Finally, the recombinant plasmid linearized by PmeI was transfected into 293 cells. Recombinant adenoviruses were generated within 2 weeks.</p><p><b>RESULTS</b>The recombinant adenoviral plasmids were cut by BamHI and PacI respectively, and the diagnostic fragments appeared in 0.8% agarose electrophoresis. The infected 293 cells showed evident cytopathic effect (CPE). The productions of PCR confirmed the presence of recombinant adenovirus. The expression of hTGFbeta1 was verified by immunohistochemical staining.</p><p><b>CONCLUSIONS</b>The successful generation of the adenoviral vector Ad-CMV-hTGFbeta1 and the confirmation of the interest gene expression make it possible for the experimental study of the reversion of the intervertebral disc degeneration by gene therapy.</p>