Construction of recombinant adenovirus vector encoding the antisense RNA of rat NF-κB and its expression in vascular smooth muscle cells

AIM: To construct the recombinant adenovirus contained antisence RNA of rat nuclear factor κB (NF-κB, p65) gene and investigate the change of the gene expression in the infected vascular smooth muscle cells (VSMCs). METHODS: Total RNA was extracted from VSMCs of spontaneously hypertensive rats (SHR) and it was used for amplifing the 1653 bp fragment of NF-κB (p65) gene by RT-PCR. The amplified product was inserted into the pMD18-T vector and identified subsequently by enzyme digestion analysis and sequencing. The downstream 269 bp of NF-κB (p65) gene was amplified from the recombinant pMD18-T/1653 plasmid and cloned in reverse orientation into the eukaryotic expression vector, pcDNA3.1(+), under the CMV promoter. The intact antisense RNA expression frame from pcDNA3.1 (+)/269 was inserted into entry vector pENTR4 to form medium recombinant pENTR4/CMV/269 plasmid, followed by homologous recombination technique, the expression frame was integrated into adenovirus. The linearized recombinant adenovirus plasmid, pAd/CMV/269, infected the monolayer 293 cells, the adenovirus packaging cell line. Western blot was employed to determine the changes of NF-κB (p65) gene expression level in the virus-infected VSMCs. RESULTS: A 1653 bp fragment of NF-κB (p65) gene was amplified from VSMCs, and its sequence analysis was documented as expected. The transition- vectors contained 269 bp of reverse sequence were identified by restrictive endonuclease and PCR analysis. Recombinant adenovirus that expressed NF-κB (p65) antisense RNA was constructed correctly and the titer of virus was generally up to 9.23×109 plaque form units per milliliter ( pfu·ml -1 ). In vitro, western blot results showed that the NF-κB (p65) expression level in the infected VSMCs was markedly reduced compare with that in the normal cells. CONCLUSION: Recombinant adenovirus that expressed NF-κB (p65) antisense RNA is constructed successfully and the virus possesses the biological feature of down-regulated expression of NF-κB (p65) in the infected VSMCs. The production of the recombinant adenovirus encoding NF-κB (p65) antisense RNA provides a foundation for investigation of its activity and further gene therapy.