关键词: 核因子-κB, 反义 RNA, 腺病毒载体, 表达调控, 蛋白免疫印迹, 大鼠

Abstract: AIM: To construct the recombinant aden-ovirus 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 wasused 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 subse-quently 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 vec-tor, pcDNA3.1(+), under the CMV promoter.The in-tact antisense RNA expression frame from pcDNA3. 1 (+) /269 was inserted into entry vector pENTR4 to form medium recombinant pENTR4/CMV/269 plasmid, fol-lowed by homologous recombination technique, the ex-pression frame was integrated into adenovirus.The lin-earized recombinant adenovirus plasmid, pAd/CMV/269, infected the monolayer 293 cells, the adenovirus packag-ing 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 analysiswas documented as expected.The tran-sition-vectors contained 269 bp of reverse sequence were identified by restrictive endonuclease and PCR analysis. Recombinant adenovirus that expressed NF-κB (p65) an-tisense 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 in-fected VSMCs was markedly reduced compare with that in the normal cells.CONCLUSION: Recombinant aden-ovirus that expressed NF-κB (p65) antisense RNA is con-structed 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 pro-vides a foundation for investigation of its activity and fur-ther gene therapy.

Key words: nuclear factorκB, antisense RNA, ex-pression regulation, adenovirus vector, western blot, rat