动物造模 z-bio 2022-03-25 279

　　Objective: To construct a recombinant adenovirus vector that specifically inhibits the Raf-1 gene in rats, and transduce it into rat cardiomyocytes in vitro for functional validation.

　　Methods: The target sequence and negative control sequence for rat Raf-1 were synthesized, the DNA double-strand formed by annealing was directionally cloned into the crossing plasmid pAdTrackCMV to obtain the pAdTrack-siRaf-1 plasmid. 1. The backbone plasmid stopped homologous recombination to obtain pAd-siRaf-1 plasmid, and then transfected HEK293 cells, packaged to obtain pAd-siRaf-1 adenovirus particles, and then infected the primary cultured cardiomyocytes. 1. The inhibition efficiency of NF-κB gene was measured by liquid scintillation counter to measure the incorporation rate of 3H-leucine ([3H]-leu), and the extracellular area was measured by HJ2000 image analysis system.

　　RESULTS: The recombinant adenovirus vector was digested and verified correctly, and the prepared virus had high infection efficiency. Viral particles carrying Raf-1 could effectively inhibit the expression of Raf-1, extracellular area and [3H] induced by Ang II in cardiomyocytes at the protein level. ]-leu increased and down-regulated the expression of Raf-1 and NF-κB.

　　Conclusion: The pAd-siRaf-1 recombinant adenovirus vector was constructed by Shengli and packaged into recombinant adenovirus in HEK293 cells. After transfection into cardiomyocytes, it can effectively inhibit the expression of Raf-1 and NF-κB, and inhibit the myocardial cells induced by Ang Ⅱ. hypertrophy.