动物实验,Raf-1基因 z-bo 2020-08-24 572

　　Objective: To construct a recombinant adenovirus vector that specifically inhibits rat Raf-1 gene and transduce it into rat cardiomyocytes for functional identification.

　　Method: Synthesize the target sequence of rat Raf-1 and the negative control sequence, and clone the annealed DNA into the shuttle plasmid pAdTrackCMV to obtain the pAdTrack-siRaf-1 plasmid. After linearization of PmeI, it binds to BJ5183 bacteria. After homologous recombination of the pAdEasy-1 skeleton plasmid, pAd-siRaf-1 plasmid was obtained, which was transfected into HEK293 cells, and pAd-siRaf-1 adenovirus particles were obtained after packaging, and then cultured into primary cultured cardiomyocytes in. I am infected. Western blot is used to detect Raf-targeted siRNA. -1. Use a liquid scintillation counter to measure the inhibition efficiency of NF-κB gene, 3H-leucine ([3H] -leu) uptake rate, and use the HJ2000 image analysis system to measure the cell surface area.

　　Result: The recombinant adenovirus vector was digested and identified, and the virus infection efficiency was high. Virus particles carrying Raf-1 can effectively inhibit AngII, cell surface area and [3H-induced Raf-1 expression in cardiomyocytes. ] -leu increases and down-regulates the expression of Raf-1 and NF-κB.

　　Conclusion: The pAd-siRaf-1 recombinant adenovirus vector has been successfully constructed and packaged into the recombinant adenovirus of HEK293 cells. After transfection of cardiomyocytes, it can effectively inhibit the expression of Raf-1 and NF-κB, and inhibit the myocardium induced by AngⅡ.