Objective: To construct a recombinant adenovirus vector carrying KATP; channel mutant subunit Kir6.2AAA and express it in rat cardiomyocytes.
Methods: Based on the primers for Kir6.2 subunits, Kir6.2 GFG amino acids were site-directed mutated into AAA by means of overlap PCR. PCR products were cloned into pShuttle vector for sequence analysis. After Pme I linearization, it was transformed into adenovirus expression vector pAdEasy-1. Then the pAdEasy-1 was packaged into liposome and transfected into primary cultured rat cardiomyocytes. The expression of Kir6.2AAA was confirmed by reverse transcription PCR(RT-PCR) and Western blotting.
Results: The recombinant adenovirus carrying the gene fragment Kir6.2AAA and EGFP was constructed successfully, and the virus titer was 2.64×10(11); VP/mL. After infected by the recombinant adenovirus expressing Kir6.2AAA, rat cardiomyocytes expressed EGFP and emitted green fluorescence under a fluorescence microscope. RT-PCR demonstrated that the expression of Kir6.2AAA was significantly up-regulated in the infected cardiomyocytes, and Western blotting also proved the over-expression of Kir6.2AAA in the cardiomyocytes.
Conclusion: The recombinant adenovirus carrying the gene fragment Kir6.2AAA and EGFP was constructed successfully and expressed correctly in rat cardiomyocytes.