重组腺病毒介导hMYH cDNA在原代培养大鼠心肌细胞中的表达及活性测定

Recombinant adenovirus-mediated hMYH cDNA gene expression and activity in primary cultured rat cardiomyocyte cells

构建携带hMYHcDNA的重组腺病毒表达载体,制备重组腺病毒,并在原代培养的大鼠心肌细胞中得到了表达,表达的hMYH蛋白具有结合并切割A∶8-OXO-G碱基错配的糖苷酶活性,对A∶G和A∶C错配具有较弱的结合活性和切割活性。该研究为进一步进行hMYH的基因治疗奠定了基础。

The replication-incompetent adenoviral vector containing the cDNA of hMYH was constructed and propagated in HEK 293A cells, the expression of hMYH and control gene LacZ were confirmed by western blot and β-Gal staining. After amplified in HEK 293A cells, the virus was purified by CsCl density gradient centrifugation and transfected to primary cultured rat cardiomyocyte cells, the expression of hMYH was detected by western blotting, nicking activity and binding activity were detected using nicking assay and gel-shift assay. The expression enzyme has nicking activity and binding activity to substrate containing A∶8-OXO-G mismatch, but only has binding activity to substrates containing A∶G and A∶C mismatch and very low nicking activity.