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目的包装人PRKAG2(R302Q)突变型的腺病毒后感染原代SD乳鼠心肌细胞,构建细胞模型。方法首先通过BP及LR重组获得人PRKAG2(R302Q)突变型的腺病毒表达载体,将其线性化转染293细胞进行腺病毒包装和扩增。收集纯化腺病毒液感染原代SD乳鼠心肌细胞后进行蛋白质印迹法检测。结果 PRKAG2(R302Q)突变型的腺病毒载体经测序验证插入序列正确,腺病毒感染乳鼠心肌细胞后蛋白质印迹法检测其过表达明显(P<0.05)。结论包装人PRKAG2(R302Q)突变型基因的腺病毒并成功感染SD乳鼠心肌细胞,为进一步研究基因PRKAG2(R302Q)突变体的功能奠定了基础。
Objective To construct a cell model by infecting primary SD neonatal rat cardiomyocytes after packaging human PRKAG2 (R302Q) mutant adenovirus. Methods The recombinant adenovirus carrying human PRKAG2 (R302Q) was first obtained by BP and LR recombination. The recombinant adenovirus was transfected into 293 cells by linearization to amplify the adenovirus. The purified adenovirus was used to infect primary SD neonatal rat cardiomyocytes for Western blotting. Results The recombinant adenovirus vector carrying PRKAG2 (R302Q) was verified by sequencing. The positive expression of PRKAG2 (R302Q) was confirmed by Western blotting. The over - expression of adenovirus was detected by Western blotting in neonatal rat cardiomyocytes (P <0.05). Conclusion The recombinant adenovirus carrying human PRKAG2 (R302Q) gene was successfully infected with cardiomyocytes of neonatal SD rats, which laid a foundation of further study on the function of PRKAG2 (R302Q) mutant.