| 摘要: |
| 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。 |
| 关键词: PRKAG2基因 突变 腺病毒包装 心肌细胞模型 |
| DOI：10.16781/j.0258-879x.2016.01.0034 |
| 投稿时间：2015-10-11修订日期：2015-12-07 |
| 基金项目:国家自然科学基金(81170092). |
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| Establishment and confirmation of neonatal rat cardiomyocyte model over-expressing mutant human PRKAG2 |
| CHEN Ting,LIU Jie,YU Yun-hua,BAO Li-zhi,KANG Bo,ZHENG Xing* |
(Department of Cardiovasology, Changhai Hospital, Second Military Medical University, Shanghai 200433, China
*Corresponding author.) |
| Abstract: |
| Objective To establish a cardiomyocyte model over-expressing mutant human PRKAG2 by infecting neonatal SD rat myocardial cells with constructed recombinant adenovirus vector Ad-PRKAG2 (R302Q)-IRES2-EGFP. Methods PRKAG2 (R302Q)-IRES2-EGFP was directly cloned into entry vector pDONR221 by using Invitrogen GatewayTM technology. Then BP and LR recombination reactions yielded the recombinant adenovirus vector containing human PRKAG2 (R302Q) gene. The pAd-PRKAG2 (R302Q)-IRES2-EGFP was digested by Pac Ⅰ, and transfected into 293 cells. After packaging, amplification and purification, the virus was used to infect neonatal rat cardiomyocytes. Then the expression of PRKAG2 protein was assayed by Western blotting analysis in the infected neonatal SD rat cardiomyocytes. Results Restriction enzyme digestion analysis and the sequence analysis confirmed that PRKAG2(R302Q) gene was successfully inserted into the adenovirus vector. The myocardial cells infected with Ad-PRKAG2(R302Q)-IRES2-EGFP gave off strikingly bright green fluorescence and PRKAG2 protein was proven significantly over-expressed by Western blotting analysis (P<0.05). Conclusion The recombinant adenovirus containing human PRKAG2(R302Q) gene has been successfully constructed and expressed in neonatal rat cardiomyocytes, which paves a way for further study of PRKAG2 (R302Q) gene mutation. |
| Key words: PRKAG2 gene mutation adenovirus package myocardial cell model |
