PRKAG2基因新突变体重组腺病毒载体的构建及其在心肌细胞中的表达

Construction of Ad-PRKAG2 G100S-EGFP Vector and Its Expression in Myocardial Cells

目的:构建携带人全长PRKAG2基因新突变体(PRKAG2 G100S)及增强型绿色荧光蛋白报告基因(EGFP)的重组腺病毒载体,并将其在乳鼠心肌细胞中表达。方法:利用Invitrogen的GatewayTM技术,通过重叠PCR技术将目的基因突变体PRKAG2G100S-IRES-EGFP及野生型PRKAG2-IRES-EGFP定向克隆至入门载体pDONR221上,然后与腺病毒载体pAd/CMV/V5-DEST重组反应,形成包含目的基因及EGFP的腺病毒表达克隆。经筛选阳性表达克隆并测序验证后,PacI酶切、包装、扩增、纯化获得携带EGFP及PRKAG2基因的重组体腺病毒。病毒PCR鉴定并将其感染原代培养的SD乳鼠心肌细胞,用Westernblot技术检测PRKAG2蛋白的表达。结果:成功构建了携带人PRKAG2 G100S及EGFP基因的重组腺病毒,病毒的滴度为8.4×108pfu/ml。荧光显微镜下可见Ad-PRKAG2 G100S重组体腺病毒感染后的乳鼠心肌细胞表达EGFP而发出绿色荧光,PCR证实重组腺病毒载体构建成功,Western blot证明PRKAG2蛋白在乳鼠心肌细胞中过表达。结论:成功构建了携带EGFP基因的Ad-PRKAG2 G100S重组腺病毒载体并将其在乳鼠心肌细胞中正确表达,为今后突变体PRKAG2基因的功能研究奠定了基础。

Objective：To construct recombinant adenovirus vector of Ad-PRKAG2 G100S-EGFP and to investigate the expression of PRKAG2 G100S in infected myocardial cells.Methods：PRKAG2 G100S-IRES-EGFP and PRKAG2-IRES-EGFP were acquired by the overlapping PCR,and were cloned directly into entry vector pDONR221 by using Invitrogen Gateway TM technology.Then,along with the BP and LR recombination reactions finished,the recombinant adenovirus vector containing human PRKAG2 G100S gene was constructed.The sequencing sequence was right.The pAd-PRKAG2 G100S-IRES-EGFP and pAd-PRKAG2-IRES-EGFP were digested by Pac I,packaging,amplification and purified.Finally,the virus was infected into myocardial cells in order to measure PRKAG2 protein by Western blot.Results：Restriction enzyme digestion analysis and the sequence analysis confirmed that PRKAG2 G100S gene was successfully inserted into the adenovirus vector.Myocardial cells which were infected with Ad-PRKAG2 G100S-EGFP gave off strikingly bright green fluorescence and PRKAG2 protein was over-expressed.The titre of purified recombinant adenovirus Ad-PRKAG2 G100S-EGFP was 8.4×108 pfu/ml.Conclusion：The recombinant adenovirus containing human PRKAG2 G100S gene is successfully constructed and also expressed in myocardial cells.It will be helpful for the further study on PRKAG2 gene mutation.