腺病毒介导的DPP6过表达载体的构建及其在大鼠心肌细胞内表达的鉴定  

Construction of adenovirus mediated DPP6 overexpression vector and its identification in rat cardiomyocytes

作　　者：王娜[1] 华乐[2] 杨甫[2] 王珏[2] 王志华[1] 王琼[1] 董梅[3] 王川[1] 

机构地区：[1]河北医科大学药理学教研室,河北石家庄050017 [2]河北医科大学临床医学系,河北石家庄050017 [3]河北科技大学校医院,河北石家庄050018

出　　处：《暨南大学学报（自然科学与医学版）》2015年第6期453-457,共5页Journal of Jinan University(Natural Science & Medicine Edition)

基　　金：国家自然科学基金项目(31171097);河北省自然科学基金项目(C2014206419);教育部留学回国人员科研启动基金项目(2013-693);河北省高等学校科学技术研究项目重点项目(ZD2015007)和自筹项目(Z2015005)

摘　　要：目的:构建腺病毒介导的DPP6过表达载体并验证其在大鼠心肌细胞内的表达.方法:应用PCR技术扩增目的基因DPP6,经酶切、连接等反应将目的基因插入穿梭载体p Shuttle-CMV中,进而转化至含有Ad Easy质粒的大肠杆菌中,进行重组;所产生的腺病毒载体转染在HEK-293细胞中,进行腺病毒的包装、分泌和扩增,所得病毒感染大鼠心肌细胞,进行荧光及实时定量PCR(q PCR)鉴定.结果:DPP6过表达腺病毒载体构建成功,经HEK293细胞包装、扩增后,所得病毒可成功感染初生大鼠心肌细胞,观测到腺病毒携带的绿色荧光蛋白表达,q PCR检测DPP6 mRNA表达明显升高.结论:成功构建了腺病毒介导的DPP6过表达载体,并在大鼠心肌细胞内成功表达,为进一步研究DPP6在心肌细胞内的功能奠定了基础.Aim：To construct adenovirus mediated DPP6 overexpression vector and to evaluate its ex-pression in rat cardiomyocytes.Methods：DPP6 gene was amplified by PCR technique and inserted into pShuttle-CMV vector by enzyme digestion and ligation.DPP6-pShuttle-CMV constructor was transformed into E.coli BJ5183 strain containing AdEasy vector for recombination.The produced adenovirus con-structor was transfected into HEK-293 cells to package,secret and amplify adenovirus,which was further infected cultured rat cardiomyocytes and identified by fluorescence and real-time PCR.Results：We suc-cessfully get DPP6 overexpression adenovirus constructor.The produced virus was identified in neonatal rat cardiomyocytes.GFP was observed in infected cardiomyocytes.DPP6 mRNA expression was markedly increased in DPP6 virus infected cells.Conclusion：Adenovirus mediated DPP6 overexpression vector was successfully constructed and its expression was evaluated in rat cardiomyocytes,which will facilitate further functional studies of DPP6 in cardiomyocytes.

关 键 词：DPP6 基因 腺病毒载体 绿色荧光蛋白 心肌细胞 

分 类 号：R33[医药卫生—人体生理学] R34[医药卫生—基础医学]