应用Ad Easy腺病毒载体系统构建人肌浆网钙离子ATP酶2a(SERCA2a)  

Construction and Expression of Recombinant Adenovirus Carrying Human Sarco(endo)plasmic Reticulum Ca～(2+) ATPase2a

作　　者：吕茜[1] 魏翠[1] 张清林[1] 王洁[1] 宫海滨[1] 

机构地区：[1]徐州市心血管病研究所徐州市中心医院,苏州徐州221009

出　　处：《现代生物医学进展》2010年第13期2409-2412,共4页Progress in Modern Biomedicine

基　　金：徐州市科技局社会发展项目(XM09B073);国家自然科学基金资助项目(30572073);江苏省自然科学基金项目(BK2005428);江苏省"六大人才高峰"第三批项目(资助类型C);江苏省医学重点人才(RC2007024)

摘　　要：目的:利用Adeasy腺病毒表达系统构建含人肌浆网钙离子ATP酶2a(SERCA2a)基因重组腺病毒,并在HEK293细胞中扩增制备重组腺病毒。方法:将人SERCA2a基因全长cDNA(3700bp)插入到腺病毒穿梭载体pAdTrack-CMV,成功构建pAd-TrackCMVSERCA2a重组质粒,经PmeI酶切线性化,采用电击法转入到已含Adeasy质粒的电感受态菌BJ5183进行重组。挑选同源重组质粒,PacI酶切线性化转染HEK293细胞包装成重组腺病毒颗粒,荧光检测有绿色荧光蛋白表达。将重组病毒和SD大鼠心肌细胞共培养,western-blot检测SERCA2a可以在大鼠心肌细胞过表达且影响了胞内SERCA2a的活性。结果:成功包装含人SERCA2a基因的重组腺病毒,并可以有效感染SD大鼠心肌细胞。结论:利用新型腺病毒载体在短时间内成功构建了携带有人SERCA2a基因的腺病毒,为以后进一步研究人SERCA2a基因治疗提供了新途径。Objective：To construct recombinant adenovirus vector bearing the human sarco（endo）plasmic reticulum Ca2＋ ATPase2a and amplify the adenovirus in 293 cells.Methods：Human SERCA2a cDNA（3700bp）was cloned into adenovirus shuttle plasmid pAdtrackCMV to generate a recombinant plasmid pAdtrackCMV-SERCA2a,then the linearized plasmid was transformed into E.coli BJ5183 cells which had been electroporated adenovirus backbone plasmid pAdEasy-1.The identified recombinant plasmid was digested with PacⅠand transfected into 293 cells to package the adenovirus,green fluorescence protein expression was detected under fluorescent microscope.Co-cultured recombinant adenovirus and SD rat cardiomyocytes,SERCA2a protein was overexpressed by western-blot.Results：Recombinant adenovirus with SERCA2a was constructed successfully,which were effectively infected to SD rat cardiac muscle cell.Conclusion：The recombinant plasmid SERCA2a was established rapidly and simply by homologous recombination.It may be a new tool for gene therapy using SERCA2a.

关 键 词：AdEasy腺病毒 人肌浆网钙离子ATP酶2a(SERCA2a) 转染 

分 类 号：Q75[生物学—分子生物学] Q78