心肌特异性α-actin启动子重组腺病毒的构建及其鉴定  

Construction and identification of recombinant adenovirus with cardiac specific α-actin promoter

作　　者：杨玉艾[1] 孙永科[1] 华进联[1] 窦忠英[1] 

机构地区：[1]西北农林科技大学陕西省干细胞工程技术中心,陕西杨陵712100

出　　处：《西北农林科技大学学报（自然科学版）》2007年第8期1-5,10,共6页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家"863"计划项目(2002AA216161);国家自然科学基金项目(30200137)

摘　　要：为了探讨心肌肌动蛋白(α-actin)基因启动子的心肌组织特异性及其表达活性,构建了心肌组织靶向性表达的基因载体,应用PCR从pEGFP-N1-α-actin-P质粒中克隆出α-actin启动子片段,将其插入到切除CMV启动子的腺病毒穿梭载体pAdTrack中,构建出pAdTrack-actin重组穿梭载体,经酶切和测序鉴定正确后用PmeI线性化,与含有腺病毒骨架DNA的pAdEasy-1质粒在BJ5183大肠杆菌细胞中同源重组成新的重组体pAd-easy-actin,抽提重组体DNA,经PacI酶切回收后以脂质体法转染人胚肾细胞株HEK293,包装出完整腺病毒pAd-actin进行PCR检测,并应用蚀斑形成试验测定病毒滴度。结果显示,重组腺病毒中含有α-actin启动子片段,病毒滴度为7×107pfu/mL。表明含心肌α-actin启动子的重组腺病毒pAd-actin构建成功。To study the tissue-specific expression ability and activity of cardiac α-actin gene promoter, and to construct a cardiac-specific gene expression vector, the cardiac α-actin promoter gene was amplified from the plasmid pEGFP-Nl-α-actin-P by PCR and cloned into the adenoviral shuttle plasmid pAdTrack which was deleted CMV,the recombinant shuttle was named pAdTrack-actin after the identification by restriction endonuclease analysis and sequence analysis,and then the pAdTrack-actin plasmid was linearized with Pme I ,and transferred into E. coli BJ5183 cells pretransformed with the pAdEasy-1 plasmid. A recombinant adenoviral genomic named pAd-easy-actin was obtained by homologous recombination. By endo- nuclease Pac I digestion,pAd-easy-actin was transfected into HEK 293 cells to produce recombinant adenovirus pAd-actin before PCR identification,and the titer was 7 × 10^7 pfu/mL. The recombinant adenovirus containing α-actin promoter had been constructed successfully,and provided a basis to monitor the differen-tiation of embryonic stem cells into cardiomyocytes.

关 键 词：腺病毒载体 α-肌动蛋白启动子 胚胎干细胞 

分 类 号：Q78[生物学—分子生物学]