| 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。 |
| 关键词: 腺病毒载体 α-肌动蛋白启动子 胚胎干细胞 |
| DOI： |
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| 基金项目:国家“863”计划项目(2002AA216161)；国家自然科学基金项目(30200137) |
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| Construction and identification of recombinant adenovirus with cardiac specific α-actin promoter |
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| Abstract: |
| 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-N1-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 PmeⅠ,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 endonuclease PacⅠdigestion,pAd-easy-actin was transfected into HEK 293 cells to produce recombinant adenovirus pAd-actin before PCR identification,and the titer was 7×107 pfu/mL.The recombinant adenovirus containing α-actin promoter had been constructed successfully,and provided a basis to monitor the differentiation of embryonic stem cells into cardiomyocytes. |
| Key words: adenovirus vector α-actin promoter embryonic stem cells |
