| 蔡旭伟,杨健,宋后燕,等.构建Egr-1启动子调控Smad7基因的重组腺病毒及启动子活性研究[J].中华放射医学与防护杂志,2005,25(1):13-17.CAI Xu-wei,YANG Jian,SONG Hou-yan,et al.Construction of recombinant adenovirus with Egr-1 promoter and Smad7 cDNA and study of the Egr-1 promoter's biological activity[J].Chin J Radiol Med Prot,2005,25(1):13-17 |
| 构建Egr-1启动子调控Smad7基因的重组腺病毒及启动子活性研究 |
| Construction of recombinant adenovirus with Egr-1 promoter and Smad7 cDNA and study of the Egr-1 promoter's biological activity |
| 投稿时间：2004-10-18 |
| DOI： |
| 中文关键词: 腺病毒 Egr-1基因启动子 Smad7 基因治疗 |
| 英文关键词:Adenovirus Egr-1 promoter Smad7 Gene therapy |
| 基金项目:国家自然科学基金资助项目(30170289) |
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| 中文摘要: |
| 目的 构建含Egr-1启动子和Smad7cDNA的重组复制缺陷型腺病毒并研究射线诱导Egr-1基因启动子调控Smad7体外表达的时间和剂量效应,为下一步的基因治疗奠定基础。方法 以AdenoX-XTM表达试剂盒为基础,应用分子克隆技术,将穿梭质粒pShuttle的CMV启动子替换为Egr-1启动子,将Smad7cDNA亚克隆至穿梭质粒内,与腺病毒DNA重组,在HEK293细胞中进行包装、扩增,纯化测定病毒滴度。通过Westernblot检测X射线照射激活Egr-1启动子调控Smad7体外表达的时间和剂量效应。结果 经酶切、PCR及测序鉴定证实成功构建了含Egr-1启动子和Smad7的重组复制缺陷型腺病毒pAdES。病毒滴度为30×1011TCID50ml。感染重组腺病毒的靶细胞经不同剂量的深部X射线照射后Smad7蛋白表达量存在一定的时间、剂量差异:实验组Smad7蛋白表达量在照射后1h即已升高,2～7h之间维持在一个较高水平,尔后表达量开始下降;实验组中Smad7蛋白表达量随着吸收剂量的增加而增加,在8～1.5Gy之间维持一个较高的表达水平。而对照组Smad7蛋白表达量无明显的时间和剂量效应。结论 成功构建了含Egr-1启动子和Smad7的重组复制缺陷型腺病毒pAdES,重组于腺病毒内的Egr-1启动子经射线激活后具有调控下游Smad7表达的活性。 |
| 英文摘要: |
| Objective To construct a recombinant replication-defective adenovirus containing Egr-1 promoter and Smad7 cDNA, then to evaluate the biological activity of Egr-1 promoter. Methods Based on Adeno-XTM expression system, CMV promoter of the pShuttle vector was replaced by Egr-1 promoter, and the Smad7 cDNA was subcloned into the MCS(multiple cloning site) of pShuttle. The recombinant pShuttle was then subcloned into the Adeno-XTM genome, which was transformed into E.coli to get recombinant Adeno-XTM plasmid DNA. The recombinant adenovirus was packaged and amplified in the transfected HEK 293 cells before it was purified and tested for viral titer. The fibroblasts (3T6 cells) infected by the recombinant adenovirus were irradiated, and the activity of Egr-1 promoter was quantitively determined by the amount of Smad7 protein expressed in the 3T6 cells using Western blot. Results Identified by restriction endonuclease analysis and PCR, the recombinant adenovirus containing Egr-1 promoter and Smad7 cDNA was constructed successfully, with a viral titer of 1.0×1011 TCID50/ml. The expressed amount of Smad7 protein varied at different dose levels and different time points post-irradiation in the 3T6 cells infected with the recombinant adenovirus. The amount of Smad7 protein increased along with the rising of the irradiation dose, and remained at a high expression level from 8 Gy to 15 Gy. The amount of Smad7 protein started to increase at 2 hours post-irradiation, and maintained a relatively high level for the next 5 hours before it descended, which was not observed in the control 3T6 cells. Conclusions With the aid of Adeno-XTM expression system and molecular cloning techniques, construction of recombinant adenovirus could be quick and efficient. The recombined Egr-1 promoter has the activity of regulating the expression of downstream Smad7 cDNA. The increase in Smad7 expression under control of Egr-1 promoter induced by ionizing radiation is time- and dose-dependent. |
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