双特异性启动子调控条件复制腺病毒载体的构建  

Construction of conditionally replicative adenovirus vector mediated by dual specific promoters

作　　者：李玮[1] 谭建[1] 

机构地区：[1]天津医科大学总医院核医学科,300052

出　　处：《中华生物医学工程杂志》2013年第2期134-139,共6页Chinese Journal of Biomedical Engineering

基　　金：国家自然科学基金（81171372）;天津市应用基础及前沿技术研究计划（12JCZDJC26000）

摘　　要：目的 构建并鉴定既能在人端粒酶反转录酶（hTERT）启动子调控腺病毒早期区域1A（E1A）基因,又能在胶质纤维酸性蛋白（GFAP）启动子引导入钠碘转染体（hNIS）基因的腺病毒载体.方法 采用免疫印迹分析检测人脑星形胶质母细胞瘤细胞U87、人神经胶质瘤细胞U251和人胚肺成纤维细胞MRC-5细胞感染病毒前自身GFAP和端粒酶蛋白的表达情况.通过PCR扩增获得hTERT启动子、GFAP启动子及hNIS基因,采用基因合成腺病毒E1A基因.采用hTERT和GFAP启动子重组质粒转染MRC-5、U251、U87细胞,转染24 h后应用荧光分析法检测hTERT启动子及GFAP启动子的启动活性.再将上述特异性启动子分别与E1A基因和hNIS基因连接,最后插入穿梭质粒pDC311中,构建重组质粒pDC311-Tp-E1A-Gp-NIS,并应用EcoR Ⅰ及Sal Ⅰ双酶切及测序鉴定.将pDC311-Tp-E1A-Gp-NIS与骨架腺病毒质粒pBHGlox△E1-3Cre共转染人胚肾293细胞,得到条件复制腺病毒Ad-Tp-E1A-Gp-NIS后,分别感染293、MRC-5、U87和U251细胞,利用病毒空斑形成实验检测条件复制腺病毒的复制能力.使用重组腺病毒Ad-Tp-E1A-Gp-NIS及Ad-CMV-EGFP（对照）感染U251、U87及MRC-5细胞后,采用γ计数器测定由hNIS基因介导的摄125I能力.结果 在U87和U251细胞中,均有相对分子质量120000的端粒酶和49000的GFAP蛋白的表达,在MRC-5中,则无表达.GFAP启动子和hTERT启动子能引导目标基因胶质瘤靶向性表达,启动效率分别为U87细胞中（62.10±6.26）％和（49.24±1.73）％,U251细胞中（59.75±34.36）％和（37.31±16.86）％.限制性内切酶能将质粒pDC311-Tp-E1A-Gp-NIS酶切成3252、5500 bp的片段,经测序鉴定后与预计序列一致.成功构建的Ad-Tp-E1A-Gp-NIS条件复制腺病毒,在U87和U251细胞能很好的产生病毒颗粒,在293细胞也能实现复制,但是在MRC-5细胞中则无病毒产生.U251和U87细胞感染Ad-Tp-E1A-Gp-NIS后均具备了较明显的摄125I能力,约为对照组的93.4倍和107.1Objective To construct and identify the conditionally replicative adenovirus vectors which may induce the human sodium iodide symporter （hNIS） expression in the early region 1A （EIA） gene of the human telomerase reverse transcriptase （hTERT） and the glial fibrillary acidic protein （GFAP） promoter regions. Methods Immunoblotting assay was employed to detect the variation in GFAP and telomerase protein expression prior to and following viral infection of the cerebral stellar glioblastoma cells （U87）, neuroglioma cells （U251 ） and human embryonic lung fibroblasts （MRC-5） cells. The hTERT and GFAP promoters and the hNIS genes were amplified by polymerase chain reaction for synthesis of adenoviral E1A genes. The recombinant plasmids containing hTERT and GFAP gene promoters were adopted for transfection into MRC-5, U25l and U87, which entailed assessment of the hTERT and GFAP promoter activity via fluorescent analysis after 24 h. This was followed by ligation with the E1A and hNIS genes and subsequent cloning into the plasmid pDC311 for construction of the recombinant plasmid pDC31 l-Tp-E 1A- Gp-NIS that was identified by double enzyme digestion （EcoR I and Sal I ） and gene sequencing. This recombinant plasmid was cotransfected with adenoviral genomic plasmid pBHGloxAE1- 3Cre into the human embryonic kidney 293 cells forming the conditionally replicative recombinant adenovirus Ad-Tp- E1A-Gp-NIS. This Ad-Tp-E1A-Gp-NIS was employed to transfect the 293, MRC-5, U87 and U251 cells, whose conditional replicability was measured by plaque forming assay. The recombinant virus Ad-Tp-E1A- Gp-NIS and Ad-CMV-EGFP controls were used to transfect the U251, U87 and MRC-5 cells for detection of the capacity of ~25I uptake by using a ~/-ray counter. Results The expression of the 120 000 telomerase and 49 000 GFAP protein could be found in U87 and U251 cells, but not in MRC-5 cells. Glioma target gene expression could be induced by the GFAP and hTERT promoters, the efficiency of which was （62.10±6.26�

关 键 词：条件复制腺病毒 人钠碘转染体基因 人端粒酶反转录酶启动子 胶质纤维酸 性蛋白启动子 腺病毒E1A基因 

分 类 号：R3[医药卫生—基础医学]