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目的 构建针对端粒酶阳性肿瘤的特异性腺病毒载体 ,研究其介导目的基因在肿瘤细胞内的定向表达能力。方法 将端粒酶逆转录酶 (hTERT )启动子克隆入质粒 pDC3 12的多克隆位点构成外源基因表达盒 ,在基因表达盒上下游分别插入 1个绝缘子序列 ,构建针对端粒酶阳性肿瘤的特异性腺病毒载体pSG TPE ;采用Luciferase报告基因进行hTERT启动子的活性分析 ;以pSG TPE携带绿色荧光蛋白 (EGFP)报告基因 ,重组腺病毒AdTPE EGFP ,观察其介导EGFP在肿瘤细胞内的定向表达能力 ,并与CMV启动子控制的腺病毒AdCMV EGFP作对照。结果 hTERT启动子在正常细胞内几乎没有活性 ,而在肿瘤细胞内的活性与SV 40启动子相近。腺病毒AdTPE EGFP能介导EGFP在肿瘤细胞内定向而稳定表达 ,在正常细胞内不表达 ,而对照腺病毒AdCMV EGFP在肿瘤和正常细胞内均表达EGFP。结论 靶向端粒酶阳性肿瘤的腺病毒载体 pSG TPE ,不但提高了对基因表达调控的特异性 ,而且降低了对正常细胞毒性作用。绝缘子的应用隔断了外源基因表达盒和病毒基因表达调控序列之间的互相干扰 ,进一步提高目的基因的表达效率。该载体对肿瘤的靶向基因治疗具有重要的应用价值。
Objective To construct a specific adenoviral vector targeting telomerase positive tumors and study its ability to mediate the targeted expression of the target gene in tumor cells. Methods The hTERT promoter was cloned into the multi-cloning site of plasmid pDC3 12 to construct an exogenous gene expression cassette. One insulator sequence was inserted into the upstream and downstream of the gene expression cassette to construct a plasmid for telomerase positive tumors Specific adenovirus vector pSG TPE; luciferase reporter gene hTERT promoter activity analysis; pSG TPE carrying green fluorescent protein (EGFP) reporter gene, adenovirus AdTPE EGFP recombinant adenovirus, observe its orientation in the EGFP tumor cells And compared with AdCMV EGFP, an adenovirus controlled by CMV promoter. Results The hTERT promoter showed almost no activity in normal cells, whereas the activity in tumor cells was similar to SV40 promoter. The adenovirus AdTPE EGFP could mediate the directional and stable expression of EGFP in tumor cells and not in normal cells, whereas the control adenovirus AdCMV EGFP expressed EGFP in both tumor and normal cells. CONCLUSION: The adenovirus vector pSG TPE targeting telomerase positive tumors not only enhances the specificity of gene expression regulation, but also reduces the effect on normal cytotoxicity. The use of insulator intercepts the mutual interference between the exogenous gene expression cassette and the viral gene expression regulatory sequences to further improve the expression efficiency of the target gene. The vector has important application value for targeted gene therapy of tumors.