肿瘤端粒酶靶向腺病毒载体的表达活性

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。

Expression Activity of Cancer-Specific Adenovirus Vector Targeting Telomerase

Objective To construct an adenovirus vector specifically targeting telomerase-positive cancers,and to investigate its capability of targeting transgene expression in cancer cells.Methods hTERT promoter was cloned into the multiple cloning sites of pDC312 plasmid to produce a transgene expression cassette.An insulator was inserted respectively into upstream and downstream of the cassette,then the adenovirus vector targeting telomerase-positive cancers was generated as pSG-TPE.The luciferase assay was applied to analyze the hTERT promoter activity.pSG-TPE carrying green fluorescent protein(EGFP) was used to recombine adenovirus AdTPE-EGFP.The restricted expression of EGFP mediated by AdTPE-EGFP was examined and compared with the control adenovirus AdCMV-EGFP regulated under the CMV promoter.Results hTERT promoter had no activity in normal cells,but nearly had as strong activity as SV40 promoter in cancer cells.AdTPE-EGFP could express EGFP specifically and steadily in cancer cells but not in normal cells,whereas AdCMV-EGFP could express EGFP both in cancer and normal cells.Conclusion The adenovirus vector pSG-TPE has a specificity of targeting transgene expression,with decreased toxicity to normal cells.The application of insulator in adenovirus vector interdicts the interference between the transgene expression cassette and the regulatory sequence of viral gene,and further increases the efficiency of transgene expression.The application of adenovirus vector pSG-TPE is a promising approach to the targeting gene therapy of tumor.