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目的:构建一种端粒酶启动子调控目的基因MDA-7/hIL24表达的复制缺陷型腺病毒,并初步探索其抗肿瘤活性。方法:通过基因操作技术将启动子(hTERTp)+目的基因(MDA-7)插入到5型腺病毒E1A区域,构建复制缺陷型腺病毒AdTP-mda7;同时构建对照病毒AdTP-EGFP。TCID50法测定病毒滴度。应用蛋白质印迹法检测MDA-7在肿瘤细胞株中的表达状态;通过结晶紫染色实验检测两种病毒对肿瘤细胞的杀伤差异。结果:成功构建携带目的基因的腺病毒载体,PCR鉴定正确;蛋白质印迹结果表明MDA-7选择性地在多种肿瘤细胞株中表达,诱导肿瘤细胞凋亡,如A549,SGC-7901等,在以相同MOI值感染原代成纤维细胞时,原代细胞中没有检测到MDA-7表达;结晶紫染色试验结果表明AdTP-mda7对肿瘤细胞株的杀伤能力明显高于对照病毒AdTP-EGFP。结论:成功构建复制缺陷型腺病毒AdTP-mda7,并证实MDA-7蛋白在肿瘤细胞中过表达诱导多种肿瘤细胞株发生凋亡。
OBJECTIVE: To construct a replication-defective adenovirus whose telomerase promoter regulates the expression of the target gene MDA-7 / hIL24, and to explore its antitumor activity. METHODS: The promoter (hTERTp) + gene of interest (MDA-7) was inserted into E1A region of adenovirus type 5 by gene manipulation to construct replication-defective adenovirus AdTP-mda7. At the same time, the control virus AdTP-EGFP was constructed. TCID50 determination of virus titer. The expression status of MDA-7 in tumor cell lines was detected by Western blotting. The difference between the two viruses in killing tumor cells was detected by crystal violet staining. Results: The adenovirus vector carrying the target gene was successfully constructed and correctly identified by PCR. Western blotting results showed that MDA-7 selectively expressed in many tumor cell lines and induced apoptosis of tumor cells, such as A549 and SGC-7901. The expression of MDA-7 in the primary cells was not detected when the primary fibroblasts were infected with the same MOI. The crystal violet staining results showed that the ability of AdTP-mda7 to kill the tumor cell lines was significantly higher than that of the control virus AdTP-EGFP. Conclusion: We successfully constructed replication-defective adenovirus AdTP-mda7 and confirmed that overexpression of MDA-7 in tumor cells induced apoptosis in many tumor cell lines.