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目的:构建一种受人端粒酶逆转录酶(h TERT)启动子和mi R-145双调控的更加安全的条件复制型腺病毒。方法:在已构建的h TERT启动子调控早期复制必需基因E1A的条件复制型腺病毒Adzq11基础上,再将四个拷贝的mi R-145的靶序列加入到E1A的3’非翻译区,构建新型溶瘤腺病毒Adzq11-145T。应用Real-time PCR测定人非小细胞肺癌细胞A549和人胚肺成纤维细胞HLF中mi R-145的表达量;然后在这两株细胞中,分别转染Adzq11和Adzq11-145T对应的穿梭质粒及感染病毒本身,用Real-time PCR检测E1A表达量以测定调控效果;用病毒空斑单位法检测两种病毒的复制情况;用CCK8检测对A549和HLF细胞的杀伤作用。结果:构建的新型溶瘤腺病毒Adzq11-145T在HLF中复制量明显低于Adzq11,其E1A m RNA水平和杀伤效应也显著降低。而在A549中,Adzq11-145T在E1A表达、病毒复制和溶瘤能力方面较Adzq11均无显著降低。结论:成功构建出一种新的受转录和转录后水平双重调控的条件复制型腺病毒Adzq11-145T,它比仅加入h TERT启动子调控早期复制必需基因E1A的腺病毒Adzq11更加安全。
Objective: To construct a safer replication-competent adenovirus that is double-regulated by hTERT promoter and mi R-145. Methods: Based on Adzq11, a conditional replication-competent adenovirus that regulates the E1A essential for early replication of hTERT promoter, four copies of mi R-145 target sequence were added to the 3 ’untranslated region of E1A to construct New oncolytic adenovirus Adzq11-145T. Real-time PCR was used to detect the expression of mi R-145 in human non-small cell lung cancer A549 cells and human embryonic lung fibroblasts HLF. Then, the two recombinant plasmids were transfected into Adzq11 and Adzq11-145T shuttle plasmids respectively And infected with the virus itself. The expression of E1A was detected by Real-time PCR to determine the regulatory effect. The replication of the two viruses was detected by the virus plaque unit assay. The cytotoxicity of A549 and HLF cells was detected by CCK8. Results: The constructed recombinant oncolytic adenovirus Adzq11-145T was significantly lower than that of Adzq11 in HLF, and its E1A m RNA level and killing effect were also significantly decreased. In A549, however, Adzq11-145T showed no significant reduction in E1A expression, viral replication and oncolytic ability compared with Adzq11. CONCLUSION: A new conditional replication-competent adenovirus Adzq11-145T successfully constructed is successfully constructed, which is more secure than Adzq11, an adenovirus that only regulates the early replication-essential gene E1A by adding h TERT promoter.