生物工程学报 25 July 2009, 25(7): 1022-1027
利用RNAi逆转录病毒载体系统对人朊病毒蛋白表达的稳定抑制
徐文婧1,2, 王娣3, 王娟1,2, 杨怀义1
1 中国科学院微生物研究所, 北京100101
2 中国科学院研究生院, 北京100039
3 北京中医药大学, 北京100029
摘 要: 朊病毒是引起可传染的致死性海绵状脑病的致病因子, 细胞中正常的朊病毒蛋白(PrPC)在该疾病病程发展中起着必不可少的作用。同时, PrPC已被证明在胃癌、乳腺癌等癌症中发挥着保护癌细胞的作用。根据人源PrPC(HuPrPC) cDNA序列, 本研究设计了4种19 nt的siRNA, 将其构建成RNAi逆转录病毒载体系统, 进行了其对HuPrPC表达的抑制效应的分析, 从中获得了能高效稳定抑制HuPrPC表达的3种靶向序列, 其中si626(5.-GGTTGAGCAGATGTGTATC-3.)的抑制效果最为明显, 其抑制效率可达85%以上。随后, 利用筛选出的si292和si626的稳定干扰细胞系进行了细胞浸润性实验, 结果发现, PrPC干扰细胞系细胞浸润能力显著下降。这为进一步研究朊病毒疾病的基因治疗、以PrPC为靶标进行PrPC相关癌症的辅助治疗研究奠定了一定的基础。
关键词: 朊病毒, RNAi, 逆转录病毒载体, 细胞浸润
Stable inhibition of human prion protein through a retrovirus-based RNAi system
Wenjing Xu1,2, Di Wang3, Juan Wang1,2, and Huaiyi Yang1
1 Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
2 Graduate University of Chinese Academy of Sciences, Beijing 100039, China
3 Beijing University of Chinese Medicine, Beijing 100029, China
Abstract: Prion leads to fatal transmissible spongiform encephalopathies. Cellular prion protein (PrPC) is necessary in prion disease. At present, it is demonstrated that PrPC plays a protective role in several carcinomas, such as gastric and breast cancer. We designed four 19-nt siRNAs according to cDNA sequence of human PrPC and constructed retrovirus-based RNAi vectors. We evaluated the inhibitive effect of these sequences on HuPrPC(human PrPC) and selected out three sequences with stable and efficient inhibition. And the efficiency of si626 reached more than 85%, which effect was significant. Next, we performed cell invasion assays of PC3M-si292 and PC3M-si626 in which PrPC was inhibited. And it showed that the cell invasive ability decreased in PrPC knock-down cell lines. This will make preparations for the further research on gene therapy of prion diseases and PrPC related carcinoma treatment and PrPC could be considered as a potential therapeutic target molecule in prostate cancer treatment.
Keywords: prion, RNAi, retroviral vector, cell invasion
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