生物工程學(xué)報(bào) 25 July 2009, 25(7): 1022-1027
利用RNAi逆轉(zhuǎn)錄病毒載體系統(tǒng)對(duì)人朊病毒蛋白表達(dá)的穩(wěn)定抑制
徐文婧1,2, 王娣3, 王娟1,2, 楊懷義1
1 中國科學(xué)院微生物研究所, 北京100101
2 中國科學(xué)院研究生院, 北京100039
3 北京中醫(yī)藥大學(xué), 北京100029
摘 要: 朊病毒是引起可傳染的致死性海綿狀腦病的致病因子, 細(xì)胞中正常的朊病毒蛋白(PrPC)在該疾病病程發(fā)展中起著必不可少的作用,。同時(shí), PrPC已被證明在胃癌,、乳腺癌等癌癥中發(fā)揮著保護(hù)癌細(xì)胞的作用。根據(jù)人源PrPC(HuPrPC) cDNA序列, 本研究設(shè)計(jì)了4種19 nt的siRNA, 將其構(gòu)建成RNAi逆轉(zhuǎn)錄病毒載體系統(tǒng), 進(jìn)行了其對(duì)HuPrPC表達(dá)的抑制效應(yīng)的分析, 從中獲得了能高效穩(wěn)定抑制HuPrPC表達(dá)的3種靶向序列, 其中si626(5.-GGTTGAGCAGATGTGTATC-3.)的抑制效果最為明顯, 其抑制效率可達(dá)85%以上,。隨后, 利用篩選出的si292和si626的穩(wěn)定干擾細(xì)胞系進(jìn)行了細(xì)胞浸潤性實(shí)驗(yàn), 結(jié)果發(fā)現(xiàn), PrPC干擾細(xì)胞系細(xì)胞浸潤能力顯著下降,。這為進(jìn)一步研究朊病毒疾病的基因治療、以PrPC為靶標(biāo)進(jìn)行PrPC相關(guān)癌癥的輔助治療研究奠定了一定的基礎(chǔ),。
關(guān)鍵詞: 朊病毒, RNAi, 逆轉(zhuǎn)錄病毒載體, 細(xì)胞浸潤
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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