利用人RNA聚合酶ⅰ構(gòu)建腸道病毒D68的反向遺傳系統(tǒng)

2018年8月，天津大學(xué)生命科學(xué)學(xué)院；環(huán)境科學(xué)與工程學(xué)院(School of Life Sciences, Tianjin University, 92 Weijin Road, Nankai District, Tianjin 300072, China；School of Environmental Science and Engineering, 92 Weijin Road, Nankai District, Tianjin 300072, China) Tao Wang老師研究團(tuán)隊(duì)在《Virus Genes》上發(fā)表論文：

“A reverse genetics system for enterovirus D68 using human RNA polymerase I”

“利用人RNA聚合酶ⅰ構(gòu)建腸道病毒D68的反向遺傳系統(tǒng)”

Abstract：

Human enterovirus D68 (EV-D68) is a highly contagious virus, which causes respiratory tract infections. However, no effective vaccines are currently available for controlling EV-D68 infection. Here, we developed a reverse genetics system to recover EV-D68 minireplicons and infectious EV-D68 from transfected plasmids using the RNA polymerase I (Pol I) promoter. The EV-D68 minireplicons contained the luciferase reporter gene, which flanked by the non-coding regions of the EV-D68 RNA. The luciferase signals could be detected in cells after transfection and Pol I promoter-mediated luciferase signal was significantly stronger than that mediated by the T7 promoter. Furthermore, recombinant viruses were generated by transfecting plasmids that contained the genomic RNA segments of EV-D68, under the control of Pol I promoter into 293T cells or RD cells. On plaque morphology and growth kinetics, the rescued virus and parental virus were indistinguishable. In addition, we showed that the G394C mutation disrupts the viral 5'-UTR structure and suppresses the viral cap-independent translation. This reverse genetics system for EV-D68 recovery can greatly facilitate research into EV-D68 biology. Moreover, this system could accelerate the development of EV-D68 vaccines and anti-EV-D68 drugs.

摘要：

人類腸道病毒D68 (EV-D68)是一種高傳染性病毒，可引起呼吸道感染。然而，目前還沒有有效的疫苗可用于控制EV-D68感染。在這里，科研人員開發(fā)了一個(gè)反向遺傳系統(tǒng)，利用RNA聚合酶I (Pol I)啟動(dòng)子從轉(zhuǎn)染的質(zhì)粒中恢復(fù)EV-D68的微型復(fù)制子和傳染性EV-D68。EV-D68微型復(fù)制子包含熒光素酶報(bào)告基因，該基因位于EV-D68 RNA的非編碼區(qū)兩側(cè)。轉(zhuǎn)染后細(xì)胞中可檢測(cè)到熒光素酶信號(hào)，且Pol I啟動(dòng)子介導(dǎo)的熒光素酶信號(hào)明顯強(qiáng)于T7啟動(dòng)子介導(dǎo)的熒光素酶信號(hào)。在Pol I啟動(dòng)子的控制下，將含有EV-D68基因組RNA片段的質(zhì)粒轉(zhuǎn)染到293T細(xì)胞或RD細(xì)胞中，生成重組病毒。在斑塊形態(tài)和生長動(dòng)力學(xué)上，獲救病毒和親本病毒沒有區(qū)別。此外，科研人員發(fā)現(xiàn)G394C突變破壞了病毒的5'-UTR結(jié)構(gòu)并抑制了病毒的帽獨(dú)立翻譯。該EV-D68的反向遺傳系統(tǒng)為EV-D68的生物學(xué)研究提供了極大的便利。此外，該系統(tǒng)可加快EV-D68疫苗和抗EV-D68藥物的開發(fā)。

該論文中，對(duì)293T(人胚胎腎293T)細(xì)胞和RD(橫紋肌肉瘤)的體外培養(yǎng)是使用Ausbian特級(jí)胎牛血清完成的。欲了解或購買Ausbian特級(jí)胎牛血清可以聯(lián)系北京締一生物400-166-8600.