转基因介导的RNA干扰抵抗口蹄疫病毒感染的研究

【作者】 焦晔；

【导师】 郑兆鑫； 钟江；

【作者基本信息】 复旦大学 ， 微生物学， 2012， 博士

【摘要】 口蹄疫病毒(FMDV)每年在全球范围内造成畜牧业的巨大损失。现有防疫手段的不足使新型抗病毒办法的研究变得十分必要。RNA干扰(RNAi)是近年来新发现的一种真核生物基因转录后调控的分子机制,并且已对其抵抗病毒感染的能力开展了大量的探索性研究。但是,如何使得RNAi在有机体内能长期而又高效地发挥作用,仍然存在很多的困难。本文尝试使用转基因的手段来让组织培养细胞和小鼠获得自身表达抗FMDV的shRNA的能力。首先构建了转基因载体质粒PB-EN3D2B和PB-N3D2B,两者都能编码针对FMDV基因组2B与3D基因保守序列的shRNAo使用PB转座子作为转基因载体系统将PB-EN3D2B质粒转入猪细胞IBRS-2和仓鼠细胞BHK-21之后,筛选得到27个稳定的转基因细胞系。在攻毒实验中,6个IBRS-2转基因细胞系中的3个表现出了显著的抗O型和AsiaI型口蹄疫病毒感染的能力：当受到20～50 TCID50的病毒攻击时,转基因细胞系和对照细胞相比,抗病毒能力提高10～1000倍,持续48小时。其中效果最好的细胞系IB32-6在所有的三次攻毒实验中均完全抵御了病毒,未发生任何细胞病变。实验证明,这种抵抗能力是由内源性的shRNA带来的,并且与细胞系的转基因拷贝数无直接的联系。之后我们将PB-N3D2B质粒中表达针对FMDV基因组保守区段shRNA的2.5 kb功能片段采用DNA直接注射或慢病毒(lenti-virus)载体介导的方法导入小鼠受精卵。两种方式一共得到27只转基因原代小鼠,使用其中三只原代小鼠F3D2B-10、N3D2B-18和N3D2B-81的后代进行了O型FMDV的攻毒实验。实验表明,来自N3D2B-18和N3D2B-81家系的转基因乳鼠具有先天的抵抗3到6个LDso O型口蹄疫病毒感染的能力,存活率提高了19到27.3个百分点。但F3D2B-10和N3D2B-18家系在后续的攻毒实验中不能抑制AsiaI型口蹄疫病毒的复制。本项工作是第一次系统地在哺乳动物组织培养细胞和个体水平上尝试由转基因手段介导RNAi抵抗口蹄疫病毒感染的研究,为未来该技术在大型易感动物,如猪和牛上的应用提供了有益的资料。更多还原

【Abstract】 Foot-and-mouth disease virus (FMDV) is responsible for the heavy economic losses across the world each year. Because of the deficiency of existing vaccines, the development of new antiviral strategies is needed. RN A interference (RNAi) is a recently found mechanism which induces gene silencing post-transcriptionally in all eukaryotes and a lot of investigative works have been performed on its potential of overcoming virus infection. However, current techniques have their own limitations of conducting RNAi persistently and effectively in vivo. In this study, we tried to endue cultured cells and mice with the ability of expressing anti-FMDV shRNAs endogenously by transgenic technology. Fisrt, two recombinant plasmids PB-EN3D2B and PB-N3D2B were constructed, both of which encode two shRNAs that target the 3D and 2B non-structural regions of FMDV genome. By employing piggyBac transposon system to integrate PB-EN3D2B into swine cell IBRS-2 or hamster cell BHK-21, we got 27 stable monoclonal transgenic cell lines.3 of 6 IBRS-2 transgenic cell lines exhibited innate resistance against both type O and type AsiaI FMDV:IB32-2 and IB32-3 displayed 10 to 1000 times of inhibition of the virus replication within 48 hours post challenge, when the cell line IB32-6 was intact in all three experiments without any CPEs. The results implied that this resistance was brought by endogenous shRNAs but not interferons and was not directly relevant to transgenic copy numbers. Then the 2.5kb shRNA-expressing fragment of PB-N3D2B was transfected into mouse fertilized eggs by microinjection or by lenti-virus vector to harvest 27 primary transgenic mice. Offspring of transgenic mouse strain F3D2B-10, N3D2B-18, and N3D2B-81 received type O FMDV challenge and the transgenic suckling mice of strain N3D2B-18 and N3D2B-81 showed 19 to 27.3 percentage of increase in surviving rates when injected with 3 to 6 LD50 of type O FMDV. But strain F3D2B-10 and N3D2B-18 failed to inhibit the replication of type AsiaI FMDV in subsequent experiments. This investigation was the first to detailedly study the feasibility and validity of introducing RNAi into mammals to overcome FMDV infection by transgenic strategy both in vitro and in vivo. We wish our work could provide useful information for similar applications on large susceptible animals like swine and bovine.更多还原