慢病毒载体介导的RNAi持续抑制草鱼呼肠孤病毒复制的研究

Grass carp hemorrhage is the most severe disease in cultured grass carp (Ctenopharygodon idellus), which is caused by the grass carp reovirus(GCRV). Currently, no effective prevention and treatment methods are available for the disease yet. RNA interference (RNAi), a natural mechanism for post-transcriptional silencing of homologous genes by doub le-stranded RNA (dsRNA), has emerged as a powerful tool being widely used in the investigation of gene functions and the inhibintion of infection of many viral pathogens. Lentiviral vector has the ability of stably integrating the foreign gene(s) carried into the chromosomes of the transduced cells, and therefore, the consistent transcription and expression of the foreign gene(s) are realized. Based on our previous study that highly effective siRNA constructs have been generated targeting the RNA dependant RNA polymerase (RdRp) coding gene and outer capsid protein (OCP) coding gene of GCRV, in this study, the lentiviral vector carrying siRNA constructs will be constructed, and then the grass carp cell line and the fertilized eggs of rare minnow (Gobiocypris rarus) will be transduced with the generated lentiviral vector to obtain the grass carp cells and rare minnow (T0 and T1) stably expressing the siRNA constructs and consistently inhibiting the GCRV replication. This project will investigate the propagation, mRNA transcription, protein expression and ultrastructural morphogenesis of GCRV in the transduced cells and the transgenic rare minnow by cytopathic effect examination, virus titration, real time qRT-PCR, in situ hybrdization, immunofluorescent staining, flow cytometry analysis and electron microscope observation. Therefore, the mechanism and efficacy of consistent inhibition of GCRV replication by the lentiviral vector mediated RNAi will be elucidated systematically.

草鱼呼肠孤病毒（GCRV）引起的草鱼出血病是草鱼养殖中最为严重的疾病，目前仍缺乏有效防治方法。RNA干扰（RNAi）是动植物抵抗外来病原生物感染、抑制其复制的一种细胞内保护机制。慢病毒载体可将携带的外源基因稳定整合在靶细胞染色体上，实现外源基因的持续表达。前期研究已筛选出高效抑制GCRV复制的siRNA分子，本研究将构建转录siRNA结构的慢病毒载体，转导草鱼细胞系和稀有鮈鲫受精卵，以获得稳定转录siRNA结构持续抗GCRV的细胞株和T0代及T1代转基因稀有鮈鲫；采用观察细胞病变效应、GCRV滴度测定、Real-time qRT-PCR检测、病毒核酸原位杂交、免疫荧光染色、流式细胞仪检测以及电镜超微结构观察，研究慢病毒载体转导的草鱼细胞与稀有鮈鲫体内不同时间段GCRV滴度、mRNA水平、GCRV蛋白合成的差异、病毒颗粒形态发生过程，系统揭示慢病毒载体介导的RNAi持续抑制GCRV复制的机制

草鱼呼肠孤病毒（GCRV）引起的草鱼出血病是草鱼养殖中最为严重的疾病，目前仍缺乏有效防治方法。本项目在前期研究已筛选出高效抑制GCRV复制的siRNA分子（GCRV-RdRp，GCRV-OCP）的基础上，针对GCRV流行株（GCRV-106和GCRV-104）构建了转录siRNA结构的慢病毒载体，建立并优化了慢病毒转导草鱼肾脏细胞系（CIK）的方法，优化后的慢病毒转导效率可达86.5%，使用不同基因型GCRV感染转导并筛选后的CIK细胞，通过观察细胞病变效应、GCRV滴度测定、Real-time qRT-PCR检测证实慢病毒介导的RNA干扰可明显抑制草鱼肾脏细胞中GCRV的感染和增殖，病毒感染72h后转导细胞内GCRVmRNA的表达量仅为正常细胞内的30.9%-45.4%。采用慢病毒载体显微注射的方法获得了可持续表达GCRV-shRNA的转基因稀有鮈鲫，检测了慢病毒载体携带的外源基因在鱼体细胞内的整合与siRNA分子的转录，但研究发现慢病毒转导的稀有鮈鲫受精卵孵化率及成活率低，其原因可能与慢病毒载体介导的shRNA随机插入鱼体染色体组，造成了插入突变，影响了稀有鮈鲫正常的发育生长。本项目进一步使用了更加高效稳定的基因编辑技术CRISPR/CAS9技术，构建抑制草鱼呼肠孤病毒的细胞模型。建立并利用CRISPR/CAS9系统体外定向敲除草鱼呼肠孤病毒受体蛋白基因（gcJAM-A），在gcJAM-A基因缺失的草鱼肾脏细胞中，不同基因型GCRV感染CIK细胞后48-72h，GCRV的病毒滴度、mRNA表达和病毒蛋白的表达均被明显抑制；本研究同时在大鲵肌肉细胞（GSM）中表达了草鱼JAM-A，可以使本来对GCRV不敏感的GSM细胞可感染并支持GCRV病毒的复制。本项目的研究证实，慢病毒介导的GCRV-shRNA可整合到鱼类细胞及受精卵染色体中并持续表达，在细胞水平实现对GCRV的抑制作用；研究还证实了在细胞水平利用CRISPR/CAS9系统构建的草鱼-GCRV受体基因缺失细胞对GCRV的敏感性明显降低，是可开发利用的新型抗病毒方法之一。