基于黄瓜花叶病毒的多价弱毒疫苗创制与突变修复机理研究

弱毒疫苗是防治植物病毒病的有效方法，但存在突变修复导致疫苗效果不稳定，是弱毒疫苗创制的限制因素。本研究将以黄瓜花叶病毒（CMV）为基础创制防治多个靶标病毒的多价弱毒疫苗，并运用病毒生物学、反向遗传学、RNA结构学等技术手段研究疫苗的突变修复的引发因素。首先对CMV的2b、CP蛋白和非翻译区的TLS元件构建突变，分析突变的稳定性和突变体致病性，筛选遗传稳定的防治CMV的单价弱毒疫苗；单价弱毒疫苗中插入不同长度的异源片段，分析插入型突变体的遗传稳定性和致病性，明确CMV容纳异源片段的能力；分析可防治相应靶标病毒的最小插入片段；在CMV中插入不同靶标病毒的组合片段，逐步获得多价弱毒疫苗；分析疫苗创制中核心调控元件的RNA结构变化、病毒蛋白的致畸突变和病毒粒子的包装限制与突变修复的关系。获得广寄主多价弱毒疫苗，并解析RNA病毒突变修复的引发机理，为超多价广寄主弱毒疫苗的开发提供材料基础和理论依据。

Cross-protection via attenuated vaccine is an effective strategy for prevention and control of plant virus diseases. However, mutation repair frequently occurred during the construction of attenuated vaccine and restricted the stability and safety of attenuated vaccine. In this study, multivalent attenuated vaccine based on Cucumber mosaic virus (CMV) will be constructed to aim at prevention and control of several important viruses. In addition, mutation repair of attenuated mutation on CMV will be analyzed to identify the initiation factors of mutation repair through virus biology, reverse genetics, RNA structure probing and so on. Firstly, mutations on 2b ORF, CP ORF and/or TLS in 3’UTR of CMV will be constructed and used to infect plants through agro-infiltration to test the pathogenicity of mutants and the stability of mutation sites. Based on the stability of mutation sites and the evaluation of cross-protection against CMV, monovalent vaccine against CMV will be screened. Secondly, different length of heterogenous fragments will be inserted into MCS (multiple clone sites) of monovalent vaccine. Through the analysis of pathogenicity of insertion mutants and the stability of inserted fragments, the intake capacity of the length of heterogenous fragment in CMV will be identified. 50-200 bp fragments of heterogenous viruses were inserted into MCS (multiple clone sites) of monovalent vaccine. Through the evaluation of cross-protection against target virus from these vaccines containing heterogenous virus fragments, the smallest insertion fragment/sequences inducing gene silencing on target virus will be identified. Subsequently, combined fragments from different target viruses were amplified through over-lapped PCR and inserted into MCS (multiple clone sites) of monovalent vaccine. Cross-protection on co-inoculation of different target viruses will be evaluated. Finally, multivalent attenuated vaccine against at least five species of target viruses will be constructed. During the construction of attenuated vaccine, mutation repair will be detection by RT-PCR on mutation sites and analyzed on the repair frequency. In addition, we will also identify the relationship between mutation repair and RNA structure change of cis-elements, mutation of viral protein and package limitation of virus particle. Through this project, we will not only get multivalent attenuated vaccine but also identify the initiation factors of mutation repair, which will provide the basis of material and theory for the construction of super-multivalent attenuated vaccine in the future.