基于共有序列、全基因合成的登革病毒蛋白质芯片快速构建及其应用

Recently, more and more viral infectious diseases were emerging. In order to know thoroughly the biological properties of these viruses, a strategy, which was quick, high effective and systematic, was in urgent need. In addition, Dengue virus was one of these viruses, whose genetic information was high mutable. Tens of million people were infected by Dengue virus every year. Up to now, besides that there was no effective vaccines and weak basic research, high effective platform tools were also short in the field of Dengue virus. At last, as a systematic platform, the conventional method for protein microarray was disadvantage in the long fabricated cycle, addiction to natural template and shortage flexible for high-mutable sequences. As a result, based on knowledge on protein microarray accumulated in our lab, the Dengue virus was taken as an example. Consensus sequences were firstly calculated from more than 3000 published ployprotein amino acid sequences. Subsequently, all the consensus sequences were transferred into the corresponding gene sequences. After optimized, all the genes were artificially synthesized and the expression clones were constructed. Further, all the proteins were isolated, purified and characterized in a high-throughput way. At last, the protein microarray of Dengue virus could be fabricated in a short time. In order to discover some new biomarkers and immunogens, a lot of sera, collected from the patients infected by Dengue virus, were used to be screened by the Dengue virus protein microarray. The Dengue virus protein microarray, fabricated in this project, would promote the basic research in the field of Dengue virus. In addition, the biomarkers and immunogens, discovered by exploiting the sera, would guide for new diagnosed methods. At last but not least, the strategy, developed in this project for fabricating protein microarray quickly, would be used for constructed protein microarray of other RNA viruses, because this procedure had two important advantages, such as commonality and no need to contact source of infection directly.

病毒性传染病愈演愈烈，亟需快速、高效、系统了解突发病毒生物学特性的整体解决方案；登革病毒每年感染数千万人，其遗传信息高度可变，目前缺乏有效的疫苗、药物及研究工具；传统蛋白质芯片构建技术具有构建周期长、严重依赖天然模板、无法应对高变异序列等不足。因此，基于所在实验室的已有基础，本项目将以登革病毒为例，计算已公开发表的3000多条登革病毒多蛋白氨基酸序列，获得共有序列，转换成对应的基因序列。经序列优化后，采用全基因合成的方式，快速获得共有序列及表达克隆，进行异源表达，高效获得大量可溶性的目标蛋白质，实现蛋白质芯片的快速制备，并将该芯片用于血清分析以发现新的标识物和免疫原。本研究所构建的蛋白质芯片将有力地促进登革病毒相关研究，基于血清分析所发现的标识物将为新型诊断技术的发展提供指引。本研究所发展的快速蛋白质芯片构建技术具有通用性、无需接触传染源等，将为其在重要RNA病毒蛋白质芯片的构建铺平道路。

病毒性传染病，愈演愈烈；登革病毒，基因组高度可变，每年感染数千万人，疫苗研制缓慢，缺乏有效治疗手段和药物；传统蛋白质芯片构建策略，极其耗费时间，严重依赖基因组材料，无法应对突变等传统构建策略的不足，严重制约其进一步推广应用。本课题针对蛋白质芯片传统构建策略的不足，以登革病毒为例，联合共有序列、基因合成等技术，发展了一套蛋白质芯片快速构建技术，成功构建了具有广泛代表性的登革病毒蛋白质芯片。通过血清筛选，ELISA验证等实验，表明登革病毒蛋白质I-E和III-E具有发展成为生物标识物的潜力。使用登革病毒蛋白质芯片进一步监测了登革热患者体内抗体的动态变化，发现了类似的病毒免疫反应，但是不同患者间存在巨大差异。