非共价糖链聚合物的构建及其在流感病毒检测上的研究

Influenza, commonly known as flu, is a contagious respiratory illness caused by influenza virus, with seasonal circulation. The seasonal influenza every year brings about 1 billion new inflections and 250,000 deaths all over the world. Accurate and rapid diagnosis of influenza is essential for appropriate patient management and anti-influenza therapy. There are several methods available for routine clinical diagnosis of influenza, such as viral cell culture, rapid antigen-based tests and real-time PCR (RT-PCR). However, the low sensitivity still troubles us due to the complexity and variable concentration of the clinical samples. To improve the current rapid test methods, easily-available purification technologies are desirable. Glycopolymers that contain sugar pendants have attracted a growing attention for their protein recognition and purification since the amplified multivalency interaction, also known as the glyco-cluster effect. Therefore, a thermosensitive diblock glycocopolymer, PNiPPAm-b-Polycation, will be prepared using RAFT polymerization protocol. It should be highlighted that the designed sialyl oligosaccharide modified with an inorganic thiosulfate leaving group at its reducing end can be linked with the PNiPPAm-b-Polycation via anion exchanging, producing a non-covalent glycopolymer which can capture the influenza virus based on the specific binding between sialyl oligosaccharide and Influenza hemagglutinin. To meet the needs of influenza rapid diagnosis, the thermo-regulated phase separable glycopolymers will release the absorbed virus through another anion-exchanging, delivering a purified virus solution. In addition, direct labeling of virus will also be studied carefully via the chemical fluorescent modification on the specific binding sugars.

流感是威胁人类健康的主要传染性疾病之一。快速准确地检测流感病毒，对于流感的早期诊疗和防控有重要意义。在临床上，生物样本有其复杂性，且病毒浓度存在差异，导致现有流感快检方法准确率较低。相对于小分子标记物筛查，糖链高分子可提供更多的作用靶点，因而在流感病毒的检测上更具优势。而通过共价键构筑的糖链高分子，虽然可以有效捕捉流感病毒，但在之后的检测应用上依然存在很多问题。本项目中，申请人计划合成一种非共价键结合的糖链高分子，利用唾液酸寡糖与流感病毒表面血凝素的特异性结合，对临床上鼻咽拭子提取液中的流感病毒进行快速分离纯化和直接荧光标记。首先，利用之前开发的预活化策略和水相糖基化反应，合成出一位硫代硫酸盐修饰的寡糖单元；再通过水溶液中的离子交换制备出非共价糖链高分子；最后经过选择性病毒捕捉、温敏相分离和糖链末端荧光修饰，达到对目标病原体快速纯化和标记的目的；从而为提高流感病毒快检准确率提供有效手段。

病毒的快速检测和疫苗研究生产是应对新出现病毒和变异病毒威胁的关键技术。然而，受到咽拭子样本中低病毒载量的影响，包括胶体金免疫层析在内的临床快检手段时常会出现假阴性的结果。此外，传统的病毒纯化工艺往往包含梯度离心沉降、超滤、离子交换色谱等多步复杂、耗时的操作，这也是制约灭活病毒疫苗和病毒载体疫苗规模化快速生产的瓶颈之一。因此，开发简单快速的病毒富集纯化技术对于提高病毒快检的灵敏度和准确性、简化和加快病毒疫苗的生产具有重要的意义。本项目基于寡糖抗原和病毒表面突刺蛋白能够特异性结合的理论基础，设计并制备了一种表面修饰唾液酸寡糖的糖基微珠材料，并利用化学手段对糖链的分子结构进行优化，从而赋予其高效捕捉流感病毒的能力。此外，通过可逆连接臂的设计，在富集病毒的同时还可以实现温和洗脱条件下，病毒颗粒的有效释放。后续临床研究表明，通过糖基磁珠的前处理操作，可将临床咽拭子样本的甲型流感胶体金快检的灵敏度提高30倍以上。同时，基于糖基琼脂糖凝胶填料的病毒亲和色谱柱可以实现培养基中目标流感病毒的快速纯化，初步病毒回收率大于50%，且洗脱后病毒保持了原有的侵染活性。这一能够捕捉释放目标病毒的糖基微珠技术平台，不仅能有效服务于临床医学检验，改善其灵敏度，而且能为开发新的、高效的病毒疫苗纯化工艺提供技术支持。