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本研究旨在制备人GⅡ.14诺如病毒的（Norovirus, NoV）病毒样颗粒（Virus-like particle, VLP），探索其抗原性与组织血型抗原结合的特征。利用杆状病毒系统表达人GⅡ.14 NoV VP1蛋白，通过超速离心和分子筛层析纯化VLP；将VLP免疫动物制备兔多抗血清，通过免疫印迹（Western blot, WB）、酶联免疫吸附（ELISA）进行抗原性评价及多抗功能验证；利用唾液结合实验检测人GⅡ.14 VLP的糖结合特征；通过蛋白序列及结构比较分析GⅡ.14P蛋白潜在的糖结合位。纯化获得人GⅡ.14 VP1蛋白，电镜观察显示可以形成结构较为均一的VLP；制备的兔多抗能与GⅡ.14 VLP特异性结合，与检测的其他基因型VLP没有交叉反应；唾液结合实验表明GⅡ.14 VLP与人A/B/O/AB型别唾液都有较好的结合；序列和结构分析显示GⅡ.14 P蛋白模拟结构与GⅡ.9最为接近，具有与GⅡ.9以及流行株GⅡ.4等相似的潜在糖结合区。本研究表明GⅡ.14具有较为广泛的唾液结合特性，通过序列比对和结构学方法分析了GⅡ.14潜在的受体结合机制，为不同型别NoVs的流行监测提供了更多依据。
Abstract：This study aimed to get the virus-like particles(VLPs) of human GⅡ. 14 norovirus(NoV) and explore its binding characteristics with histo-blood group antigens(HBGAs). The VP1 protein of human GⅡ.14NoV was expreesed by baculovirus system. VLPs were purified by ultracentrifugation and gel-filtration chromatography. Rabbit polyclonal antisera were generated by immunizing animals with GⅡ. 14 VLPs. The function of polyclonal antibodies was verified by western blotting and enzyme-linked immunosorbent assay. The saliva-binding assay was carried out to detect the glycan-binding characteristics of human GⅡ. 14 VLPs.Potential glycan binding sites of the GⅡ. 14 P protein were analyzed by sequence alignment and structural comparison. The purified GⅡ. 14 VP1 protein formed relatively uniform VLPs when observed by electron microscopy. Rabbit polyclonal antibodies could specifically bind to GⅡ.14 VLPs with no obvious cross-reactivity with other tested VLPs. GⅡ.14 VLPs bound well to human saliva of A/B/O/AB types according to the salivabinding assay. Sequence and structural analyses showed that the simulated structure of the GⅡ.14 P protein was closest to GⅡ.9 P protein, and possessed similar potential glycan-binding sites to that of GⅡ.9 and the epidemic GⅡ.4. Our data indicate that GⅡ.14 has relatively broad binding characteristics to saliva. The potential glycanbinding mechanism of GⅡ.14 was illustrated through sequence alignment and structural comparison. Our results provide more evidence for the epidemic monitoring of NoVs of different genotypes.
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基本信息:
DOI：10.13242/j.cnki.bingduxuebao.004528
中图分类号:R392
引用信息:
[1]欧阳瑄泽,柴鹏弟,宋敬东等.人GⅡ.14诺如病毒VLP抗原性及组织血型抗原结合特征[J].病毒学报,2024,40(04):705-715.DOI:10.13242/j.cnki.bingduxuebao.004528.
基金信息:
北京市自然科学基金（项目号：L232011）,题目：我国诺如病毒基因变异变迁及所致胃肠炎流行特征和经济负担研究~~