我国食源性诺如病毒GII.2[P2]型毒株基因组结构与衣壳蛋白功能研究

Genome Structure and Capsid Protein Function of the Foodborne Norovirus GII.2[P2] Strain in China

摘要:

食源性诺如病毒（Norovirus）是引起全球食品安全事件的主要微生物病原，具有丰富的遗传多样性，基因型较复杂，近年来GII.2逐渐成为我国主要流行基因型之一。为了解食源性诺如病毒GII.2型变异特点，该研究以2015年在广州地区检出的GII.2[P2]型GZ2015-L335毒株为对象，对其基因组结构及衣壳蛋白功能进行了系统表征。该毒株基因全长为7 496 bp，在线基因分型结果表明其为GII.2[P2]基因型。通过克隆其衣壳蛋白VP1基因，借助杆状病毒表达系统获取重组VP1蛋白，并采用氯化铯密度梯度离心法进行纯化。SDS-PAGE和Western blot实验结果表明重组VP1蛋白分子量约为58 ku；透射电镜结果显示该蛋白可自组装形成直径约为30 nm的病毒样颗粒（Virus-like Particle，VLP）；ELISA结果显示该VLP与抗GII.2[P]衣壳P颗粒血清具有较高结合活性。此外，受体结合实验证实该VLP与A/B/O等分泌型及非分泌型唾液呈现出广泛的结合作用。该研究对GII.2[P2]型GZ2015-L335毒株基因组结构进行了系统分析，成功制备并表征了其病毒样颗粒，结果将为食源性诺如病毒的高效抗体研制及新型检测技术研发提供支撑。

Abstract:

Foodborne norovirus is the leading microbial pathogens that causes food safety incidents worldwide and has abundant genetic diversity. In recent years, GII.2 has gradually become one of the main prevalent genotypes in China. In order to understand the variation characteristics of this food-borne norovirus GII.2, the GZ2015-L335 strain of GII.2[p2] isolated in Guangzhou in 2015 was used as the research objective, and the genome structure and capsid protein function of this strain were characterized. The full length of the gene of this strain was 7 496 bp. The results of online genotyping showed that it was GII.2[P2] genotype. The recombinant capsid protein VP1 was obtained by cloning the capsid protein VP1 gene and using the recombinant baculovirus expression system, then purified by cesium chloride density gradient centrifugation. The results of SDS-PAGE and Western blot showed that the molecular weight of the recombinant protein VP1 was about 58 ku. The transmission electron microscopy examinations showed that the VP1 could self-assemble into virus-like particles (VLP) with a diameter of approximately 30 nm. The results of ELISA showed that the VLP had high binding activity for the serum against GII.2[P2] capsid P particles. In addition, the receptor binding assay showed that the VLP exhibited broad binding affinities to both secretory salivary receptors (including A/B/O) and non-secretory salivary receptors. In summary, this study systematically analyzed the genome structure of the GII.2[P2] strain, and successfully prepared and characterized its virus-like particles. The obtained results would provide support for the development of highly effective antibody and novel detection technology for food-borne norovirus.