广州地区GII.4 Sydney 2012[P31]型诺如病毒GZ2013-L10毒株病毒样颗粒功能和免疫原性鉴定

Identification of the function and immunogenicity of virus-like particles of GII.4 Sydney 2012[P31] norovirus GZ2013-L10 strain in Guangzhou

摘要:

【背景】诺如病毒(Norovirus，NoV)是全球范围内引起急性胃肠炎暴发的主要病原体之一，其中GII.4型通过不断变异在人群中持续存在并占据诺如病毒感染的主导地位，尤其GII.4 Sydney2012[P31]变异株自2012年出现以来在全球各地持续流行至今。【目的】制备广州地区GII.4 Sydney2012[P31]型诺如病毒毒株GZ2013-L10的病毒样颗粒(virus like particle，VLP)，并系统表征其功能及免疫原性特点。【方法】从毒株GZ2013-L10中扩增ORF2基因并克隆构建重组转座载 PFastBac1-L10-ORF2，进一步转化至大肠杆菌DH10Bac构建重组杆状病毒质粒，进而在昆虫细胞sf9中表达病毒样颗粒并通过超速离心纯化，最后经透射电镜、Western blotting和受体结合实验对病毒样颗粒进行表征。此外，将免疫小鼠获得的病毒抗血清通过间接酶联免疫吸附测定(enzyme-linked immunosorbent assay，ELISA)和受体结合阻断试验进行验证。【结果】成功构建了重组杆状病毒质粒Bacmid-L10-ORF2并获得病毒样颗粒，电镜结果表明病毒样颗粒直径约为30 nm，SDS-PAGE和Western blotting显示蛋白大小约为58 kDa。受体结合实验结果显示，病毒样颗粒能与A/B/O等分泌型唾液受体及猪胃黏膜蛋白结合，而与非分泌型唾液受体均不结合。免疫小鼠获得效价为1.3×105的抗血清，但ELISA结果显示其与不同基因型诺如病毒衣壳蛋白无交叉免疫活性。此外，抗血清对同型病毒样颗粒具有受体中和阻断作用，但对不同型别病毒样颗粒(包括GII.8、GII.17和GII.3)无中和效果。【结论】本研究制备并系统表征了广州地区GZ2013-L10毒株的病毒样颗粒及其抗血清，其研究结果可为解析其流行原因以及疫苗研发提供参考。

Abstract:

[Background] Norovirus (NoV) is one of the main causes of global acute gastroenteritis. Among the viruses, GII.4 genotype persists through continuous mutation and is responsible for the majority of NoV infections. Particularly, since the emergence in 2012, GII.4 Sydney 2012[P31] variant has been prevalent all over the word. [Objective] To prepare virus-like particle (VLP) of GII.4 Sydney 2012[P31] NoV strain GZ2013-L10 in Guangzhou and systematically characterize its function and immunogenicity. [Methods] The ORF2 gene of GZ2013-L10 was amplified and cloned to construct the recombinant transposon vector pFastBac1-L10-ORF2. The vector was further transformed into Escherichia coli DH10Bac to develop the recombinant baculovirus plasmid, which was then transfected into insect cells sf9 for the expression of VLPs. The yielded VLPs were purified by ultracentrifugation. Finally, transmission electron microscopy, Western blotting, and receptor binding experiment were performed to characterize the VLPs. In addition, indirect enzyme-linked immunosorbent assay (ELISA) and test of the blocking of receptor binding were carried out verify the virus antiserum of the immunized mice. [Results] We successfully constructed the recombinant baculovirus plasmid Bacmid-L10-ORF2 and obtained VLPs. Electron microscopy demonstrated that the VLPs were about 30 nm in diameter. SDS-PAGE and Western blotting showed that the proteins were 58 kDa. Salivary receptor experiment results showed that the VLPs of GZ2013-L10 can bind to secretory salivary receptors such as A/B/O and porcine gastric mucosa protein (PGM) rather than non-secretory salivary receptors. Antiserum with a titer of 1.3×10⁵ was detected in the immunized mice. However, ELISA results showed no cross-immunoreactivity with capsid proteins of different NoV genotypes. In addition, the antiserum blocked the receptor binding of VLP of the same genotype but had no neutralizing effect on VLP of a different genotype (such as GII.8, GII.17, and GII.3). [Conclusion] VLP of GZ2013-L10 in Guangzhou and its antiserum were prepared and systematically characterized. The result can serve as a reference for elucidating the cause of the epidemic and developing vaccines.