人源诺如病毒GII.3[P12]型病毒样颗粒的免疫原性及受体结合能力

Immunogenicity and receptor binding ability of the virus-like particle of the GII.3[P12]human norovirus

目的制备GII.3[P12]型人源诺如病毒(human norovirus,HuNoV)广州株GZ2013-L20的病毒样颗粒(virus-like particle,VLP)和多克隆抗体,系统表征其免疫原性特征和受体结合能力,为HuNoV防控提供数据支撑。方法以GZ2013-L20毒株基因组为模板,扩增ORF2并构建重组转座载体,转化至大肠埃希菌DH10Bac获得重组杆状病毒质粒Bacmid-L20-ORF2,在昆虫细胞sf9中表达目的VLP,采用透射电镜、SDS-PAGE、Western blot(WB)和受体结合实验等方法对纯化后VLP进行鉴定。此外,将VLP免疫小鼠获得多克隆抗体,通过间接ELISA和受体结合阻断试验进行评价。结果构建了重组杆状病毒Bacmid-L20-ORF2,并成功获得目的毒株VLP;透射电镜显示颗粒直径约为30 nm;SDS-PAGE和WB结果显示蛋白相对分子质量(Mr.×10^(3))约58;受体结合实验表明,目的VLP与分泌型唾液受体(A型、B型、AB型和O型)、O型非分泌型唾液受体以及猪胃黏蛋白均能有效结合;VLP多克隆抗体效价达到2×10^(5),且与20种(20/28)基因型HuNoV的衣壳蛋白具有交叉免疫反应;受体结合阻断实验显示,多克隆抗体仅对同型VLP具有中和阻断效果,而对GII.2、GII.4、GII.8和GII.17等基因型VLP没有中和作用。结论GII.3[P12]型HuNoV广州株VLP与分泌型和非分泌型唾液受体均呈现较强的结合能力,其多克隆抗体具有免疫结合广谱性,但中和阻断效果仅对同型病毒有效,其结果为疫苗研发的理性设计提供基础数据。

Objective To prepare the virus-like particle(VLP)of the GII.3[P12]human norovirus(HuNoV)strain GZ2013-L20 in Guangzhou and its polyclonal antibody,and systematically characterize its immunogenicity and receptor binding ability,which would provide data for prevention and control of HuNoV.Methods ORF2 gene was amplified from the genome of the GZ2013-L20 strain to construct the recombinant transposon vector,which was further transformed into Escherichia coli DH10Bac to develop the recombinant baculovirus Bacmid-L20-ORF2.VLP was expressed in the sf9 insect cells and then purified.Transmission electron microscopy,SDS-PAGE,Western blot(WB),and receptor binding experiments were performed to characterize the purified VLP.In addition,the polyclonal antibody from the immunized mice was evaluated by indirect enzyme-linked immunosorbent assay(ELISA)and the blocking test of receptor binding.Results The recombinant baculovirus plasmid Bacmid-L20-ORF2 was constructed,and the target VLP was successfully obtained.The result by the transmission electron microscope demonstrated that the VLP were about 30 nm in diameter.SDS-PAGE and WB analyses showed that the protein’s relative molecular mass(Mr.×10^(3))was about 58.The result of receptor binding experiments showed that the VLP could bind to the secretory salivary receptors(types of A,B,AB and O),non-secretory salivary receptors(O type)and the porcine gastric mucin.The polyclonal antibody with a titer of 2×10^(5) was detected in the immunized mice,which showed strong cross-immunoreactivity with capsid proteins of 20(20/28)HuNoV genotypes.In addition,the result of blocking tests of receptor binding showed that the VLP polyclonal antibody only blocked the viral VLP of the same genotype,but had no neutralizing effects on the VLPs of GII.2,GII.4,GII.8 and GII.17.Conclusions The VLP of GII.3[P12]HuNoV Guangzhou strain showed strong binding ability to both secretory and non-secretory salivary receptors,and its polyclonal antibody showed a broad spectrum of immunobinding,but its neutralization blocking feature was effective only against the virus of the same genotype.The result provide basic data for rational design of vaccine development.