广州地区GII.17型诺如病毒GZ-L343毒株病毒样颗粒的构建与免疫特征的鉴定

Construction and immune characterization of virus-like particles of GII.17 norovirus strain GZ-L343 in Guangzhou

【背景】人源诺如病毒是急性胃肠炎暴发的主要原因,GII.4是过去几十年的主要流行基因型。2014/2015年出现的GII.17型变异株是中国首例导致大规模暴发的非GII.4流行株。通过对来自华南地区的诺如病毒GII.17型毒株的完整基因组序列进行分析,证实了该GII.17型突变株与先前确定的GII型变异株不同。【目的】制备广州地区GII.17型诺如病毒GZ-L343的病毒样颗粒,并系统表征其免疫原性及功能特性。【方法】借助杆状病毒表达系统制备GII.17-GZ-L343的病毒样颗粒,并通过氯化铯梯度超速离心对其进行纯化,制备抗血清并对其免疫功能进行评价。【结果】聚丙烯酰胺凝胶电泳和蛋白质免疫印迹结果表明所得蛋白分子量大小约为58 kDa;透射电镜结果表明病毒样颗粒直径约为30 nm;酶联免疫吸附测定结果显示该病毒样颗粒具有较好的免疫原性;唾液组织血型抗原的体外受体结合测定表明,该病毒样颗粒与部分A型、B型、O型及AB型分泌及非分泌血型样本存在阳性结合;效价测定结果表明免疫所得血清效价在104以上;交叉反应结果表明该抗血清与异型病毒样颗粒不存在交叉反应。此外,体外阻断结果表明,该抗血清仅能阻断相应型别病毒样颗粒与受体的结合作用,与异型病毒样颗粒不存在交叉阻断。【结论】GII.17-GZ-L343具有广泛的结合谱,其抗血清对异型病毒样颗粒不具有广谱性,且只对同型病毒样颗粒存在阻断作用,结果将为进一步揭示该病毒的宿主适应特性、进化机制及研发多价疫苗提供理论支持。

[Background]Human norovirus is regarded as the leading cause of the outbreak of acute gastroenteritis with GII.4 being the predominant genotype during the past decades.The GII.17 variant that emerged in 2014/2015 was the first non-GII.4 epidemic strain causing a large-scale epidemic in China.The full-length genome sequencing of the GII.17 strain from South China confirmed that it was different from the previously identified GII variants.[Objective]To prepare the virus-like particles and systematically characterize the immunogenicity and functions of GII.17-GZ-L343 in Guangzhou.[Methods]The baculovirus expression system was employed to produce GII.17-GZ-L343 in Sf9 insect cells.The virus-like particle was purified by cesium chloride gradient ultracentrifugation.Finally,the antiserum was prepared and its immune function was evaluated.[Results]The results of SDS-PAGE and Western blotting showed that the molecular weight of the obtained protein was about 58 kDa.The prepared virus-like particle had a diameter of about 30 nm,good immunogenicity,and binding activities to the saliva of type A/B/O/AB secretors and non-secretors.The serum titer after immunization was above 104,and the antiserum had no inhibitory effects on other variants.In addition,the antiserum only blocked the binding of homotypic virus-like particle to the receptor,and did not block the binding of heterotypic virus-like particle.[Conclusion]GII.17-GZ-L343 has a broad binding pattern.Its antiserum is not broad-spectrum for heterotypic virus-like particle and only has blocking effect on homotypic virus-like particle.The results provide theoretical support for further revealing the host adaptation characteristics and evolution mechanism of the virus and developing multivalent vaccines.