P22 VLPs载体多表位幽门螺杆菌疫苗的构建及表征分析  

Construction and characterization of P22 VLPs Vector Multi-epitope Helicobacter pylori vaccine

作　　者：朱娅岚 杨加雪 方楚斌 何流 汪川[1] 唐田 ZHU Ya-lan;YANG Jia-xue;FANG Chu-bin;HE Liu;WANG Chuan;TANG Tian(West China School of Public Health and West China Fourth Hospital,Sichuan University,Chengdu,Sichuan 610041,China;不详)

机构地区：[1]四川大学华西公共卫生学院/四川大学华西第四医院,四川成都610041 [2]北京大学肿瘤医院

出　　处：《现代预防医学》2023年第1期169-176,共8页Modern Preventive Medicine

基　　金：四川省科技厅重点研发项目(2021YFQ0060);四川省科技厅重点研发项目(2021YFS0179)。

摘　　要：目的利用生物信息学软件分析幽门螺杆菌主要抗原蛋白的T/B淋巴细胞表位,评估不同表位组合的抗原性与致敏性,预测优势表位组合与P22噬菌体病毒样颗粒(VLPs)融合后的三级结构,构建基于P22 VLPs的多表位幽门螺杆菌疫苗。方法利用IEDB数据库、NetMHCIIpan-3.2和BepiPred 2.0在线工具对抗原蛋白的T/B淋巴细胞表位进行搜索和预测;利用VaxiJen v2.0、AllerTOP v2.0和ProtParam在线服务器分别对随机串联组成的不同表位组合进行抗原性、致敏性和理化性质进行分析;采用AlphaFold2、ProSA-web、RAMPAGE及ERRAT等软件对融合蛋白的三级结构进行预测、评估;构建P22 VLPs载体多表位幽门螺杆菌疫苗P22-C3,通过诱导表达及纯化获得疫苗实体;运用SDS-PAGE、透射电子显微镜和动态光散射技术对疫苗实体进行表征分析。结果将筛选得到的高保守、非致敏优势抗原表位进行随机串联,获得了20个表位组合(C1-C20),这20个表位组合均具有良好的抗原性和非致敏性。其中,表位组合C3不仅抗原评分高、理化性质稳定且预测的结构模型符合天然构象。将C3序列融合至P22 VLPs衣壳蛋白(Gp5)的C端,获得了基于P22 VLPs的幽门螺杆菌疫苗P22-C3。表征分析显示P22-C3较野生型的P22 VLPs,衣壳更厚,直径更大。结论本研究通过多维生物信息学软件分析了不同Hp表位组合的理化特性和三维结构,成功构建了基于P22 VLPs的幽门螺杆菌疫苗,为疫苗后期的动物试验和I期临床试验奠定了基础。Objective To analyze the T and B lymphocyte epitopes of the main antigen proteins of Helicobacter pylori(Hp)by bioinformatics software,to evaluate the antigenicity and sensitization of different epitope combinations,to predict the tertiary structure of the fusion dominant epitope combinations with P22 phage virus-like particles(VLPs)and to construct a multi-epitope Hp vaccine based on P22VLPs.Methods IEDB database,NetMHCIIpan-3.2 and BepiPred2.0 online tools were used to search and predict T and B lymphocyte epitopes of antigenic protein.The antigenicity,allergenicity and physicochemical properties of different epitope combinations randomly connected in series were analyzed by VaxiJen v2.0,AllerTOP v2.0 and ProtParam online tools.The tertiary structure of the fusion protein was predicted and evaluated by AlphaFold2,ProSA-web,RAMPAGE and ERRAT software.The P22VLPs vector multi-epitope Hp vaccine P22-C3 was constructed,the vaccine entity was obtained by induced expression and purification,and the vaccine entity was characterized by SDS-PAGE,transmission electron microscope and dynamic light scattering.Results The selected highly conserved and non-sensitized dominant epitopes were randomly linked together to obtain 20 epitope combinations(C1-C20),all of which had good antigenicity and non-allergenicity.Among them,the epitope combination C3 not only has high antigen scores,stable physical and chemical properties,but also the predicted structural model accords with the natural conformation.The C3 sequence was fused into the C terminal of P22VLPs capsid protein(Gp5),and the P22VLPs-based Hp vaccine P22-C3 was obtained.Characterization analysis showed that P22-C3 had a thicker capsid and a larger diameter than P22-WT.Conclusion In this study,the physicochemical properties and three-dimensional structure of different Hp epitope combinations are analyzed by multi-dimensional bioinformatics software,and the Hp vaccine based on P22VLPs is successfully constructed,which lays the foundation for later animal trials and phase I

关 键 词：幽门螺杆菌 病毒样颗粒 多价表位疫苗 生物信息学 

分 类 号：R181.3[医药卫生—流行病学]