牛病毒性腹泻病毒E2蛋白多表位疫苗筛选及生物信息学验证  

Screening of multi-epitope vaccine of bovine viral diarrhea virus E2 protein and bioinformatics verification

作　　者：何金科 杨亚军 邓肖玉 何延华 张超 马忠臣 王震[2] 陈创夫[2] HE Jin-ke;YANG Ya-jun;DENG Xiao-yu;HE Yan-hua;ZHANG Chao;MA Zhong-chen;WANG Zhen;CHEN Chuang-fu(College of Life Sciences,Shihezi University,Shihezi,Xinjiang,832000,China;College of Animal Science and Technology,Shihezi University,Shihezi,Xinjiang,832000,China)

机构地区：[1]石河子大学生命科学学院,新疆石河子8320001 [2]石河子大学动物科技学院,新疆石河子832000

出　　处：《中国兽医学报》2020年第9期1690-1698,共9页Chinese Journal of Veterinary Science

基　　金：国家自然科学基金资助项目(U1803236);兵团重大科技资助项目(2017AA003)。

摘　　要：为了筛选牛病毒性腹泻病毒(BVDV)E2蛋白的多表位亚单位疫苗,本研究选用4种B细胞预测软件(immunomedicine group、ABCpred、BepiPred、SMVTriP)和2种T细胞预测软件(NetBoLApan、NetMHCIIpan)筛选出重叠肽作为优势表位,通过柔性linker连接成多表位疫苗,使用免疫信息学方法对抗原性、过敏源、理化性质、糖基化位点、二级和三级结构进行评估。通过二硫化物工程提高疫苗稳定性。使用分子对接评估疫苗构建体与免疫受体的结合能力,最后进行silico克隆。结果表明,成功构建17000相对分子质量的可溶性蛋白质,免疫信息学结果表明,疫苗构建体是非过敏性良好抗原,具有一个潜在的N-糖基化位点,在二级结构中α螺旋占约3.2%,β折叠占约43.2%,无规卷曲占约53.6%。三级结构Ramachandran作图发现优势区域中含有88.2%残基,进行细化后优势区域的残基增加到93.5%,3D模型表位作图也证明多表位疫苗具有良好的免疫原性,二硫化物工程确定出3对残留物可以形成二硫键,分子对接表明疫苗构建体与TLR3受体具有高亲和力,最后密码子优化和silico克隆确保设计的亚单位疫苗在大肠杆菌K12表达系统中更高表达。免疫信息学分析表明这种多表位疫苗可以有效表达并能够诱导强烈的T细胞和B细胞免疫应答。本研究为BVDV E2多表位疫苗的设计提供了一种新的方法。For screening multi-epitope subunit vaccine of the bovine viral diarrhea virus(BVDV)E2 protein,in this study,screening of overlapping peptides using four B cell prediction software and two T cell prediction software were used for the dominant epitope,then connected to a multi-epitope vaccine with a flexible linker.Immunological informatics was used to evaluate antigenicity,allergens,physicochemical properties,glycosylation sites,secondary and tertiary structures.Vaccine stability was improved through disulfide engineering.Molecular docking was used to evaluate the ability of the vaccine construction to bind to the immune receptor,and then silico cloning was performed.The results showed that the soluble protein with relative molecular quality of 17000 was successfully constructed.The immunological informatics showed that the vaccine construct is a non-allergic antigen with a potential N-glycosylation site.In the secondary structure,theαhelix accounted for 3.2%,β-folded accounted for 43.2%,and random coil accounted for 53.6%.The tertiary structure Ramachandran showed that there were 88.2%residues in the dominant region.After refinement,the residues in the dominant region were increased to 93.5%.The 3 D model epitope mapping also proved that the multi-epitope vaccine has good immunogenicity.The disulfide project identified three pairs of residues that could form disulfide bonds.Molecular docking indicated that the vaccine construct has high affinity to the TLR3 receptor,and finally codon optimization and silico cloning ensure that the designed subunit vaccine is higher expression in the Ec.oli K12 expression system.In summary,immunoinformatic analysis indicated that this multi-epitope vaccine can be efficiently expressed and capable of inducing strong T cell and B cell immune responses.This study provides a new approach to the design of BVDV E2 multi-epitope vaccine.

关 键 词：牛病毒性腹泻病毒 E2蛋白 B细胞表位 T细胞表位 分子对接 二硫化物工程 silico克隆 

分 类 号：S852.65[农业科学—基础兽医学]