A型塞内卡病毒VP2蛋白多表位抗原免疫原性分析  

Immunogenicity Analysis of Multi-epitopes Antigen of Senecavirus A VP2 Protein

作　　者：陶世宇 茹毅 陈娇 郝荣增 李亚军 卢炳州 石正旺 刘学荣 郑海学[2] 魏彦明[1] TAO Shi-Yu;RU Yi;CHEN Jiao;HAO Rong-Zeng;LI Ya-Jun;LU Bing-Zhou;SHI Zheng-Wang;LIU Xue-Rong;ZHENG Hai-Xue;WEI Yan-Ming(College of Veterinary Medicine,Gansu Agricultural University,Lanzhou 730070,China;State Key Laboratory of Veterinary Etiological Biology,Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China;China Agricultural Vet.Bio.Science and Technology Co.,Ltd.,Lanzhou 730046,China)

机构地区：[1]甘肃农业大学动物医学院,兰州730070 [2]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室,兰州730046 [3]中农威特生物科技股份有限公司,兰州730046

出　　处：《农业生物技术学报》2023年第6期1284-1295,共12页Journal of Agricultural Biotechnology

基　　金：甘肃省科技重大专项计划(21ZD3NA001);中国农业科学院兰州兽医研究所基本科研业务费(1610312021013);国家生猪产业技术体系(CARS-35)。

摘　　要：A型塞内卡病毒(Senecavirus A,SVA)是一种新型的小RNA病毒,严重危害我国养猪业的发展。目前尚无有效的SVA疫苗,为探究SVA新型亚单位疫苗,本研究以SVA的病毒结构蛋白2(viral structural protein 2,VP2)基因序列为基础,选取利用生物信息学方法预测的B细胞表位序列及已报道的口蹄疫病毒(Foot-and-mouth disease virus,FMDV)3A蛋白的T细胞表位基因,密码子优化后通过柔性连接肽进行串联连接,分别构建重组表达质粒pET28a-rSVP2-B和pET28a-rSVP2-BT。将原核可溶性表达并纯化的重组串联表位蛋白rSVP2-B和rSVP2-BT,经SDS-PAGE和Western blot鉴定后,免疫BALB/c小鼠(Mus musculus),评价其免疫原性。结果显示,重组质粒转化至大肠杆菌(Escherichia coli)BL21(DE3),经诱导表达后,目标蛋白均正确表达,SDS-PAGE分析显示,其相对分子质量分别为34和38 kD。Western blot结果显示,rSVP2-B和rSVP2-BT与SVA阳性血清均具有良好的反应原性。纯化后rSVP2-B和rSVP2-BT免疫小鼠产生的体液免疫水平无显著差异,均诱导产生了较强的特异性IgG抗体及IgG1抗体,且主要诱导偏向于Th2型的体液免疫应答。病毒中和实验结果显示,2种重组蛋白的中和抗体效价介于1∶64~1∶128。同时,rSVP2-B和rSVP2-BT免疫小鼠也产生了较强的细胞免疫,其中rSVP2-BT免疫组的脾淋巴细胞增殖水平、细胞因子白细胞介素-2(interleukin-2,IL-2)和干扰素-γ(interferon-γ,IFN-γ)水平显著高于rSVP2-B。以上结果表明,成功制备了具有良好免疫原性的重组串联多表位蛋白rSVP2-B和rSVP2-BT,rSVP2-BT的免疫原性优于rSVP2-B。本研究为SVA多表位疫苗研制提供理论依据。Senecavirus A(SVA),a new RNA virus belonging to the Picornaviridae family,which seriously endangers the development of China's pig industry.At present,there is no effective SVA vaccine.In order to study the new SVA subunit vaccine,this study was based on the viral structural protein 2(VP2)gene sequences of SVA,and the B cell epitope sequences predicted by bioinformatic methods and the T cell epitope genes in the reported Foot-and-mouth disease virus(FMDV)3A protein.The recombinant expression plasmids pET28a-rSVP2-B and pET28a-rSVP2-BT were constructed respectively after optimization codon and connecting in series with the flexible linkers.The recombinant tandem epitope proteins rSVP2-B and rSVP2-BT,which were expressed in prokaryotic system and purified by Ni+ion chromatographic method,were identified by SDS-PAGE and Western blot,and then immunized BALB/c mice(Mus musculus)to evaluate their immunogenicity.The results showed that the target proteins were expressed correctly after the recombined plasmid pET28a-rSVP2-B and pET28a-rSVP2-BT were transformed into Escherichia coli BL21(DE3)and induced by 1 mmol/L isopropy-β-D-thiogalactoside(IPTG).SDS-PAGE analysis showed that the relative molecular weights were 34 and 38 kD,respectively.Western blot results showed that rSVP2-B and rSVP2-BT had good reactivity with SVA positive sera.There was no significant difference in humoral immune levels between rSVP2-B and rSVP2-BT immunized mice with purification protein.Both of them induced strong specific IgG and IgG1 antibodies,and mainly induced humoral immune response biased towards Th2 type.The results of virus neutralization test showed that the neutralizing antibody titers induced by the two recombinant proteins ranged from 1∶64 to 1∶128.Meanwhile,mice immunized rSVP2-B and rSVP2-BT also produced strong cellular immunity.The mice spleen lymphocytes proliferation levels and interleukin-2(IL-2)and interferon-γ(IFN-γ)expression levels in the mice vaccinated with the rSVP2-BT protein group was significantly higher than

关 键 词：A型塞内卡病毒(SVA) 病毒结构蛋白2(VP2) 重组串联表位蛋白 可溶性表达 免疫原性 

分 类 号：S855.3[农业科学—临床兽医学]