融合蛋白E^(MP)-VP2在杆状病毒表达系统中的表达与免疫原性鉴定  

Baculovirus Expression and Immunogenicity of Fusion Protein E^(mp)-VP2 of Japanese Encephalitis Virus and Porcine Parvovirus

作　　者：张欣雅 杨扬 田羽彤 李宗杰 李蓓蓓[1] 刘珂[1] 邵东华[1] 邱亚峰[1] 魏建超[1] 马志永[1] ZHANG Xinya;YANG Yang;TIAN Yutong;LI Zongjie;LI Beibei;LIU Ke;SHAO Donghua;QIU Yafeng;WEI Jianchao;MA Zhiyong(Shanghai Veterinary Research Institute,CAAS,Shanghai 100141,China;不详)

机构地区：[1]中国农业科学院上海兽医研究所,上海200241 [2]长江大学,荆州434023

出　　处：《中国动物传染病学报》2024年第6期25-35,共11页Chinese Journal of Animal Infectious Diseases

基　　金：国家重点研发计划项目(2022YFD1800801);上海市科委农业领域项目(22N41900400)。

摘　　要：日本脑炎病毒(JEV)和猪细小病毒(PPV)是引起猪繁殖障碍的2种重要病原体,严重影响养猪业经济效益。本研究将日本脑炎病毒E基因上的多个B细胞及T细胞抗原表位与猪细小病毒VP2基因进行融合表达,构建pFastBac Dual-E^(MP)-VP2载体,转化到DH10Bac感受态细胞后,通过蓝白斑筛选成功构建了rBacmid E^(MP)-VP2重组杆粒,并在昆虫细胞Sf9中成功拯救了重组杆状病毒,实现了E^(MP)-VP2高效表达。SDS-PAGE和Western blot分析表明,表达产物分子量与预期相符,且可以和JEV及PPV阳性血清反应,表明重组E^(MP)-VP2蛋白具有良好的免疫反应性。表达的蛋白可以通过亲和层析成功纯化,用纯化后的E^(MP)-VP2蛋白免疫小鼠和豚鼠,可以分别激活小鼠和豚鼠JEV和PPV特异性体液免疫反应,证明E^(MP)-VP2蛋白具有免疫原性,为后续JEV和PPV的防控以及二联亚单位疫苗的研发奠定了基础。Japanese encephalitis virus(JEV)and Porcine parvovirus(PPV)cause reproductive disorders in pigs,which seriously affect the economic benefits of the pig industry.In this study,multiple B cell and T cell antigenic epitopes on the E gene of Japanese encephalitis virus(JEV)were fused with the VP2 gene of porcine parvovirus(PPV)to construct the pFastBac Dual-E^(MP)-VP2 vector.After transforming into DH10Bac competent cells,the rBacmid E^(MP)-VP2 recombinant bacmid was successfully constructed through blue-white screening.The recombinant baculovirus was then successfully rescued in Sf9 insect cells,achieving efficient expression of E^(MP)-VP2.SDS-PAGE and Western blot showed that the molecular weight of the expressed product was in line with expectations.The expressed recombinantE^(MP)-VP2 protein reacted with JEV and PPV positive serum samples,indicating its good immunoreactivity.Subsequently,the expressed protein was purified through affinity chromatography and used to immunize mice and guinea pigs.These animals produced specific humoral immune responses to JEV and PPV,which proved that the E^(MP)-VP2 protein had immunogenicity,laid the foundation for development of a binary subunit vaccine for prevention and control of JEV and PPV.

关 键 词：日本脑炎病毒 猪细小病毒 杆状病毒表达系统 免疫反应性 免疫原性 

分 类 号：S859.797[农业科学—临床兽医学]