CN113527516B - 一种a型塞内卡病毒基因工程复合表位蛋白、疫苗及其应用 - Google Patents
一种a型塞内卡病毒基因工程复合表位蛋白、疫苗及其应用 Download PDFInfo
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Abstract
本发明提供了一种A型塞内卡病毒基因工程复合表位蛋白、疫苗及其应用，属于兽医生物制品技术领域。本发明提供了A型塞内卡病毒基因工程复合表位蛋白的氨基酸序列如SEQ ID NO：14所示。经体外重组表达得到重组复合表位蛋白后，与佐剂混合制备的疫苗免疫健康易感仔猪，28天时可检测到特异性抗体和中和抗体的产生，且本申请保护的复合表位蛋白rP2攻毒保护率为80％以上，与对照复合表位蛋白相比具有较高的攻毒保护作用。制备的新型A型塞内卡病毒基因工程复合表位蛋白疫苗，在防控A型塞内卡病毒方面具有广阔的应用前景。
Description
技术领域
本发明属于兽医生物制品技术领域，具体涉及一种A型塞内卡病毒基因工程复合表位蛋白、疫苗及其应用。
背景技术
塞内卡病毒病是由A型塞内卡病毒(Seneca virus A，SVA)引起的一种主要感染猪的病毒性传染病。临床症状与口蹄疫、猪水泡病和水泡性口炎极为相似，以猪鼻吻部、口腔粘膜和蹄冠等部位发生水疱、溃烂甚至蹄壳脱落为特征，偶见腹泻症状，病情急促，新生仔猪(4日龄以内)发病以后死亡率高达30％～70％。该病对养猪业的生产和经济效益具有很大地潜在威胁，因此应重视该病的防控。2018年6月20日，农业农村部发布了《关于做好塞内卡病毒病防治工作的通知》，引起了业界对塞内卡病毒病的关注。
A型塞内卡病毒在分类上属于小RNA病毒科，塞内卡病毒属，A型塞内卡病毒是其唯一成员。SVA具有典型的小RNA病毒结构特点。结构蛋白VP1和VP2上存在着许多不规则的环状结构，这些结构与病毒吸附、入侵、保护性免疫应答和血清型特异性有关。研究显示，SVA可以诱导机体产生B细胞和T细胞免疫应答，而VP1蛋白含有多个中和结构域，是最重要的免疫原性蛋白。
疫苗接种是特异性预防和控制各类疫病的可靠和有效手段，但目前，只有少数研究评价了宿主对SVA感染的免疫应答，对于SVA疫苗的研究更是处于探索阶段，只有个别研究者如yang等进行了SVA灭活疫苗的初步研究，尚无商品化疫苗问世，也无特效的治疗方法。而表位疫苗作为一种更加安全可靠、便于规模化生产的新型疫苗，在口蹄疫、疟疾和肝炎等疫病的防治过程中有着广泛而成功的应用。目前关于SVA表位疫苗的研究尚未见文献报道，研究和开发安全高效的SVA表位疫苗具有重要的科学和应用价值。
发明内容
有鉴于此，本发明的目的在于提供一种A型塞内卡病毒基因工程复合表位蛋白、疫苗及其应用，所述复合表位蛋白能够产生良好的免疫保护效果，为制备一种安全、高效的塞内卡病毒病新型基因工程疫苗提供了基础。
本发明提供了一种A型塞内卡病毒基因工程复合表位蛋白，包括B细胞表位和T细胞表位；
所述B细胞表位包括A型塞内卡病毒的VP1蛋白、VP2蛋白和VP3蛋白；所述VP1蛋白包括氨基酸序列依次如SEQ ID NO：1～SEQ ID NO：4所示的片段1～片段4；
所述VP2蛋白包括氨基酸序列依次如SEQ ID NO：5～SEQ ID NO：9所示的片段5～片段9；
所述VP3蛋白包括氨基酸序列依次如SEQ ID NO：10所示的片段10；
所述T细胞表位包括PADRE和invasin；
所述PADRE的氨基酸序列如SEQ ID NO：11所示；
所述invasin的氨基酸序列如SEQ ID NO：12所示。
优选的，复合表位蛋白为PADRE-片段5-片段6-片段7-片段8-片段9-片段10-片段1-片段2-片段3-片段4-invasin。
优选的，所述复合表位蛋白还包括连接肽；
所述B细胞表位之间用连接肽GGSSGG连接；所述B细胞表位与T细胞表位之间用连接肽GGC连接。
优选的，所述复合表位蛋白的氨基酸序列如SEQ ID NO：14所示。
本发明提供了一种编码所述A型塞内卡病毒基因工程复合表位蛋白的基因，所述基因的核苷酸序列如SEQ ID NO：15所示。
本发明提供了一种A型塞内卡病毒基因工程复合表位蛋白疫苗，包括所述A型塞内卡病毒基因工程复合表位蛋白和佐剂。
优选的，所述A型塞内卡病毒基因工程复合表位蛋白的浓度为250μg/mL。
本发明提供了所述A型塞内卡病毒基因工程复合表位蛋白疫苗的制备方法，包括以下步骤：
将所述A型塞内卡病毒基因工程复合表位蛋白用PBS缓冲液溶解，与佐剂混合，乳化，得到疫苗。
本发明提供了一种A型塞内卡病毒的中和抗体，由所述A型塞内卡病毒基因工程复合表位蛋白疫苗免疫仔猪获得。
本发明提供了所述A型塞内卡病毒基因工程复合表位蛋白或所述中和抗体在制备预防和/或控制猪A型塞内卡病毒病的药物或制备诊断A型塞内卡病毒病的试剂或试剂盒中的应用。
本发明提供的A型塞内卡病毒基因工程复合表位蛋白，包括B细胞表位和T细胞表位。本发明通过筛选A型塞内卡病毒VP1、VP2和VP3蛋白的B细胞表位，通过采用不同表位片段的B细胞表位配合一个或两个通用T细胞表位形成3种复合表位蛋白(rP1、rP2和rP3)，经体外重组表达免疫仔猪，28天时可检测到显著的特异性抗体和不同水平的中和抗体；同时3种复合表位蛋白(rP1、rP2和rP3)表现出不同的攻毒保护能力，其中本申请保护的复合表位蛋白rP2攻毒保护率为80％以上，而复合表位蛋白rP1攻毒保护率仅为40％，rP3攻毒保护率仅为60％，这表明本发明保护的复合表位蛋白rP2具有较高的攻毒保护作用，是一种新型A型塞内卡病毒基因工程疫苗，具有广阔的应用前景。
附图说明
图1为重组菌表达产物的SDS-PAGE分析；图1A：IPTG诱导后重组抗原表达情况，M：蛋白分子量，1：未诱导的重组菌对照，2：重组菌诱导5小时产物；图1B：重组抗原表达可溶性分析，M：蛋白分子量，3：重组菌表达上清，4：重组菌表达沉淀；
图2为重组菌表达蛋白纯化后SDS-PAGE分析；
图3为重组表位蛋白的Western Blotting检测；M：蛋白分子量，图3A：一抗为兔抗SVA VP1多克隆抗体(1:10000稀释)，二抗为羊抗兔IgG(1:20000稀释)；图3B：一抗为兔抗SVA VP2多克隆抗体(1:10000稀释)，二抗为羊抗兔IgG(1:20000稀释)；图3C:一抗为猪SVA阳性血清(1:2000稀释)，二抗为山羊抗猪IgG(1:10000稀释)；
图4为间接ELISA法检测免疫猪的特异性抗体水平。
具体实施方式
本发明提供了一种A型塞内卡病毒基因工程复合表位蛋白，包括B细胞表位和T细胞表位。
在本发明中，所述B细胞表位包括A型塞内卡病毒的VP1蛋白、VP2蛋白和VP3蛋白。
其中，所述VP1蛋白中片段1位于VP1上7～26aa，氨基酸序列如SEQ ID NO：1(TGVIEAGNTDTDFSGELAAP)所示。
所述VP1蛋白中片段2位于VP1上48～74aa，氨基酸序列如SEQ ID NO：2(VLEKDAVFPRPFPTATGAQQDDGYFCL)所示。
所述VP1蛋白中片段3位于VP1上92～109aa，氨基酸序列如SEQ ID NO：3(YVNPSDSGVLANTSLDFN)所示。
所述VP1蛋白中片段4位于VP1上183～213aa，氨基酸序列如SEQ ID NO：4(PWNSVSSVLPVRWGGASKLSSATRGLPAHAD)所示。
所述VP2蛋白包括片段5～片段9。
其中，所述VP2蛋白中片段5位于VP2上12～18aa，氨基酸序列如SEQ ID NO：5(DRVITQT)所示。
所述VP2蛋白中片段6位于VP2上38～57aa，氨基酸序列如SEQ ID NO：6(EDPTKSDPPSSSTDQPTTTF)所示。
所述VP2蛋白中片段7位于VP2上137～172aa，氨基酸序列如SEQ ID NO：7(PETTLDVKPDGKAKSLQELNEEQWVEMSDDYRTGKNM)所示。
所述VP2蛋白中片段8位于VP2上193～208aa，氨基酸序列如SEQ ID NO：8(FINPYQVTVFPHQILN)所示。
所述VP2蛋白中片段9位于VP2上249～284aa，氨基酸序列如SEQ ID NO：9(KEGATTDPEITFSVRPTSPYFNGLRNRFTTGTDEEQ)所示。
所述VP3蛋白为氨基酸序列依次如SEQ ID NO：10(AFGRVSEPEPASDAYVPYV)所示的片段10，位于VP3上55～73aa；
所述T细胞表位包括PADRE和invasin。所述PADRE的氨基酸序列如SEQ ID NO：11(AKFVAAWTLKAAA)所示。所述invasin的氨基酸序列如SEQ ID NO：12(TAKSKKFPSYTATYQF)所示。
在本发明中，所述复合表位蛋白优选按照SVA衣壳蛋白VP2-VP3-VP1的排列顺序连接所选B细胞表位，同时将T细胞表位的多肽连接至两端。复合表位蛋白的具体连接顺序优选为PADRE-片段5-片段6-片段7-片段8-片段9-片段10-片段1-片段2-片段3-片段4-invasin。
在本发明中，所述复合表位蛋白优选还包括连接肽。B和T细胞表位间以GGC连接，以增强表位的正确折叠和结构的稳定性。B-B细胞表位间以GGSSGG(SEQ ID NO:13)连接，确保不会干扰表位的独立性。在本发明实施例中，所述复合表位蛋白的氨基酸序列如SEQ IDNO：14所示。
本发明对A型塞内卡病毒基因工程复合表位蛋白的制备方法优选采用体外重组方法制备即可。本发明对所述体外重组方法没有特殊限制，采用本领域所熟知的体外重组方法即可。
本发明提供了一种编码所述A型塞内卡病毒基因工程复合表位蛋白的基因，所述基因的核苷酸序列如SEQ ID NO：15(gctaaatttgtagcggcatggacactaaaggctgctgctggcggctgcgatcgcgtgatcacccagaccggcggctcgtcaggaggtgaagatccgaccaagagtgacccaccgagcagcagcacggatcagccgacaaccacgtttggcggaagctcgggcggccctgagaccactctggatgtaaaaccggatggcaaggcgaagagcctgcaagaactgaacgaggaacaatgggttgaaatgagcgacgactatcgtacgggcaagaacatgggtggttcgtccggtggttttatcaacccgtaccaagtgaccgtttttccgcaccagattttaaacggtggcagctccggcggcaaagagggtgccacgactgacccggaaattacctttagtgtgcgcccaaccagcccgtactttaacggtctgcgcaatagattcaccaccggtactgacgaagagcaaggtggctcctctggcggggcgttcggtcgtgtcagcgagccggagccggcaagcgacgcgtacgttccgtatgttggtggtagctctggtggcaccggcgttatcgaggcaggtaataccgacaccgacttcagcggtgagctggccgccccgggtggtagctccggtggcgttttggaaaaagatgcggtgttcccgcgtccgtttccgaccgcgacgggtgcgcagcaggatgacggttacttctgcctgggtggctcctcgggcggctacgtgaatccgtccgacaacggcgtcctcgctaataccagcctggatttcaacgggggttcttctgggggtccatggaacagcgtcagctctgtgttgccggttcgttggggcggtgcgagcaaactgagcagcgcaacccgtggtcttccggcgcatgcagatggcgggtgtaccgcgaagtcaaagaaattcccgagctataccgcgacctatcagttc)所示。
本发明提供了一种A型塞内卡病毒基因工程复合表位蛋白疫苗，包括所述A型塞内卡病毒基因工程复合表位蛋白和佐剂。所述A型塞内卡病毒基因工程复合表位蛋白和佐剂的体积比优选为1:0.8～1.2，更优选为1：1。所述A型塞内卡病毒基因工程复合表位蛋白的浓度优选为250～300μg/mL，更优选为250μg/mL。本发明对所述佐剂的种类没有特殊限制，采用本领域所熟知的佐剂种类即可。在本发明实施例中，所述佐剂优选为Montanide ISA-201油佐剂。
本发明提供了所述A型塞内卡病毒基因工程复合表位蛋白疫苗的制备方法，包括以下步骤：
将所述A型塞内卡病毒基因工程复合表位蛋白用PBS缓冲液溶解，与佐剂混合，乳化，得到疫苗。
在本发明中，所述溶解后，A型塞内卡病毒基因工程复合表位蛋白溶液的浓度为500～600μg/mL，更优选为500μg/mL。所述A型塞内卡病毒基因工程复合表位蛋白溶液和佐剂的体积比为优选为1:1。本发明对所述乳化的方法没有特殊限制，采用本领域所熟知的乳化方法即可。
本发明还提供了一种A型塞内卡病毒的中和抗体，由所述A型塞内卡病毒基因工程复合表位蛋白疫苗免疫仔猪获得。
本发明提供了所述A型塞内卡病毒基因工程复合表位蛋白或所述中和抗体在制备预防和/或控制猪A型塞内卡病毒病的药物中的应用。
本发明对所述药物的剂型没有特殊限制，采用本领域所熟知的药物剂型即可。
本发明提供了所述A型塞内卡病毒基因工程复合表位蛋白或所述中和抗体在制备诊断A型塞内卡病毒病的试剂或试剂盒中的应用。
本发明对制备的试剂或试剂盒的种类没有特殊限制，采用本领域所熟知的基于免疫检测技术原理来检测A型塞内卡病毒病的试剂或试剂盒即可，例如酶联免疫试剂盒或胶体金检测试剂盒。
下面结合实施例对本发明提供的一种A型塞内卡病毒基因工程复合表位蛋白、疫苗及其应用进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
实施例1
1、SVA复合表位基因的设计
表位是蛋白质抗原性的基础,是抗原分子中诱生特异性免疫应答的基本结构和功能单位。利用定位精确、氨基酸序列较短的抗原表位，既能够有效地被免疫系统识别、递呈，又能诱发机体产生特异性体液和细胞免疫应答，表位疫苗作为一种基因工程疫苗，其特异性强、安全可靠、便于规模化生产，具有广阔的应用前景。
根据文献报道，SVA VP1蛋白全长264个氨基酸，包含1个BC环、1个CD环和1个GH环；SVA VP2蛋白全长284个氨基酸，包含1个EF环、1个位于VP2蛋白141-143位的关键基序LDV以及位于146-148位的关键基序DGK；SVA VP3蛋白全长239个氨基酸，包含1个knob结节。这些结构与病毒吸附、入侵、血清型特异性和保护性免疫应答有关，抗原表位大多位于这些衣壳蛋白的环结构上。具体氨基酸序列及位置见表1。
表1 SVA衣壳蛋白上环结构位置及其序列
同时对SVA VP1蛋白和VP2蛋白的B细胞表位筛选结果，VP1蛋白上筛选到3个优势B细胞表位，分别为：
7-26aa：TGVIEAGNTDTDFSGELAAP(SEQ ID NO:1)；
48-74aa：VLEKDAVFPRPFPTATGAQQDDGYFCL(SEQ ID NO:2)；
92-109aa：YVNPSDNGVLANTSLDFN(SEQ ID NO:3)；
VP2蛋白上筛选到7个B细胞表位，分别为：
1-16aa：DHNTEEMENSADRVIT(SEQ ID NO:20)；
17-32aa：QTAGNTAINTQSSLGV(SEQ ID NO:21；
38-57aa：EDPTKSDPPSSSTDQPTTTF(SEQ ID NO:6)；
137-160aa：PETTLDVKPDGKAKSLQELNEEQW(SEQ ID NO:22)；
154-172aa：ELNEEQWVEMSDDYRTGKN(SEQ ID NO:23)
193-208aa：FINPYQVTVFPHQILN(SEQ ID NO:8)；
249-264aa：EGATTDPEITFSVRP(SEQ ID NO:24)；
265-284aa：TSPYFNGLRNRFTTGTDEEQ(SEQ ID NO:25)。
其中38-57aa、154-172aa、249-264aa和265-284aa为优势B细胞表位。
根据对SVA VP1蛋白T细胞表位筛选结果，VP1蛋白上193-208aa可能存在一个T细胞表位。此外，PADRE(AKFVAAWTLKAAA，SEQ ID NO:11)和invasin(TAKSKKFPSYTATYQF，SEQID NO:12)为两个通用T细胞表位，已被广泛应用于多种表位疫苗的设计中，起到增强T细胞免疫反应的作用。
此外，根据文献报道，Hui Fan等人利用单抗筛选法共筛出位于SVA VP1和VP2蛋白上的6个B细胞表位，分别为VP1：21-26aa、VP2：12-18aa(SEQ ID NO:5)、VP2：71-76aa、VP2:98-103aa、VP2：150-156aa和VP2：:248-253aa。
本发明根据以上确定的表位信息，进行合理的组合、搭配及连接，按照SVA衣壳蛋白VP4-VP2-VP3-VP1的排列顺序连接所选B细胞表位，以尽可能的模拟SVA衣壳蛋白的原有结构；B-T细胞表位间以两个赖氨酸(Lysine-lysine,KK)或GGC连接，以增强表位的免疫原性以及维持结构的稳定性。B细胞表位间以GGSSGG连接，确保不会干扰表位的独立性。根据以上思路共设计出3组SVA复合表位基因：
rP1：PADRE-KK-VP2(38-57aa)-VP2(141-148aa)-VP2(154-172aa)-VP2(249-284aa)-VP1(7-26aa)-VP1(48-74aa)-VP1(92-109aa)；其氨基酸序列SEQ ID NO:26所示：
AKFVAAWTLKAAAKKEDPTKSDPPSSSTDQPTTTFGGSSGGLDVKPDGKGGSSGGELNEEQWVEMSDDYRTGKNGGSSGGKEGATTDPEITFSVRPTSPYFNGLRNRFTTGTDEEQGGSSGGTGVIEAGNTDTDFSGELAAPGGSSGGVLEKDAVFPRPFPTATGAQQDDGYFCLGGSSGGYVNPSDNGVLANTSLDFN；
rP2：PADRE-GGC-VP2(12-18)-VP2(38-57)-VP2(137-172)-VP2(193-208)-VP2(249-284)-VP3(55-73)-VP1(7-26)-VP1(48-74)-VP1(92-109)-VP1(183-213)-GGC-invasin；其编码氨基酸序列如SEQ ID NO：14所示：
AKFVAAWTLKAAAGGCDRVITQTGGSSGGEDPTKSDPPSSSTDQPTTTFGGSSGGPETTLDVKPDGKAKSLQELNEEQWVEMSDDYRTGKNMGGSSGGFINPYQVTVFPHQILNGGSSGGKEGATTDPEITFSVRPTSPYFNGLRNRFTTGTDEEQGGSSGGAFGRVSEPEPASDAYVPYVGGSSGGTGVIEAGNTDTDFSGELAAPGGSSGGVLEKDAVFPRPFPTATGAQQDDGYFCLGGSSGGYVNPSDNGVLANTSLDFNGGSSGGPWNSVSSVLPVRWGGASKLSSATRGLPAHADGGCTAKSKKFPSYTATYQF。
rP3：PADRE-KK-VP2(38-57aa)-VP2(141-148aa)-VP2(154-172aa)-VP2(249-284aa)-VP2(249-284aa)-VP2(249-284aa)-VP1(7-26aa)-VP1(48-74aa)-VP1(48-74aa)-VP1(48-74aa)-VP1(92-109aa)-VP1(92-109aa)-VP1(92-109aa)；其氨基酸序列SEQ ID NO:27所示：
AKFVAAWTLKAAAKKEDPTKSDPPSSSTDQPTTTFGGSSGGLDVKPDGKGGSSGGELNEEQWVEMSDDYRTGKNGGSSGGKEGATTDPEITFSVRPTSPYFNGLRNRFTTGTDEEQGGSSGGKEGATTDPEITFSVRPTSPYFNGLRNRYTTGTDEEQGGSSGGKEGATTDPEITFSVRPTSPYFNGLRNRYKTGTDEEQGGSSGGTGVIEAGNTDTDFSGELAAPGGSSGGVLEKDAVFPRPFPTATGAQQDDGYFCLGGSSGGVLEKDAVFPRPLPTATGAQQDDGYFCLGGSSGGVLEKDAVFPRPFPTATGTQQDDGYFCLGGSSGGYVNPSDSGVLANTSLDFNGGSSGGYVSPSDSGVLANTSLDFNGGSSGGYVSPSDNGVLANTSLDFN。
对不同组合表位基因进行生物信息学的验证分析，确保目的片段具有较高亲水性、抗原指数性和表面可能性。
实施例2
SVA复合表位蛋白的表达、纯化及鉴定方法
SVA复合表位蛋白的小量表达与鉴定：将设计好的3组SVA多表位基因委托南京金斯瑞生物科技有限公司进行密码子优化及合成，并将其两端分别加入NdeⅠ和XhoⅠ酶切位点，合成序列通过NdeⅠ/XhoⅠ酶切位点连至pET30(a)载体，用于复合表位蛋白的表达，重组质粒命名为pET30(a)-rP1、pET30(a)-rP2和pET30(a)-rP3。将重组质粒转化BL21(DE3)感受态细胞进行小量表达与鉴定，即：挑选含重组质粒的单菌落至5mL LB液体培养基(卡那抗性)中，37℃培养过夜后-20℃保种；再挑选含重组质粒的单菌落至5mL LB液体培养基(卡那抗性)中，37℃震荡培养至OD600约0.6；取部分菌液作为对照组，余下菌液加入IPTG诱导剂(终浓度1mM)，37℃震荡培养5h；分别取两组菌液0.15mL，12000×g离心2min，菌体沉淀以40μL 1×loading buffer重悬裂解，取10ul进行SDS-PAGE检测。结果如图1A所示，表达的rP1蛋白大小介于35～40kD之间、rP2蛋白大小介于40～55kD之间，rP3蛋白大小介于55～70kD之间，经质谱分析鉴定，表达蛋白确为目的蛋白。
SVA复合表位蛋白大量表达及破菌检测：取鉴定为阳性的过夜培养转化菌按1:1000接种到2000mL LB液体培养基中，待OD600值约0.6时，加入终浓度为1mM IPTG，37℃震荡培养5h，得诱导表达后的重组蛋白；5000rpm离心10min，收集菌体，用预冷的PBS洗两遍，每次4℃、8000rpm离心10min。最后将菌体重悬于1/20体积的PBS中，冰上超声破碎菌体，4℃、10000rpm离心10min，分离上清以及沉淀；分别取10ul上清及沉淀进行SDS-PAGE检测，剩余上清及沉淀置于4℃备用。结果如图1B所示，3组蛋白主要在上清中以可溶性蛋白形式表达。
SVA复合表位蛋白的纯化及浓度测定：将收集的表达蛋白上清与镍柱填料(Ni-NTASefinose Resin，BBI)在室温低速振摇1～2h，使二者发生完全的特异性结合；上柱，流干流穿液，用5倍柱体积的Binding Buffer洗净未结合杂蛋白。用含40mM咪唑的Elution buffer洗脱非特异性结合杂蛋白，用含500mM咪唑的Elution buffer洗脱目的蛋白，每次洗脱5个柱体积。将收集的纯化蛋白进行透析换液到PBS中，再进行超滤浓缩，用Bradford蛋白质定量试剂盒(碧云天公司)测定纯化后重组蛋白的浓度，所得纯化rP1蛋白的浓度为1.2mg/mL，rP2蛋白的浓度为1.4mg/mL，rP3蛋白的浓度为1.3mg/mL。取部分纯化后的rP1、rP2和rP3蛋白溶液进行SDS-PAGE电泳分析，结果如图2所示，均成功纯化到所需目的蛋白。
SVA复合表位蛋白的抗原性检测：用Western blotting方法检测制备的重组蛋白分别与VP1、VP2兔多克隆抗体以及猪SVA阳性血清的反应性。将纯化后的重组rP1、rP2和rP3蛋白做SDS-PAGE电泳，并转印至NC膜上，用含5％奶粉的PBST溶液室温封闭2h，加入1:10000稀释的兔抗SVA-VP1多克隆抗体或兔抗SVA-VP2多克隆抗体或猪SVA阳性血清(1:2000)，4℃孵育过夜，洗膜后加入1:20000稀释的HRP标记的山羊抗兔IgG或山羊抗猪IgG(1:10000)，室温孵育1h，洗膜后加入ECL显色液避光反应，用Laser Jet Pro MFP M227fdw成像系统扫描NC膜。结果，制备的重组rP1、rP2和rP3蛋白与兔抗SVA VP1、VP2多克隆抗体、猪SVA阳性血清均呈显著阳性反应(见图3)。
实施例3
疫苗制备方法
将上述实施例2所制备的重组rP1、rP2和rP3蛋白分别用PBS稀释至浓度为500μg/ml，与等体积油佐剂(Montanide ISA 201油佐剂，Seppic，France)混合吸入一支10ml注射器中，然后用双母鲁尔接头(Disposable Syringe Valve)连接至另一注射器，互相推注约20次，使得水相与油相充分混合乳化成双向油乳剂(W/O/W)疫苗，其中rP1、rP2和rP3蛋白终浓度均为250μg/ml。
实施例4
动物免疫方法
取来源相同、品种相同的18头2月龄大小健康易感猪(猪塞内卡病毒、口蹄疫病毒抗原阴性，猪塞内卡病毒血清中和抗体效价不高于1:4)，随机分为4组，阴性对照组3头，rP1、rP2和rP3复合表位蛋白疫苗免疫组分别为5头。颈部肌肉注射，每头猪的免疫剂量为2ml；阴性对照组注射等量的PBS。在0天免疫1次。于0d、14d、21d和28d颈静脉采血。采集的血液置于37℃放置30min，4℃放置2h，充分析出血清；4000r/min离心10min，小心吸出血清，用塞尼卡病毒间接ELISA抗体检测试剂盒(购自中国农业科学院兰州兽医研究所)检测SVA特异性抗体，S/P值≥0.4判为阳性；用细胞中和试验方法检测免疫猪血清中和抗体效价。在免疫后第28天，通过颈部肌肉注射(3.0ml，107.3TCID50)和鼻内接种(3.0ml，每鼻孔1.5ml，107.3TCID50)两种方式联合攻毒(SVA A/ZJ/2015株)，攻毒后连续观察10天，详细记录发病情况。对照猪应至少1个蹄部出现水疱，免疫猪口唇及四蹄任何一部位出现水疱，即判定为不保护。
试验结果显示，3组重组表位疫苗免疫猪均在第14天就可检测到显著的SVA特异性抗体，S/P值﹥0.4，抗体效价随接种时间缓慢升高，免疫第28天时抗体效价最高，rP3免疫组SVA特异性抗体效价显著高于其他两组。而PBS免疫猪的SVA特异性抗体一直呈阴性，S/P值<0.4(见图4)。28日后试验猪血清中和抗体检测结果表明，rP1、rP3免疫组均只有1头猪中和抗体效价达1:32以上，rP2免疫组3/5猪塞内卡病毒中和抗体效价达1:32以上，PBS对照组猪的中和抗体效价均小于1:8；攻毒后10天，rP2疫苗免疫组有4头猪抵抗了SVA攻击，没有表现出任何临床症状。而rP1免疫组只有2头猪抵抗了SVA攻击，rP3免疫组有3头猪抵抗了SVA攻击，对照组(PBS免疫组)3头猪于攻毒后10天内全部发病(结果见表2)。本发明所述rP2复合表位蛋白是一种很好的免疫原，rP2复合表位蛋白疫苗是一种新型的安全有效的塞内卡病毒疫苗。
表2猪免疫效力试验
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 中国农业科学院兰州兽医研究所
<120> 一种A型塞内卡病毒基因工程复合表位蛋白、疫苗及其应用
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ggtggctcct cgggcggcta cgtgaatccg tccgacaacg gcgtcctcgc taataccagc 780
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Claims (9)
1.一种A型塞内卡病毒基因工程复合表位蛋白，其特征在于，包括B细胞表位和T细胞表位；所述B细胞表位包括A型塞内卡病毒的VP1蛋白、VP2蛋白和VP3蛋白；所述T细胞表位包括PADRE和invasin；
所述复合表位蛋白为PADRE-片段5-片段6-片段7-片段8-片段9-片段10-片段1-片段2-片段3-片段4-invasin；
所述VP1蛋白包括氨基酸序列依次如SEQ ID NO：1~SEQ ID NO：4所示的片段1~片段4；
所述VP2蛋白包括氨基酸序列依次如SEQ ID NO：5~SEQ ID NO：9所示的片段5~片段9；
所述VP3蛋白包括氨基酸序列依次如SEQ ID NO：10所示的片段10；
所述PADRE的氨基酸序列如SEQ ID NO：11所示；
所述invasin的氨基酸序列如SEQ ID NO：12所示。
2.根据权利要求1所述A型塞内卡病毒基因工程复合表位蛋白，其特征在于，所述复合表位蛋白还包括连接肽；
所述B细胞表位之间各片段用连接肽GGSSGG连接；所述B细胞表位与T细胞表位之间用连接肽GGC连接。
3.根据权利要求2所述A型塞内卡病毒基因工程复合表位蛋白，其特征在于，所述复合表位蛋白的氨基酸序列如SEQ ID NO：14所示。
4.一种编码权利要求1~3任意一项所述A型塞内卡病毒基因工程复合表位蛋白的基因，其特征在于，所述基因的核苷酸序列如SEQ ID NO：15所示。
5.一种A型塞内卡病毒基因工程复合表位蛋白疫苗，其特征在于，包括权利要求1~3任意一项所述A型塞内卡病毒基因工程复合表位蛋白和佐剂。
6.根据权利要求5所述A型塞内卡病毒基因工程复合表位蛋白疫苗，其特征在于，所述A型塞内卡病毒基因工程复合表位蛋白的浓度为250μg/mL。
7.权利要求5或6所述A型塞内卡病毒基因工程复合表位蛋白疫苗的制备方法，其特征在于，包括以下步骤：
将所述A型塞内卡病毒基因工程复合表位蛋白用PBS缓冲液溶解，与佐剂混合，乳化，得到疫苗。
8.一种A型塞内卡病毒的中和抗体，其特征在于，由权利要求5或6所述A型塞内卡病毒基因工程复合表位蛋白疫苗免疫仔猪获得。
9.权利要求1~3任意一项所述A型塞内卡病毒基因工程复合表位蛋白或权利要求8所述中和抗体在制备预防和/或控制猪A型塞内卡病毒病的药物或制备诊断A型塞内卡病毒病的试剂或试剂盒中的应用。
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