CN114891121A - 一种抗pedv和prv的二联病毒样颗粒疫苗及其制备方法 - Google Patents

一种抗pedv和prv的二联病毒样颗粒疫苗及其制备方法 Download PDF

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CN114891121A
CN114891121A CN202210513835.1A CN202210513835A CN114891121A CN 114891121 A CN114891121 A CN 114891121A CN 202210513835 A CN202210513835 A CN 202210513835A CN 114891121 A CN114891121 A CN 114891121A
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贺笋
闫鹏先
王清华
肖升东
杜久斌
饶婷婷
潘毅平
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Tiankang Pharmaceutical Suzhou Co ltd
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Abstract

本发明涉及生物医药领域，具体涉及一种抗PEDV和PRV的二联病毒样颗粒疫苗及其制备方法。本发明的病毒样颗粒含有猪轮状病毒的VP6蛋白和VP2蛋白，且所述VP6蛋白的loop区连接有猪流行性腹泻病毒的RBD。本发明所提供的二联病毒样颗粒及疫苗对猪流行性腹泻病毒和猪轮状病毒均具有很好的免疫原性，能诱导机体产生强效免疫应答，并形成较强的免疫保护。其不仅有利于在相关疾病的防控中节省劳力，提高工作效率，而且还解决了之前弱毒疫苗和灭活疫苗所具有的制备工艺复杂、有效性不佳、容易散毒等问题。

Description

一种抗PEDV和PRV的二联病毒样颗粒疫苗及其制备方法
技术领域
本发明涉及生物医药领域，具体涉及一种抗PEDV和PRV的二联病毒样颗粒疫苗及其制备方法。
背景技术
目前市场上用于预防、控制猪流行性腹泻的疫苗基本都是传统猪流行性腹泻病毒(porcine epidemic diarrhea virus，PEDV)灭活疫苗和弱毒疫苗。但单价或多联灭活疫苗不能刺激动物机体产生足够量的sIgA抗体，保护效果不佳，且制苗成本也比较昂贵。同时，灭活疫苗在猪体内产生免疫力时间长达二周，需要提前接种，以保证预防效果。弱毒疫苗虽然可引起黏膜免疫，但由于存在着成本高、易返祖、有潜在毒力增强风险等缺陷，限制了其在生产中的应用。
发明内容
本发明利用猪轮状病毒(porcinerotavims，PRV)的病毒样颗粒(virus-likeparticle，VLP)作为核心VLP骨架，挑选出骨架表面不影响VLP结构的区域进行改造，并利用基因工程的手段将猪流行性腹泻病毒(porcine epidemic diarrhea virus，PEDV)的受体结合结构域通过蛋白质超级胶水的技术偶联到VLP表面待改造区，研制抗PEDV和PRV的二价VLP疫苗，能诱导机体产生强效的免疫应答，刺激机体产生足够量的抗猪流行性腹泻病毒和猪轮状病毒的保护性抗体。
具体而言，本发明首先提供了一种病毒样颗粒，其含有猪轮状病毒的VP6蛋白，且所述VP6蛋白的loop区连接有猪流行性腹泻病毒的RBD。
作为优选，所述VP6蛋白的loop区为loop1、loop3或loop4；
其中，所述loop1为VP6蛋白的第169-176位，所述loop3为VP6蛋白的第240-245位，所述loop4为VP6蛋白的第296-301位。
更优选为loop4时，VP6蛋白嵌合spytag后的表达量和可溶性更佳。
作为优选，所述VP6蛋白的氨基酸序列如SEQ ID NO.6所示。
作为本发明的一个实施方式，所述loop1的氨基酸序列为SQPAHDNL，所述loop3的氨基酸序列为SAGGTT，所述loop4的氨基酸序列为RPPNMT。
作为本发明的一个实施方式，所述猪流行性腹泻病毒的RBD的氨基酸序列如SEQID No.3所示。
作为本发明的一个实施方式，所述的病毒样颗粒还含有猪轮状病毒的VP2蛋白。
作为本发明的一个实施方式，所述VP2蛋白的氨基酸序列如SEQ ID NO.9所示。
作为本发明的一个实施方式，所述的病毒样颗粒含有VP6-spytag蛋白、VP2蛋白和spycatcher-RBD蛋白。
其中，所述VP6-spytag蛋白通过在猪轮状病毒的VP6蛋白的loop区嵌合spytag而得到，所述spycatcher-RBD蛋白通过将所述猪流行性腹泻病毒的RBD连接至spycatcher上而得到。
在本发明的一种优选实施方式中，所述spycatcher的氨基酸序列如SEQ ID NO.1所示。
在本发明的一种优选实施方式中，所述spytag的氨基酸序列如SEQ ID NO.2所示。
作为优选方案，所述VP6-spytag蛋白的氨基酸序列如SEQ ID NO.7所示。
作为优选方案，所述spycatcher-RBD蛋白的氨基酸序列如SEQ ID NO.4所示。
本领域人员可对上述方案进行组合，得到有关本发明病毒样颗粒的较优实施例。
作为本发明的一种实施方式，所述病毒样颗粒含有VP6-spytag蛋白、VP2蛋白和spycatcher-RBD蛋白；
其中，所述VP6-spytag蛋白的氨基酸序列如SEQ ID NO.7所示，所述spycatcher-RBD蛋白的氨基酸序列如SEQ ID NO.4所示。
进一步的，本发明还提供一种核酸，其编码所述的病毒样颗粒中所含有的蛋白。
作为本发明的一个实施方式，编码所述VP6-spytag蛋白的核苷酸序列如SEQ IDNO.8所示。
作为本发明的一个实施方式，编码所述spycatcher-RBD蛋白的核苷酸序列如SEQID NO.5所示。
本发明进一步提供一种生物材料，其为表达盒、载体、宿主细胞或重组菌，其包含所述的核酸。
本发明进一步提供一种药物组合物，其包含：(a)所述的病毒样颗粒和/或所述的核酸和/或所述的生物材料；以及(b)药学上可接受的载体或赋形剂。
本发明进一步提供一种疫苗组合物，其包含：(a)所述的病毒样颗粒和/或所述的核酸和/或所述的生物材料；以及(b)药学上可接受的载体或赋形剂。
在具体实施时，所述的疫苗组合物中还包含佐剂。
本发明进一步提供所述的病毒样颗粒的制备方法，其步骤包括：
将所述猪轮状病毒的VP6蛋白的loop区与猪流行性腹泻病毒的RBD连接。
作为优选，所述制备方法的步骤包括：
将所述VP6-spytag蛋白与所述VP2蛋白组装成双层VLP，而后与所述spycatcher-RBD蛋白偶联。
作为一种优选的实施方案，将spycatcher-RBD和所述双层VLP蛋白按照1：(1～3)的摩尔比混匀后于进行孵育偶联；更优选其摩尔比为1：2。
作为本发明的一个实施方式，其步骤还包括：在宿主中分别表达VP6蛋白、VP2蛋白、RBD，并回收表达产物。
在具体实施时，所述的宿主包括大肠杆菌、酵母、昆虫细胞、植物或哺乳动物细胞。
优选地，所述的VP2和VP6-spytag蛋白的表达宿主为毕赤酵母。
优选地，所述的spycatcher-RBD蛋白的表达宿主为ExpiCHO-S细胞或CHO-S细胞。
上述宿主在本发明中均能进行高效稳定持续的蛋白表达。
本发明还提供所述的病毒样颗粒和/或所述的核酸和/或所述的生物材料在治疗或预防以下至少一种疾病中的应用：
(1)由猪流行性腹泻病毒感染所引起的疾病；
(2)由猪轮状病毒感染所引起的疾病。
本发明还提供所述的病毒样颗粒和/或所述的核酸和/或所述的生物材料在制备药物中的应用，所述药物被用于以下至少一方面：
(1)治疗或预防由猪流行性腹泻病毒感染所引起的疾病；
(2)治疗或预防由猪轮状病毒感染所引起的疾病。
本发明的有益效果至少在于：
本发明所提供的二联病毒样颗粒及疫苗同时对猪流行性腹泻病毒和猪轮状病毒均具有很好的免疫原性，能诱导机体产生强效免疫应答，并形成较强的免疫保护，免疫生效时间短。不仅在相关疾病的防控中省时省力，提高工作效率，而且还解决了弱毒疫苗制备工艺复杂、生产成本高、有散毒风险的问题，也克服了灭活疫苗免疫生效时间长、免疫原性差等缺陷，具有成本低、生产简单、存储方便，且安全性好、保护效果好等优势。
附图说明
图1为本发明实施例1中嵌合VP6-spytag蛋白与VP2蛋白共转后SDS-PAGE图。
图2为本发明实施例1中spycatcher-RBD融合蛋白SDS-PAGE图。
图3为本发明实施例1中spycatcher-RBD融合蛋白与嵌合了spytag的双层VLP偶联后蛋白SDS-PAGE图。
图4为本发明实施例1中最终的偶联RBD的轮状病毒双层VLP示意图。
图5为本发明实施例3中轮状病毒VP6蛋白表面不同loop区嵌合spytag后表达结果；其中，泳道3～6为本发明实验结果，具体包括：3、RV-L1-ST裂解上清；4、RV-L1-ST裂解沉淀；5、RV-L3-ST裂解上清；6、RV-L3-ST裂解沉淀；各泳道均为10μL样品的结果。
图6为本发明实施例3中轮状病毒VP6蛋白表面不同loop区嵌合spytag后表达结果；其中，泳道7～10为本发明实验结果，具体包括：7、RV-L1-ST上清；8、RV-L1-ST沉淀；9、RV-L4-ST上清；10、RV-L4-ST沉淀；各泳道均为10μL样品的结果。
图7为不同RBD蛋白的表达和纯化结果；其中，1、A分子培养基上清；2、A分子Ni柱穿透；3、A分子Ni柱样品-reduced；4、A分子Ni柱样品-Nonreduced；5、B分子培养基上清；6、B分子Ni柱穿透；7、B分子Ni柱样品-reduced；8、B分子Ni柱样品-Nonreduced。
图8为不同RBD蛋白纯化后的HPLC图；其中，A图为A分子的HPLC图；B图为B分子的HPLC图。
具体实施方式
以下实施例用于说明本发明，但不用来限制本发明的范围。
实施例中未注明具体技术或条件者，按照本领域内的文献所描述的技术或条件，或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可通过正规渠道商购买得到的常规产品。
实施例1
本实施例提供一种病毒样颗粒，其制备过程如下：
(1)将合成的VP6-spytag融合蛋白基因序列(spytag连接于VP6蛋白的loop4区，融合蛋白的氨基酸序列如SEQ ID NO.7所示，对应的核苷酸序列如SEQ ID NO.8所示)与VP2基因序列(氨基酸序列如SEQ ID NO.9所示)分别构建入载体，然后线性化后共同电转毕赤酵母感受态细胞，对此二蛋白进行表达(图1)；
(2)在细胞裂解后，对VP2及VP6-spytag蛋白进行纯化、颗粒自组装和定量，并利用电镜鉴定组装效果；
(3)将合成的spycatcher-RBD融合蛋白的基因序列(氨基选序列如SEQ ID NO.4所示，对应的核苷酸序列如SEQ ID NO.5所示)构建入载体，然后转入ExpiCHO细胞进行瞬转表达；
(4)对表达出的spycatcher-RBD蛋白进行纯化和定量(图2)；
(5)将spycatcher-RBD和VP2-VP6-spytag蛋白分别按照1：1、1：2、1：3的摩尔比混匀后于25℃下在缓冲液(300mmNaCl，50mmtris，pH8.0)中孵育偶联8h以上(图3)；
(6)将步骤(5)中偶联好的的病毒样颗粒再次进行纯化，去除掉未结合的蛋白，检测偶联效果和效率，结果表明spycatcher-RBD和VP2-VP6-spytag蛋白摩尔比为1:2时，偶联效率最高，进一步通过电镜鉴定发现原有VLP结构完整，未被破坏(图4)。
实施例2
将实施例1中最优方案制备得到的病毒样颗粒分别按照0.5ug/头份、5ug/头份，10ug/头份和30ug/头份的剂量与佐剂混合后得到PRV-PEDV疫苗，进行疫苗效力检测，具体方法及结果如下：
1、抗体检测
使用实施例1中的疫苗免疫50头仔猪，一次免疫前、二次免后14日、21日和28日采血，分离血清，按PEDV IgG抗体检测试剂盒说明书进行PEDV ELISA抗体检测，使用猪轮状病毒抗体检测试剂盒进行猪轮状病毒ELISA抗体检测，计算抗体S/P几何平均值，检测结果见表1～2。
表1实验仔猪PEDV抗体检测情况
Figure BDA0003638827260000071
备注：S/P≥0.5为抗体阳性。
表2实验仔猪轮状病毒抗体检测情况
Figure BDA0003638827260000072
在表2中，仔猪二免后14日，5ug/头剂量组所有仔猪抗体水平均转阳(S/P值>0.3)。10ug/头剂量组和30ug/头剂量组所有仔猪，二免后14日抗体水平持续升高，明显优于其他低剂量组。
由上述结果可知，随着疫苗中抗原含量的增加，对应抗体效价相应的升高，说明抗原免疫仔猪后能够产生良好的免疫原性。
2、免后观察
仔猪免疫后体重与对照组仔猪体重增长无明显差异，也无观察到发热、厌食等现象，表明本发明的疫苗安全，具体结果见表3。
表3疫苗免疫后安全性试验临床观察情况
Figure BDA0003638827260000073
Figure BDA0003638827260000081
实施例3
针对VP6蛋白的不同loop区的选择进行大量实验，其中部分结果如下：
按照实施例1中的步骤将RBD序列分别连接于VP6蛋白的loop1(SEQ ID NO.11：SQPAHDNL)、loop3(SEQ ID NO.12：SAGGTT)和loop4(SEQ ID NO.13：RPPNMT)区，以合成RV-L1-ST、RV-L3-ST和RV-L4-ST融合蛋白的基因序列，而后将其分别构建入载体，而后转入毕赤酵母感受态细胞，对其蛋白进行表达。在细胞裂解后，通过SDS-PAGE对细胞裂解后的上清和沉淀进行检测，结果见图5～图6。
由结果可知，从表达量结果来看，RV-L3-ST≈RV-L4-ST>RV-L1-ST，从可溶性结果来看，RV-L4-ST>RV-L1-ST>RV-L3-ST。可见，将spytag-RBD连接于VP6蛋白的loop3、loop4区均能获得较高表达量，其中将spytag-RBD连接于VP6蛋白的loop4区域能够获得可溶性好、更利于后续纯化收集的蛋白。
实施例4
针对PEDV S的RBD序列，发明人经过大量涉及和验证进行确定，以下列举A序列(本发明的RBD序列，具体氨基酸序列如SEQ ID No.3所示)和B序列(氨基酸序列如SEQ IDNo.10所示)进行表达、纯化结果验证，其中，A序列和B序列的具体信息见表4。
表4
Figure BDA0003638827260000082
将合成的RBD蛋白的基因序列构建入载体，然后转入ExpiCHO细胞进行瞬转表达，并对表达出的RBD蛋白进行纯化和定量。其间，分别收集A分子和B分子的培养基上清样品、纯化Ni柱穿透样品、Ni柱样品以及Ni柱样品Non-reduced，并进行SDS-PAGE检测，结果见图7。同时，通过HPLC对纯化得到的A分子和B分子进行检测，结果见图8。图中结果表明本发明中的A序列在表达量、纯化过程、纯化结果上都要优于B序列。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之作一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
序列表
<110> 天康制药（苏州）有限公司
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atggaggtat tgtattcctt gtctaaaaca ttgaaggacg cacgtgacaa aatagtggaa 60
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tttccacaag cacctccctt catattccat gccacggtgg gactaaccct acgtacggag 1020
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gttgcatcca ttcgttccat gcttattaaa 1230
<210> 9
<211> 890
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Met Ala Tyr Arg Lys Arg Gly Ala Lys Arg Glu Asn Leu Pro Gln Gln
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Leu Lys Lys Leu Glu Asp Ile Lys Pro Glu Gln Ala Lys Lys Gln Thr
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Asp Met Ala Val Glu Asn Lys Asn Ser Arg Asp Ala Gly Lys Val Val
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Asp Ser Glu Thr Ala Ser Ile Cys Asp Ala Ile Phe Gln Asp Glu Glu
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Thr Glu Gly Val Ile Arg Arg Phe Ile Ala Asp Met Arg Gln Gln Val
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Gln Ala Asp Arg Asn Val Val Asn Tyr Pro Ser Ile Leu His Pro Ile
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Asp His Ala Phe Asn Glu Cys Phe Leu Asn His Gln Leu Val Glu Pro
260 265 270
Leu Asn Asn Glu Ile Ile Phe Asn Tyr Ile Pro Glu Arg Ile Arg Asn
275 280 285
Asp Val Asn Tyr Ile Leu Asn Met Asp Met Asn Leu Pro Ser Thr Ala
290 295 300
Arg Tyr Ile Arg Pro Asn Leu Leu Gln Asp Arg Leu Ser Leu His Asp
305 310 315 320
Asn Phe Glu Ser Leu Trp Asp Thr Ile Thr Thr Ser Asn Tyr Ile Leu
325 330 335
Ala Arg Ser Val Val Pro Asp Leu Lys Glu Lys Glu Leu Val Ser Thr
340 345 350
Glu Ala Gln Ile Gln Lys Met Ser Gln Asp Leu Gln Leu Glu Ala Leu
355 360 365
Thr Ile Gln Ser Glu Thr Gln Phe Leu Ala Gly Ile Asn Ser Gln Ala
370 375 380
Ala Asn Asp Cys Phe Lys Thr Leu Ile Ala Ala Met Leu Ser Gln Arg
385 390 395 400
Thr Met Ser Met Glu Phe Val Thr Thr Asn Tyr Met Ser Leu Ile Ser
405 410 415
Gly Met Trp Leu Leu Thr Val Ile Pro Asn Asp Met Phe Leu Arg Glu
420 425 430
Ser Leu Val Ala Cys Glu Leu Ala Ile Ile Asn Thr Ile Val Tyr Pro
435 440 445
Ala Phe Gly Met Gln Arg Met His Tyr Arg Asn Gly Asp Pro Gln Thr
450 455 460
Pro Phe Gln Ile Ala Glu Gln Gln Ile Gln Asn Phe Gln Val Ala Asn
465 470 475 480
Trp Leu His Phe Ile Asn Asn Asn Arg Phe Arg Gln Val Val Ile Asp
485 490 495
Gly Val Leu Asn Gln Thr Leu Asn Asp Asn Ile Arg Asn Gly Gln Val
500 505 510
Ile Asn Gln Leu Met Glu Ala Leu Met Gln Leu Ser Arg Gln Gln Phe
515 520 525
Pro Thr Met Pro Val Asp Tyr Lys Arg Ser Ile Gln Arg Gly Ile Leu
530 535 540
Leu Leu Ser Asn Arg Leu Gly Gln Leu Val Asp Leu Thr Arg Leu Leu
545 550 555 560
Ser Tyr Asn Tyr Glu Thr Leu Met Ala Cys Ile Thr Met Asn Met Gln
565 570 575
His Val Gln Thr Leu Thr Thr Glu Lys Leu Gln Leu Thr Ser Val Thr
580 585 590
Ser Leu Cys Met Leu Ile Gly Asn Thr Thr Val Ile Pro Ser Pro Gln
595 600 605
Thr Leu Phe His Tyr Tyr Asn Val Asn Val Asn Phe His Ser Asn Tyr
610 615 620
Asn Glu Arg Ile Asn Asp Ala Val Ala Ile Ile Thr Ala Ala Asn Arg
625 630 635 640
Leu Asn Leu Tyr Gln Lys Lys Met Lys Ser Ile Val Glu Glu Phe Leu
645 650 655
Lys Arg Leu Gln Ile Phe Asp Val Pro Arg Val Pro Asp Asp Gln Met
660 665 670
Tyr Arg Leu Arg Asp Arg Leu Arg Leu Leu Pro Val Glu Arg Arg Arg
675 680 685
Leu Asp Ile Phe Asn Leu Ile Leu Met Asn Met Glu Gln Ile Glu Arg
690 695 700
Ala Ser Asp Lys Ile Ala Gln Gly Val Ile Ile Ala Tyr Arg Asp Met
705 710 715 720
Gln Leu Glu Arg Asp Glu Met Tyr Gly Tyr Val Asn Ile Ala Arg Asn
725 730 735
Leu Asp Gly Tyr Gln Gln Ile Asn Leu Glu Glu Leu Met Arg Thr Gly
740 745 750
Asp Tyr Gly Gln Ile Thr Asn Met Leu Leu Asn Asn Gln Pro Val Ala
755 760 765
Leu Val Gly Ala Leu Pro Phe Val Thr Asp Ser Ser Val Ile Ser Leu
770 775 780
Ile Ala Lys Leu Asp Ala Thr Val Phe Ala Gln Ile Val Lys Leu Arg
785 790 795 800
Lys Val Asp Thr Leu Lys Pro Ile Leu Tyr Lys Ile Asn Ser Asp Ser
805 810 815
Asn Asp Phe Tyr Leu Val Ala Asn Tyr Asp Trp Ile Pro Thr Ser Thr
820 825 830
Thr Lys Val Tyr Lys Gln Val Pro Gln Pro Phe Asp Phe Arg Ala Ser
835 840 845
Met His Met Leu Thr Ser Asn Leu Thr Phe Thr Val Tyr Ser Asp Leu
850 855 860
Leu Ala Phe Val Ser Ala Asp Thr Val Glu Pro Ile Asn Ala Val Ala
865 870 875 880
Phe Asp Asn Met Arg Ile Met Asn Glu Leu
885 890
<210> 10
<211> 311
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 10
Phe Val Thr Leu Pro Ser Phe Asn Asp His Ser Phe Val Asn Ile Thr
1 5 10 15
Val Ser Ala Ser Phe Gly Gly His Ser Gly Ala Asn Leu Ile Ala Ser
20 25 30
Asp Thr Thr Ile Asn Gly Phe Ser Ser Phe Cys Val Asp Thr Arg Gln
35 40 45
Phe Thr Ile Ser Leu Phe Tyr Asn Val Thr Asn Ser Tyr Gly Tyr Val
50 55 60
Ser Asn Ser Gln Asp Ser Asn Cys Pro Phe Thr Leu Gln Ser Val Asn
65 70 75 80
Asp Tyr Leu Ser Phe Ser Lys Phe Cys Val Ser Thr Ser Leu Leu Ala
85 90 95
Ser Ala Cys Thr Ile Asp Leu Phe Gly Tyr Pro Glu Phe Gly Ser Gly
100 105 110
Val Lys Phe Ala Ser Leu Tyr Phe Gln Phe Thr Lys Gly Glu Leu Ile
115 120 125
Thr Gly Thr Pro Lys Pro Leu Glu Gly Val Thr Asp Val Ser Phe Met
130 135 140
Thr Leu Asp Val Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
145 150 155 160
Gly Gly Ser Phe Val Thr Leu Pro Ser Phe Asn Asp His Ser Phe Val
165 170 175
Asn Ile Thr Val Ser Ala Ser Phe Gly Gly His Ser Gly Ala Asn Leu
180 185 190
Ile Ala Ser Asp Thr Thr Ile Asn Gly Phe Ser Ser Phe Cys Val Asp
195 200 205
Thr Arg Gln Phe Thr Ile Ser Leu Phe Tyr Asn Val Thr Asn Ser Tyr
210 215 220
Gly Tyr Val Ser Asn Ser Gln Asp Ser Asn Cys Pro Phe Thr Leu Gln
225 230 235 240
Ser Val Asn Asp Tyr Leu Ser Phe Ser Lys Phe Cys Val Ser Thr Ser
245 250 255
Leu Leu Ala Ser Ala Cys Thr Ile Asp Leu Phe Gly Tyr Pro Glu Phe
260 265 270
Gly Ser Gly Val Lys Phe Ala Ser Leu Tyr Phe Gln Phe Thr Lys Gly
275 280 285
Glu Leu Ile Thr Gly Thr Pro Lys Pro Leu Glu Gly Val Thr Asp Val
290 295 300
Ser Phe Met Thr Leu Asp Val
305 310
<210> 11
<211> 8
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 11
Ser Gln Pro Ala His Asp Asn Leu
1 5
<210> 12
<211> 6
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 12
Ser Ala Gly Gly Thr Thr
1 5
<210> 13
<211> 6
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 13
Arg Pro Pro Asn Met Thr
1 5

Claims (10)

1.一种病毒样颗粒，其特征在于，其含有猪轮状病毒的VP6蛋白，且所述VP6蛋白的loop区连接有猪流行性腹泻病毒的RBD。
2.根据权利要求1所述的病毒样颗粒，其特征在于，所述VP6蛋白的loop区为loop1、loop3或loop4；更优选为loop4；
其中，所述loop1为VP6蛋白的第169-176位，所述loop3为VP6蛋白的第240-245位，所述loop4为VP6蛋白的第296-301位。
3.根据权利要求1或2所述的病毒样颗粒，其特征在于，还含有猪轮状病毒的VP2蛋白；
优选地，所述VP6蛋白的氨基酸序列如SEQ ID NO.6所示；
优选地，所述VP2蛋白的氨基酸序列如SEQ ID NO.9所示；
优选地，所述猪流行性腹泻病毒的RBD的氨基酸序列如SEQ ID No.3所示。
4.根据权利要求1～3中任一项所述的病毒样颗粒，其特征在于，其含有VP6-spytag蛋白、VP2蛋白和spycatcher-RBD蛋白；
优选地，所述VP6-spytag蛋白的氨基酸序列如SEQ ID NO.7所示；
优选地，所述spycatcher-RBD蛋白的氨基酸序列如SEQ ID NO.4所示。
5.一种核酸，其特征在于，其编码权利要求1～4中任一项所述的病毒样颗粒中所含有的蛋白；
优选地，编码所述VP6-spytag蛋白的核苷酸序列如SEQ ID NO.8所示；
优选地，编码所述spycatcher-RBD蛋白的核苷酸序列如SEQ ID NO.5所示。
6.一种生物材料，其为表达盒、载体、宿主细胞或重组菌，其特征在于，其包含权利要求5所述的核酸。
7.一种药物或疫苗组合物，其特征在于，其包含：
(a)权利要求1～4中任一项所述的病毒样颗粒和/或权利要求5中所述的核酸和/或权利要求6所述的生物材料；以及
(b)药学上可接受的载体或赋形剂。
8.权利要求1～4中任一项所述的病毒样颗粒的制备方法，其特征在于，其步骤包括：
将所述猪轮状病毒的VP6蛋白的loop区与猪流行性腹泻病毒的RBD连接。
9.根据权利要求8中所述的病毒样颗粒的制备方法，其特征在于，其步骤具体包括：
将所述VP6-spytag蛋白与所述VP2蛋白组装成双层VLP，再与所述spycatcher-RBD蛋白偶联；
优选地，其步骤还包括：在宿主中分别表达VP6、VP2、RBD序列并回收表达产物。
10.权利要求1～4中任一项所述的病毒样颗粒和/或权利要求5中所述的核酸和/或权利要求6所述的生物材料在制备药物中的应用，所述药物被用于以下至少一方面：
(1)治疗或预防由猪流行性腹泻病毒感染所引起的疾病；
(2)治疗或预防由猪轮状病毒感染所引起的疾病。
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Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2014218358A1 (en) * 2008-10-24 2014-09-18 Boehringer Ingelheim Animal Health USA Inc. Vaccine against african horse sickness virus
CN104459156A (zh) * 2014-12-01 2015-03-25 周丽华 一种诊断猪病毒性腹泻的蛋白芯片
CN106061503A (zh) * 2014-02-19 2016-10-26 英特维特国际股份有限公司 液体稳定的猪病毒疫苗
CN106573988A (zh) * 2014-08-07 2017-04-19 基亚生物科技股份有限公司 病毒样颗粒疫苗
US20180021258A1 (en) * 2014-12-31 2018-01-25 The Usa, As Represented By The Secretary Department Of Health And Human Services Novel multivalent nanoparticle-based vaccines
CN109182596A (zh) * 2018-08-06 2019-01-11 南阳师范学院 一种猪流行性腹泻病毒的反转录聚合酶螺旋反应等温检测试剂及检测方法
CN109535233A (zh) * 2018-12-05 2019-03-29 诺华生物科技（武汉）有限责任公司 猪瘟病毒嵌合型病毒样颗粒、制备方法及其应用和疫苗
CN110093357A (zh) * 2019-04-17 2019-08-06 仲恺农业工程学院 猪流行性腹泻病毒多表位抗原、编码基因、制备方法及应用
CN111217916A (zh) * 2020-01-15 2020-06-02 扬州大学 猪三种致腹泻病毒中和抗原表位融合蛋白及其构建方法和应用
CN111253494A (zh) * 2020-03-10 2020-06-09 天康生物(上海)有限公司 猪口蹄疫病毒和猪轮状病毒的融合蛋白、类病毒颗粒和疫苗及制备方法
CN111518222A (zh) * 2020-07-06 2020-08-11 天康生物(上海)有限公司 牛轮状病毒融合蛋白和犊牛腹泻多联疫苗
CN114163505A (zh) * 2021-11-17 2022-03-11 天康制药(苏州)有限公司 猪瘟、猪伪狂犬病毒二联亚单位疫苗及其制备方法

Patent Citations (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2014218358A1 (en) * 2008-10-24 2014-09-18 Boehringer Ingelheim Animal Health USA Inc. Vaccine against african horse sickness virus
CN106061503A (zh) * 2014-02-19 2016-10-26 英特维特国际股份有限公司 液体稳定的猪病毒疫苗
CN106573988A (zh) * 2014-08-07 2017-04-19 基亚生物科技股份有限公司 病毒样颗粒疫苗
CN104459156A (zh) * 2014-12-01 2015-03-25 周丽华 一种诊断猪病毒性腹泻的蛋白芯片
US20180021258A1 (en) * 2014-12-31 2018-01-25 The Usa, As Represented By The Secretary Department Of Health And Human Services Novel multivalent nanoparticle-based vaccines
CN109182596A (zh) * 2018-08-06 2019-01-11 南阳师范学院 一种猪流行性腹泻病毒的反转录聚合酶螺旋反应等温检测试剂及检测方法
CN109535233A (zh) * 2018-12-05 2019-03-29 诺华生物科技（武汉）有限责任公司 猪瘟病毒嵌合型病毒样颗粒、制备方法及其应用和疫苗
CN110093357A (zh) * 2019-04-17 2019-08-06 仲恺农业工程学院 猪流行性腹泻病毒多表位抗原、编码基因、制备方法及应用
CN111217916A (zh) * 2020-01-15 2020-06-02 扬州大学 猪三种致腹泻病毒中和抗原表位融合蛋白及其构建方法和应用
CN111253494A (zh) * 2020-03-10 2020-06-09 天康生物(上海)有限公司 猪口蹄疫病毒和猪轮状病毒的融合蛋白、类病毒颗粒和疫苗及制备方法
CN111518222A (zh) * 2020-07-06 2020-08-11 天康生物(上海)有限公司 牛轮状病毒融合蛋白和犊牛腹泻多联疫苗
CN114163505A (zh) * 2021-11-17 2022-03-11 天康制药(苏州)有限公司 猪瘟、猪伪狂犬病毒二联亚单位疫苗及其制备方法

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
M MALM 等: "Rotavirus capsid VP6 tubular and spherical nanostructures act as local adjuvants when co-delivered with norovirus VLPs", 《CLINICAL AND EXPERIMENTAL IMMUNOLOGY》, vol. 189, no. 3, pages 331 - 341, XP071086509, DOI: 10.1111/cei.12977 *
王萍: "嵌合型轮状病毒与肠道病毒71型病毒样颗粒在杆状病毒表达系统中的表达与组装", 《中国优秀硕士学位论文全文数据库基础科学辑》, pages 006 - 326 *
贺笋等: "猪流行性腹泻基因工程疫苗研究进展", 《畜牧兽医科技信息》, no. 8, pages 10 - 12 *

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