CN114805599B - 一种基于ADDomer嵌合猪O型口蹄疫病毒抗原表位的VLPs及应用 - Google Patents
一种基于ADDomer嵌合猪O型口蹄疫病毒抗原表位的VLPs及应用 Download PDFInfo
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
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- C—CHEMISTRY; METALLURGY
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- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
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- C07K2319/00—Fusion polypeptide
- C07K2319/70—Fusion polypeptide containing domain for protein-protein interaction
- C07K2319/735—Fusion polypeptide containing domain for protein-protein interaction containing a domain for self-assembly, e.g. a viral coat protein (includes phage display)
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Abstract
本发明属于分子生物技术领域，具体涉及一种基于ADDomer嵌合猪O型口蹄疫病毒抗原表位的VLPs及应用。一种基于ADDomer嵌合猪O型口蹄疫病毒抗原表位的重组蛋白，其特征在于，所述重组蛋白是在ADDomer载体VL区、RGD1区和RGD2区中插入以下猪O型口蹄疫病毒抗原表位的任一或串联的任意组合：所述猪O型口蹄疫病毒抗原表位为T细胞表位第16～44位氨基酸、B细胞表位第129～160位氨基酸和B细胞表位第200～213位氨基酸。所述重组蛋白的氨基酸序列如SEQ ID No.3所示。利用重组蛋白组装成新型嵌合病毒样颗粒，并用于制备亚单位疫苗，免疫BALB/c雌鼠后，可诱导机体产生特异性免疫反应，能有效预防FMDV的感染，对控制和净化我国猪O型口蹄疫具有重要意义。
Description
技术领域
本发明属于分子生物技术领域，具体涉及一种基于ADDomer嵌合猪O型口蹄疫病毒抗原表位的VLPs及应用。
背景技术
口蹄疫(food and mouth disease，FMD)是由口蹄疫病毒(food and mouthdisease virus， FMDV)引起的一种烈性传染性疾病，能够导致牛、羊、猪等偶蹄动物发病死亡。FMD的临床症状主要表现为感染动物的蹄部、口腔黏膜、乳房皮肤等多处发生水泡，进而引发溃烂现象。临床症状最明显的是高产奶牛和集中饲养的猪。FMD平均致死率虽很低，感染动物发病率却为100％，且传播速度极快，引发畜产量减少，将对畜牧业造成极大的经济损失。由于FMD危害极大，影响范围极广，因此，世界动物卫生组织(Office Internationaldes Epizoofies，OIE)将其列为“A类烈性传染病”第一位。目前，我国主要以接种灭活疫苗预防FMD感染。但传统的灭活疫苗存在病毒灭活不彻底而引发疫苗毒株流行的危险。正是由于传统灭活疫苗的不安全性加速了口蹄疫新型疫苗的研发。
完整的蛋白抗原在亚单位疫苗中通常被使用，但存在结构复杂、蛋白表达效率低等问题。抗原表位是蛋白抗原中的短氨基酸序列，更容易被MHC分子识别，与整个蛋白抗原相比，能直接诱导有效的免疫。目前，我国主要流行猪O型口蹄疫，其中研究最多并效果最好的FMDV抗原表位主要位于VP1蛋白。Pan等利用FMDV VP1第21～40aa、141～160aa 和200～213aa插入PPV的VP2外环中，构建重组腺病毒来表达PPV：VLPs(FMDV)，在实验动物中发现无佐剂下可诱导高水平的FMDV特异性体液和细胞免疫。Fang等构建了3个片段，选择O型FMDVVP1结构蛋白中B细胞表位第130～140aa和第141～160aa，以及T细胞表位第16～44aa。以不同方式串联6种多肽，结果发现抗原表位的排序对体内产生中和抗体水平是有相关性的。大量研究结果表明，抗原表位对亚单位疫苗的研发具有广阔前景。本课题组前期研究表明，选用猪O型FMDV O/BY/CHA/2010毒株中VP1蛋白的2个B细胞优势抗原表位(129～160aa和200～213aa)和1个T细胞优势抗原表位(16～ 44aa)串联后表达的重组蛋白能刺激动物机体产生特异性抗体。本发明在此基础上，选用这三个优势抗原表位作为研究对象，制备嵌合型VLPs疫苗。
病毒样颗粒(Virus-like particles，VLPs)是由一种或多种不含病毒核酸的病毒蛋白组装成的空心颗粒，VLPs能模拟天然病毒诱导机体产生有效的免疫应答，这一特性为新型疫苗的研究提供了基础。近年来，法国SNRS科研团队开发了一种能自组装形成VLPs的纳米元件，并命名为ADDomer。该元件可在其VL区和RGD区嵌入并展示病原体的免疫原性表位，从而组装成VLPs。此外，该元件具备使用Multibac系统快速装配，可展示各种抗原多肽，能自发形成稳定的VLPs，具有类似病毒大小并有免疫原性且不携带遗传物质等优点，被选为代表杆状病毒表达系统的下一代疫苗。
Multibac表达系统是通过将Bac to Bac系统进一步改造，在保留原来Bacmid的Tn7 转座基础上，增加Cre-loxP重组位点，构建能表达多基因病毒载体(Multi-bacmid)，目前报道中，运用Multibac杆状病毒表达系统可高产量获得ADDomer，并易于在工业规模中大量生产。
VLPs作为疫苗的优点是能够使用树突状细胞作为主要靶细胞，从而刺激先天性免疫和后天性免疫中产生相应物质。有的VLPs与感染性病毒类似且保留了原本的受体结合区，因此就能通过正常的受体识别途径进入细胞，经MHC-Ⅰ便可递呈外源抗原。
发明内容
为了克服现有技术的不足，本发明的第一个目的在于提供一种基于ADDomer嵌合猪 O型口蹄疫病毒抗原表位的重组蛋白，所述重组蛋白是在ADDomer载体VL区、RGD1 区和RGD2区中插入以下猪O型口蹄疫病毒抗原表位的任一或串联的任意组合；所述猪O 型口蹄疫病毒抗原表位为T细胞表位第16～44位氨基酸、B细胞表位第129～160位氨基酸和B细胞表位第200～213位氨基酸。
所述T细胞表位第16～44位氨基酸序列如SEQ ID No.4所示，B细胞表位第129～160 位氨基酸序列如SEQ ID No.5所示，B细胞表位第200～213位氨基酸序列如SEQ IDNo.6 所示。
作为本发明优选地技术方案，所述重组蛋白的氨基酸序列如SEQ ID No.3所示。
上述重组蛋白是在ADDomer载体VL区、RGD1区和RGD2区中依次插入以下猪O 型口蹄疫病毒抗原表位组合得到：
(1)以“GGGGS”为连接子，串联所述的B细胞表位第129～160位氨基酸与T细胞表位第16～44位氨基酸，串联后氨基酸序列如SEQ ID No.7所示；
(2)所述的T细胞表位第16～44位氨基酸，其氨基酸序列如SEQ ID No.4所示；
(3)以“GGGGS”为连接子，串联所述的T细胞表位第16～44位氨基酸与B细胞表位第200～213位氨基酸，串联后氨基酸序列如SEQ ID No.8所示。
本发明运用ADDomer新型载体，具有热稳定性好、结构稳定等优点，可作为FMDV 亚单位候选优良疫苗的新型载体，为研究FMD防控开辟新思路。
本发明第二个目的提供了一种上述重组蛋白的编码基因，所述编码基因的核苷酸序列如SEQ ID No.2所示。
本发明的第三个目的是提供一种含有上述重组蛋白编码基因的转移载体，所述转移载体是将上述重组蛋白编码基因克隆至表达载体所得，所述表达载体为pFBDM。
本发明的第四个目的在于提供了一种含有上述重组蛋白基因的重组病毒，是将含有上述重组蛋白编码基因的转移载体转化感受态细胞，转座重组，获得重组病毒质粒，再将重组病毒质粒转染昆虫细胞，培养后获得重组病毒。
本发明的第五个目的是提供了一种基于ADDomer嵌合猪O型口蹄疫病毒抗原表位的 VLPs，由上述基于ADDomer嵌合猪O型口蹄疫病毒抗原表位的重组蛋白组装而成。
本发明还提供了上述基于ADDomer嵌合猪O型口蹄疫病毒抗原表位的VLPs的制备方法，通过以下方法制备：扩增上述重组病毒，将扩增后的重组病毒感染到昆虫细胞表达重组蛋白，收集，采用蔗糖梯度离心纯化目的蛋白，即得所述VLPs。
本发明还提供了一种抗FMDV亚单位疫苗，活性成分为上述基于ADDomer嵌合猪O型口蹄疫病毒抗原表位的VLPs。
本发明还提供了上述抗FMDV亚单位疫苗在制备用于治疗和预防FMDV感染的药物中的应用。
本发明制备的新型嵌合病毒样颗粒制备亚单位疫苗，免疫BALB/c雌鼠后，可诱导机体产生特异性免疫反应，能有效预防FMDV的感染，对控制和净化我国猪O型口蹄疫具有重要意义。
与现有技术相比，本发明的有益效果体现在：
(1)本发明根据杆状病毒密码子的偏爱性，人工设计合成了ADDomer和插入猪O型FMDV单个表位或者不同抗原表位组合的重组ADDomer核苷酸序列，其序列如SEQ ID NO.1和SEQ ID NO.2所示，该序列提高了目的基因在杆状病毒中表达水平和免疫原性。
(2)本发明通过生物信息学软件模拟重组蛋白的空间结构，预测其在体外能自组装形成VLPs，避免体外组装VLPs的失败性。通过实施例7验证了VLPs能在体外组装，证明生物信息学软件的预测的有效性。后续研究可以借鉴验证自己的实验设计能否形成VLPs。。
(3)本发明将人工合成的重组基因ADDomer和ADDomer-RBT运用Multibac表达系统表达目的蛋白，构建至转移载体的pH启动子下表达。表达出来的重组蛋白能够与阳性血清发生特异性结合，具有良好的反应原性。
(4)本发明的重组VLPs制备亚单位疫苗，免疫BALB/c雌鼠后，可诱导机体产生体液免疫和细胞免疫反应，对控制和净化我国猪O型口蹄疫和具有重要意义。
(5)自组装纳米颗粒支架ADDomer的研究具有开发前景，可作为FMDV亚单位候选优良疫苗的新型载体，为研究FMD防控开辟新思路。
附图说明
图1是重组蛋白空间结构预测：a:ADDomer空间结构预测；b:ADDomer-RBT空间结构预测(黄色、蓝色和红色分别代表B1T、T和TB2细胞抗原表位颜色)。
图2是重组转移载体构建图。
图3是重组转移质粒的构建图。
图4是重组转移质粒的鉴定(M：DL5000 DNA marker；1:ADDomer；2：ADDomer-RBT)。
图5是重组杆状病毒质粒PCR鉴定方法图示。
图6是重组杆状病毒质粒的鉴定(M：DL10000 DNA marker；1：ADDomer；2：ADDomer-RBT)。
图7是重组杆状病毒的包装(a：正常的sf9细胞；b：转染rMultibac-PFBDM-ADDomer的sf9细胞；c：转染rMultibac-PFBDM-ADDomer-RBT的sf9细胞)。
图8是标准品质粒pMD-19-T-Ac的双酶切鉴定(M：DL5000DNA marker；1：质粒 pMD-19-T-Ac BamH I/Pst I酶切产物)。
图9是重组蛋白的Western Blot检测(1：感染Ac-ADDomer的sf9细胞；2：感染 Ac-ADDomer-RBT的sf9细胞；3：阴性对照)。
图10是ADDomer二级质谱图。
图11是ADDomer-RBT二级质谱图。
图12是蔗糖梯度离心产物的SDS-PAGE结果(1：蔗糖梯度离心前的ADDomer蛋白；2：蔗糖梯度离心前的ADDomer-RBT重组蛋白；3：蔗糖梯度离心后的ADDomer蛋白；4：蔗糖梯度离心后的ADDomer-RBT重组蛋白)。
图13电镜观察到的VLPs(92000×；标尺：100nm)，a：ADDomer-VLPs；b： ADDomer-RBT-VLPs。
图14是小鼠血清中FMDV特异性抗体ELISA检测结果。
图15是小鼠脾脏中T淋巴细胞亚群变化(*代表每组间与PBS组比具有显著差异(p＜0.05)，其中*代表p＜0.05，**代表p＜0.01；ns代表每组间与PBS组比无显著差异(p ＞0.05))。
图16是小鼠血清中IL-2、IL-4和IFN-γ检测情况(*代表每组间与PBS组比具有显著差异(p＜0.05)，其中*代表p＜0.05，**代表p＜0.01；***代表p＜0.001；****代表p＜0.0001；ns代表每组间与PBS组比无显著差异(p＞0.05))。
具体实施方式
下面将结合本发明实施例，对本发明的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。本发明实施例中所使用的试验方法如无特殊说明，均为常规方法；所使用的材料、试剂等，如无特殊说明，均为可从商业途径得到的试剂和材料。
实施例中，表达载体pFBDM、大肠杆菌感受态细胞DH10Multibac、DH5a均由华南农业大学兽医学院微生物学与免疫学教研室保存；猪O型口蹄疫灭活疫苗 Re-O/MYA98/JSCZ/2013株为金宇保灵生物药品有限公司产品；猪口蹄疫阳性血清由广东永顺生物制药股份有限公司馈赠。
实施例1重组蛋白基因的设计与合成
根据GenBank中登录的猪O型FMDV O/BY/CHA/2010株(GenBank number：JN998085.1)中选取FMDV VP1蛋白中免疫原性好的一个T细胞优势表位(16～44aa)和两个B细胞优势表位(129～160aa、200～213aa)，所述T细胞表位第16～44位氨基酸序列如SEQ IDNo.4所示，B细胞表位第129～160位氨基酸序列如SEQ ID No.5所示，B 细胞表位第200～213位氨基酸序列如SEQ ID No.6所示。
根据专利号：US201716088905提供的ADDomer序列为VLPs载体，在其两端加上酶切位点后，其核苷酸序列如SEQ ID No.1所示。
选取的猪O型FMDV抗原表位组合之间用“GGGGS”连接子相连，串联B细胞表位 (129～160aa)与T细胞表位(16～44aa)，串联基因命名为B1T；T细胞表位(16～44aa)，基因命名为T；串联T细胞表位(16～44aa)与B细胞表位(200～213aa)，串联基因命名为TB2；用“GGGGS”连接子相连，以VL(B1T)RGD1(T)RGD2(TB2)方式于ADDomer VL区、RGD1区和RGD2区中依次插入上述猪O型FMDV抗原表位，获得重组蛋白 ADDomer-RBT，其核苷酸序列如SEQ IDNo.2所示，为了确保该框架的稳定性，本实验没有插入标签。密码子优化的重组蛋白ADDomer-RBT，其氨基酸序列如SEQ ID No.3所示。
实施例2重组蛋白亚单位结构及3D模型分析
为了保证设计的重组蛋白能形成VLPs，本说明运用SWISS-MODEL (http://SWISS-Model.expasy.org/)和Modeller(https://salilab.org/modeller/download.insta
llation.html)生成五种重组蛋白的同源性模型。使用PyMoL软件显示了五种重组蛋白的亚单位和3D结构(http//www.pymol.org/)。结果如图1所示。
实施例3重组杆状病毒转移载体的构建
本发明的构建图谱如图2所示，在pFBDM的polyhedrin(PH)启动子下构建目的基因。上述设计好的重组基因和对照组ADDomer基因经上海生工生物股份有限公司合成后，会克隆入pUC-19中，获得重组质粒pUC-19-ADDomer和pUC-19-ADDomer-RBT。使用 Primer5设计一对特异性引物ADDomer/ADDomer-RBT-P1 (5’-CGGGATCCGCGATGAGGAGAGAGCGA-3’)和ADDomer/ADDomer-RBT-P2(5’-GCGAAGCTTATCAGAAAGTGCGGCT-3’)，分别用于扩增ADDomer和ADDomer-RBT基因，在ADDomer/ADDomer-RBT-P1端引入BamH I酶切位点，在 ADDomer/ADDomer-RBT-P2端引入Hind III酶切位点，引物由上海生工生物股份有限公司合成。
(1)在本发明中，将上述合成的基因作为模板，经PCR的方式扩增目的基因ADDomer和ADDomer-RBT(反应体系如表1所示)，以ADDomer/ADDomer-RBT-P1 /ADDomer/ADDomer-RBT-P2为引物，扩增目的基因ADDomer和ADDomer-RBT，配好的 PCR体系在PCR仪按照以下条件扩增：98℃预变性3min，98℃变性10s，55℃退火20s， 72℃延伸30s，共30个循环；72℃延伸5min。反应结束后，加5μL 10×loading buffer，以110V 30min，进行1％核酸琼脂电泳。
表1 PCR反应体系
(2)将步骤(1)回收的带BamH I和Hind III酶切位点的目的片段和pFastBac TMDual 载体分别用BamH I和Hind III限制性核酸内切酶进行双酶切，酶切体系如下：10×FastDigest Buffer 2.0μL，FastDigest BamH I 1.0μL，FastDigest Hind III 1.0μL，PCR回收产物/载体10.0 μL/2μL，双蒸水加至总体积20μL；37℃水浴双酶切30min，产物分别胶回收纯化。
(3)用T4 DNA连接酶在16℃条件下连接16h，将反应产物转化至DH5α感受态细胞中，涂板后放置37℃培养16h后挑取阳性菌落在5mL LB液体培养基(含Amp)中，震荡培养16h后用质粒小提试剂盒提取阳性菌落过夜培养菌液质粒。提取的重组质粒进行双酶切以及PCR鉴定，送至北京擎科生物科技有限公司测序。将测序正确的重组转移质粒命名为pFBDM-ADDomer和pFBDM-ADDomer-RBT，重组转移质粒构建策略如图3所示。
(4)在载体pFBDM的PH启动子侧的多克隆位点MSC1处，运用Primer5设计引物pFBDM-PH-F(5’-AAATGATAACCATCTCGC-3’)；pFBDM-PH-R(5’-GAAATTTGTGATG CTATTGC-3’)，用于重组转移质粒的PCR鉴定。重组质粒PCR扩增产物长度应为插入序列总长度+297bp,即重组杆状病毒转移载体pFBDM-ADDomer PCR扩增产物长度应为 1702+297＝1999bp；重组杆状病毒转移载体pFBDM-ADDomer-RBT PCR扩增产物长度应为2039+297＝2336bp。如图4所示。电泳结果显示扩增产物长度与预期相符。
(5)对重组转移质粒进行双酶切鉴定，pFBDM-ADDomer和pFBDM-ADDomer-RBT 使用限制性内切酶BamH I和Hind III分别进行双酶切鉴定，酶切产物进行1％的琼脂糖凝胶电泳。
实施例4重组杆状病毒质粒的构建
将鉴定正确的重组杆状病毒转移载体pFBDM-ADDomer和pFBDM-ADDomer-RBT分别转化到DH10Multibac感受态细胞中。具体实施方式：①从-80℃冰箱中取出DH10Multibac感受态细胞，加入5μL的重组转移质粒，混合后，冰上静置30min；②从冰浴中取出，迅速放置于42℃水浴中热激90s后取出于冰浴中孵育2min；③在超净台中向EP管中加 900μL SOC培养基，于37℃细菌培养摇床以200r/min振荡培养4h；④在超净台中用SOC 培养基对转化菌进行10-1、10-2、10-3梯度稀释后，分别吸取100μL上述稀释梯度的转化菌涂布于含7μg/mLGen，50μg/mL Kan，50μg/mL Amp，40μg/mLIPTG和100μg/mL X-Gal LB固体培养基上；⑤将平板倒置于37℃培养箱中培养48h后区分蓝白斑，其中白色菌落即为目标菌落；⑥挑取白色菌落后重新在含7μg/mL Gen，50μg/mL Kan，50μg/mL Amp，40μg/mL IPTG和100μg/mLX-GalLB固体培养基上进行划线纯化，37℃培养箱中培养48 h。
该实验原理是借由质粒上的Tn5转座单位和细胞内辅助质粒的功能将目的基因转座到杆状病毒载体Bacmid上(图5)，由于重组后的杆状病毒质粒分子量较大，不能使用常规的酶切鉴定检验外源基因片段是否插入成功，所以使用M13-F和M13-R通用引物(M13F：CCCAGTCACGACGACGTTGTAAAACG，M13R：AGCGGATAACAATTTCACACAGG) 对重组杆粒进行PCR鉴定，结果如图6所示。将鉴定正确的重组杆状病毒质粒命名rMultibac-ADDomer、rMultibac-ADDomer-RBT。
实施例5重组杆状病毒的获得
(1)转染细胞病变
利用阳离子脂质体cellfectin II介导转染，将重组杆状病毒质粒rMultibac-ADDomer和 rMultibac-ADDomer-RBT分别转染sf9单层细胞(购自武汉大学中国典型物保藏中心)，于27℃培养箱中孵育3～5h；转染完成后，去除细胞孔中的上清，并补充2mL sf900III培养基，27℃培养箱中静置培养72h以上；
转染后，每隔24h对转染后细胞观察一次，当转染细胞开始出现了典型的细胞病变特征，如细胞停止增长、细胞边缘粗糙、胞内出现大量囊泡等特征(见图7)时，表明可能成功拯救出重组杆状病毒。继续培养可观察到细胞开始大量脱壁并裂解破碎(约转染后4～6d)，收获细胞培养物，室温1000rpm离心5min，上清即为重组杆状病毒(P1代)，于收获的病毒液中加入终浓度为2％的FBS，-80℃保存备用。将重组杆粒分别转染后获得的重组杆状病毒分别命名为Ac-ADDomer和Ac-ADDomer-RBT。由于P1代重组杆状病毒的病毒滴度较低，需要对其进行传代及扩增，以增加病毒滴度，参照Bac-to-Bac Baculoviruse ExpressionSystem操作说明书，分别获得第二代重组杆状病毒(P2代)和第三代重组杆状病毒(P3代)。
(2)重组杆状病毒滴度测定
运用Primer 5软件，以杆状病毒DNA为模板，设计一对引物F/R用于实时荧光定量PCR，设计一对引物Ac-F/R用于扩增杆状病毒DNA部分AcMK107序列，引物Ac-F/R扩增区域包含引物F/R扩增片段。引物序列见表2。
表2引物序列
以杆状病毒DNA为模板，Ac-F/Ac-R为引物，扩增目的基因经1％的琼脂糖凝胶电泳，回收纯化目的片段。参照pMD18-T载体的说明书，将胶回收纯化的目的片段和pMD18-T载体进行16℃连接12～16h。反应体系如表3所示：
表3连接反应体系
将连接产物转化DH5α大肠杆菌感受态细胞，将阳性单菌落接种于5mL含Amp的LB液体培养基，放置37℃恒温摇床按200r/min振荡培养16h，按OMEGA质粒提取试剂盒步骤提取质粒，并以BamH I/PstΙ对质粒进行双酶切鉴定(图8)，将鉴定正确的质粒送至北京擎科生物科技有限公司进行测序。将测序正确的质粒命名为pMD18T-Ac，于-20℃保存备用。用微量紫外分光光度计测量所提质粒浓度，按照下面公式计算标准质粒DNA 拷贝数。
(3)绝对荧光定量PCR
将构建好的标准品质粒进行梯度稀释，稀释为108、107、106、105、104copies/μL，把稀释好的不同梯度的标准品质粒作为模板，每个浓度做三个重复，在绝对荧光定量PCR中以标准品质粒跑出来的曲线作为标准曲线；按照Viral DNA Kit抽提试剂盒提取重组杆状病毒DNA，做好相应标记后，每一个待测的重组杆状病毒做两个梯度稀释，即稀释10倍和100倍，并做三个重复进行检测，本实验以Elution Buffer作为阴性对照，做三个复孔保证实验的一致性。荧光定量PCR按照上海近岸科技有限公司的SYBR qPCR SuperMixPlus产品说明书进行操作，荧光定量PCR反应条件：95℃预变性1min；95℃变性20s，60℃延伸1min，共40个循环反应；60℃采集荧光信号。反应体系如表4所示。
表4荧光定量PCR反应体系
根据荧光定量PCR结果绘制标准曲线，计算重组病毒DNA拷贝数，并按以下公式计算病毒滴度：
重组杆状病毒Ac-ADDomer和Ac-ADDomer-RBT经连续两代扩增，获得P3代重组杆状病毒各50mL。通过步骤(3)的方法测量重组杆状病毒的滴度，结果显示重组杆状病毒 Ac-ADDomer和Ac-ADDomer-RBT拷贝数分别为3.69×106copies/μL和2.70×107copies/μL，计算病毒滴度分别为为6.96×107pfu/mL和5.09×108pfu/mL。
实施例6重组蛋白的鉴定
(1)Western Blot鉴定重组蛋白ADDomer-RBT
将Ac-ADDomer和Ac-ADDomer-RBT分别以MOI＝1的剂量感染sf9细胞，以野生型杆状病毒作为阴性对照。采用超声破碎裂解细胞，加入相应体积的5×SDS-PAGE上样缓冲液，通过金属浴100℃10min变性蛋白。按照SDS-PAGE凝胶快速配置试剂盒说明书配制 SDS-PAGE凝胶，将上述处理后的样品经SDS-PAGE凝胶电泳后，转印PVDF膜后以猪源 FMDV阳性血清为一抗进行Western blot鉴定。
结果如图9所示，感染Ac-ADDomer的细胞样品检测不到特异性条带，感染 Ac-ADDomer-RBT的细胞样品在70kDa左右存在特异性条带，感染野生杆状病毒的细胞样品检测不到特异性条带。该结果表明在ADDomer中成功嵌入FMDV抗原表位，且具有良好的反应原性。
(2)QE质谱鉴定重组蛋白ADDomer和ADDomer-RBT
为了不影响ADDomer和ADDomer-RBT的组装效率，故在设计本发明时没有加入标签蛋白，所以这两个蛋白通过QE质谱鉴定，其操作步骤如下：①按照MOI＝1接种P3代 Ac-ADDomer和Ac-ADDomer-RBT到6cm细胞培养皿的sf9细胞中，72h后待细胞出现明显病理变化，收集相应样品进行SDS-PAGE凝胶电泳；②电泳后的凝胶放到装100mL 考马斯亮蓝染色液的盒子中，在50r/min转速摇床中常温染胶1h；③回收染色液，加入脱色液在50r/min摇床中过夜脱色；③切胶、酶解：将预期大小的蛋白条带切至1.5mL EP 管中，经过还原和烷基化后，加入胰蛋白酶(质量比1:50)，在37℃条件下酶消化20h。将酶解产物脱盐并冻干，再溶解0.1％FA溶液中，-20℃保存待用；④质谱分析：A液为0.1％甲酸的水溶液，B液为0.1％甲酸的乙腈水溶液(乙腈为84％)。色谱柱以95％的A液平衡后，样品由自动进样器上样至Trap柱；⑤质谱数据采集：多肽和多肽的碎片的质量电荷比按照下列方法采集：每次全扫描后采集20个碎片图谱；⑥数据分析：质谱测试原始文件用Mascot2.2软件检索相应的数据库，最后得到鉴定的蛋白质结果。搜库参数如下(表5)：
表5数据库检索参数表
目的胶条ADDomer经酶解后，通过QE质谱分析，得到每一段的肽段信息，与目的蛋白氨基酸比对，得分大于20分的肽段表明肽段成功鉴定，挑取得分最高的一段肽段 (244～275aa)绘制二级质谱图(图10)，根据峰图结果可知肽段通过QE质谱被成功检测，表明ADDomer蛋白成功表达。
同理，取ADDomer-RBT第72～80aa制作二级峰图(图11)，根据峰图结果可知通过QE质谱能检测该肽段，结合原始二级图谱结果可知，在QE质谱水平上，ADDomer-RBT 蛋白成功表达。
实施例7蔗糖梯度离心纯化病毒样颗粒
(1)收获重组蛋白
本发明是根据专利：US201716088905提供的表达条件，表达重组蛋白。将P3代重组杆状病毒Ac-ADDomer和Ac-ADDomer-RBT按照MOI＝1.0接种到体积为50mL的悬浮 HighFive细胞中，120h后收获表达重组蛋白。
超声破碎处理细胞：按照重组蛋白收获时间，收集细胞以1000r/min室温离心10min，弃去上清，按照M细胞/VPBS＝1：5比例加入PH＝7.4的PBS，涡旋混匀后，放置冰上进行超声波破碎。超声条件为超声5s，停止9s，超声功率采用40％。
超声结束后，以11000r/min 4℃离心15min，将上清收集到新的离心管中并做好相应标记。
(2)浓缩重组蛋白
使用Millipore超滤管对收集的细胞裂解上清进行离心浓缩，离心条件为4℃，4000 r/min离心60min。
(3)蔗糖梯度超速离心
①在12.5mL的超速管中依次加入2mL 80％、50％、30％的蔗糖溶液。
②加入步骤(2)的浓缩样品，体积不足用PBS补足。
③用电子天平将超速管进行严格配平后，在超速离心机使用SW41转子，按照超速离心机的使用说明严格操作，以30000r/min，4℃离心3.5h。
④超速离心结束后，用1mL注射器吸取不同密度梯度的蛋白环进行考马斯亮蓝染色检测重组蛋白(根据QE质谱结果可知目的蛋白表达的正确位置)，结果发现在各蛋白环都能检测到重组蛋白。
⑤采用Thermo Scientific PierceTM BCA Protein Assay Kit试剂盒测量不同密度梯度的蛋白环的蛋白浓度，发现在30％～50％这一条带中浓度最高，取这部分样品经SDS-PAGE 分析，如图12所见，纯化产物ADDomer和ADDomer-RBT在64kDa和70kDa左右有明显的条带，周边杂带较少。
(4)TEM透射电镜检测
用0.22μm滤器对收集的蛋白环溶液过滤后，取10μL用PBS稀释100倍后，用1％醋酸双氧铀负染样品，在TEM透射电镜观察重组病毒粒子组装情况，结果如图13所示， ADDomer形成的VLPs大小为17nm左右，外观由多面五边形组合而成，边缘有突起，与文献报道相似；嵌入猪O型FMDV RBT的ADDomer VLPs大小在30nm～35nm之间，颗粒表面突起明显，形态与ADDomer相比变大。以上结果表明本实验成功在体外自组装形成VLPs。
实验例重组VLPs制备的亚单位疫苗的免疫效力实验
(1)疫苗的制备
将实施例7中收集的重组病毒粒子用PBS将浓度调整到每毫升50μg，并将其分别与ISA 201VG佐剂按照1：1比例进行乳化，相关研究指出核酸佐剂作为免疫刺激分子，可更好的提高疫苗的保护效力。本发明设计VLPs疫苗+核酸佐剂NAA免疫组来验证NAA 的加入是否会增强VLPs疫苗的免疫原性，随后进行理化性能的检测。
(2)疫苗质量检测
本次实验制备的疫苗外观为乳白色液体，用注射器吸取少量乳液滴于水中，第一滴会迅速散开，第二滴不会扩散，取疫苗1mL到1.5mLEP管中，以3000r/min，4℃离心15min后，疫苗分层但是不破乳。将制备好的疫苗接种BALB/c小鼠后，在免疫后28d中小鼠全部健活，每组小鼠精神状况良好，免疫部位没有发炎现象。
(3)重组VLPs免疫效力检测
①BALB/c雌鼠鼠分组情况
将30只4周龄的SPF级BALB/c雌鼠随机分成6组，每组5头，采用皮下多点注射相应疫苗，各组免疫时间、剂量和注射试剂见表6。
表6实验动物分组
所有小鼠共免疫2次，每次免疫间隔两周，免疫方式为皮下注射。在首免后第7d、14d、 21d和28d眶下采血收集每组小鼠血液，收集的血液量为每只500μL，刚收集的血液做好标记后，先放37℃培养箱中1h，接着在4℃冰箱中过夜斜放收集的小鼠血清，次日将析出的血清分装，做好标记后放-20℃保存，用于ELISA抗体检测。同时观察免疫组小鼠存活情况，接种部位有无异常等以进行亚单位疫苗安全性检查。
②ELISA抗体检测
用FMDV ELISA Antibody Test Kit(来自深圳芬德生物技术有限公司)检测待检血清抗体水平。
根据1：1000倍稀释待检BALB/c小鼠血清，将HRP标记的羊抗鼠IgG按照1：250 的稀释比进行稀释，参照试剂盒说明书检测免疫后各组小鼠血清中FMDV抗体水平，结果如图14所示：首免后28d，FMDV特异性抗体水平达到最高水平，与商品化疫苗组相比，各组间差异显著(p＜0.05)，对照组血清(ADDomer组和PBS组)无特异性抗体。由图 13可见嵌合猪O型FMDV抗原表位的免疫原性虽然没有商品化灭活疫苗好，但均能诱导体液免疫应答。
综上，本实验制备的含FMDV抗原表位的VLPs能有效诱导小鼠产生FMDV特异性抗体，具有良好的免疫原性，但加入核酸佐剂组没有取得预期效果，查阅相关文献后，其原因可能是是NAA序列是参考对畜禽有免疫增强作用序列而设计的，故对BALB/c小鼠无明显的免疫增强作用，但在猪体上实验是有良好的增强作用的。
③小鼠脾脏T淋巴细胞的流式细胞仪检测
在二免后14d，取免疫组和空白对照组小鼠脾脏淋巴细胞进行CD4+、CD8+T细胞水平分析。结果如图15所示，在首次免疫28d后，实验组小鼠的CD4+和CD8+T淋巴细胞百分率与PBS组相比有升高趋势，在CD4+T淋巴细胞水平中，ADDomer-RBT+NAA组的百分率与其他几个组相比是最高的；在CD8+T淋巴细胞水平中，各组都比PBS组的百分率高，但是差异不显著。
④细胞因子检测
在二免后14d采集各个实验组小鼠血清，采用按照江苏酶免ELISA试剂盒说明书对小鼠血清中Th1型(IFN-γ和IL-2)和Th2型(IL-4)细胞因子浓度进行检测。结果由图16 所示，各免疫组小鼠血清中IFN-γ、IL-2和IL-4分泌水平均高于PBS组。其中， ADDomer-RBT-VLPs组IFN-γ、IL-2和IL-4分泌水平高于商品化疫苗组，表明ADDomer-RBT-VLPs作为免疫原能刺激Th1、Th2型细胞因子产生。
综上，VLPs疫苗在免疫过程中可介导体液免疫和细胞免疫两个方面。ADDomer作为一个新型病毒样颗粒载体，具有广阔的应用前景，本发明是首次运用ADDomer新型载体插入猪O型FMDV抗原表位，并成功在体外组装形成VLPs，由此可见自组装纳米颗粒支架ADDomer的研究具有开发前景，可作为FMDV亚单位候选优良疫苗的新型载体，为研究FMD防控开辟新思路。此外，在未来研究中，该新型载体可应用于开发更多新型VLPs 疫苗，为VLPs疫苗的发展开辟一个新思路。
本发明的上述实施例仅仅是为了清楚地说明本发明技术方案的所作的举例，而并非是对本发明的具体实施方式的限定。凡在本发明权利要求书的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明权利要求的保护范围之内。
序列表
<110> 华南农业大学
<120> 一种基于ADDomer嵌合猪O型口蹄疫病毒抗原表位的VLPs及应用
<130> ZM221110CS
<160> 8
<170> SIPOSequenceListing 1.0
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<211> 1702
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 1
cgcggatccg cgatgaggag acgagccgtg ctaggcggag cggtggtgta tccggagggt 60
cctcctcctt cttacgagag cgtgatgcag caacaggcgg cgatgataca gcccccactg 120
gaggctccct tcgtaccccc acggtacctg gcgcctacgg aagggagaaa cagcattcgt 180
tactcggagc tgtcgcccct gtacgatacc accaagttgt atctggtgga caacaagtcg 240
gcggacatcg cctccctgaa ctatcagaac gaccacagca acttcctgac cacggtggtg 300
cagaacaatg actttacccc cacggaggct agcacccaga ccatcaactt tgacgagcgg 360
tcgcgatggg gcggtcagct gaagaccatc atgcacacca acatgcccaa cgtgaacgag 420
tacatgttca gcaacaagtt caaggcgagg gtgatggtgt ccagaaaagc tcctgaaggt 480
gaattcgtta cagtcaatga cggtccggtc aatgacacct atgatcataa agaggatatc 540
ttgaagtatg agtggtttga gttcatttta ccagaaggca acttttcagc caccatgacg 600
atcgacctga tgaacaatgc catcattgac aactacctgg aaattggcag acagaatgga 660
gtgctggaaa gtgacattgg tgttaagttt gacactagaa atttcaggct cgggtgggac 720
cccgaaacta agttgattat gccaggtgtc tacacttatg aggcattcca tcctgacatt 780
gtattgctgc ctggttgcgg ggtagacttt actgaaagcc gacttagcaa cttgcttggc 840
atcaggaaga gacatccatt ccaggagggt ttcaaaatca tgtatgaaga tcttgaaggg 900
ggtaatattc ctgccctttt ggatgtcact gcctatgagg aaagcaaaaa ggataccact 960
actgcgcgcg aaacaaccac actggctgtt gcagaggaaa ctagtgaaga tgtcgacgat 1020
gatataacta gaggagatac ctatataact gagctcgaaa aacaaaaacg tgaagctgct 1080
gctgctgaag tttctagaaa aaaagagtta aagatccaac ctctggaaaa agacagcaag 1140
agtagaagct acaatgtctt ggaagacaaa atcaacacag cctaccgcag ttggtatctg 1200
tcctacaatt acggtaaccc tgagaaagga ataaggtctt ggacactgct caccacttca 1260
gatgtcacct gtggggcaga gcaggtctac tggtcgctcc ctgacatgat gcaagaccca 1320
gtcaccttcc gctccacaag acaagtcaac aactacccag tggtgggtgc agagcttatg 1380
cccgtcttct caaagagttt ctacaatgag caagccgtgt actctcagca gctccgacag 1440
gccacttcgc tcacgcacgt cttcaaccgc ttccctgaga accagatcct catccgcccg 1500
ccggcaccca caattaccac cgtcagtgaa aacgttcctg ctctcacaga tcacgggacc 1560
ctgccgttac gcagcagtat ccggggagtc cagcgcgtga ccgttactga cgccagacgc 1620
cgcacctgtc cctacgttta caaggccctg ggcatagtcg cgccgcgcgt tctttcaagc 1680
cgcactttct gataagcttc gc 1702
<210> 2
<211> 2039
<212> DNA
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 2
cgcggatccg cgatgaggag acgagccgtg ctaggcggag cggtggtgta tccggagggt 60
cctcctcctt cttacgagag cgtgatgcag caacaggcgg cgatgataca gcccccactg 120
gaggctccct tcgtaccccc acggtacctg gcgcctacgg aagggagaaa cagcattcgt 180
tactcggagc tgtcgcccct gtacgatacc accaagttgt atctggtgga caacaagtcg 240
gcggacatcg cctccctgaa ctatcagaac gaccacagca acttcctgac cacggtggtg 300
cagaacaatg actttacccc cacggaggct agcacccaga ccatcaactt tgacgagcgg 360
tcgcgatggg gcggtcagct gaagaccatc atgcacacca acatgcccaa cgtgaacgag 420
tacatgttca gcaacaagtt caaggcgagg gtgatggtgt ccagaaaagc tcctgaaggt 480
cttaagaata ccacactgaa agtgtttaga caggttatac ttactctgca gacacaccac 540
cgcggagacc tggacacaga gcggtggcat taagagcggc ggtccgtcgc cggagcgacg 600
gaagactcgg tcgtgaacct ctagcggaga gtgcaacccg tcactcgggg gccgcataaa 660
cgtcaagggc aacatttggt agcctggcgt caatgacacc tatgatcata aagaggatat 720
cttgaagtat gagtggtttg agttcatttt accagaaggc aacttttcag ccaccatgac 780
gatcgacctg atgaacaatg ccatcattga caactacctg gaaattggca gacagaatgg 840
agtgctggaa agtgacattg gtgttaagtt tgacactaga aatttcaggc tcgggtggga 900
ccccgaaact aagttgatta tgccaggtgt ctacacttat gaggcattcc atcctgacat 960
tgtattgctg cctggttgcg gggtagactt tactgaaagc cgacttagca acttgcttgg 1020
catcaggaag agacatccat tccaggaggg tttcaaaatc atgtatgaag atcttgaagg 1080
gggtaatatt cctgcccttt tggatgtcac tgcctatgag gaaagcaaaa aggataccac 1140
tactgcgcgc atggagaatt acggtggcga gacacaggtc cagaggcgcc accacacaga 1200
cgtctcattc atattggaca gatttgtgaa agtcacacca taagtcgacg atgatataac 1260
tagaggagat acctatataa ctgagctcat ggagaattac ggtggcgaga cacaggtcca 1320
gaggcgccac cacacagacg tctcattcat attggacaga tttgtgaaag tcacaccagg 1380
cggcagacac aaacagaaaa tagtggcgcc tgtaaagcag tccttgtaat ctagaaaaaa 1440
agagttaaag atccaacctc tggaaaaaga cagcaagagt agaagctaca atgtcttgga 1500
agacaaaatc aacacagcct accgcagttg gtatctgtcc tacaattacg gtaaccctga 1560
gaaaggaata aggtcttgga cactgctcac cacttcagat gtcacctgtg gggcagagca 1620
ggtctactgg tcgctccctg acatgatgca agacccagtc accttccgct ccacaagaca 1680
agtcaacaac tacccagtgg tgggtgcaga gcttatgccc gtcttctcaa agagtttcta 1740
caatgagcaa gccgtgtact ctcagcagct ccgacaggcc acttcgctca cgcacgtctt 1800
caaccgcttc cctgagaacc agatcctcat ccgcccgccg gcacccacaa ttaccaccgt 1860
cagtgaaaac gttcctgctc tcacagatca cgggaccctg ccgttacgca gcagtatccg 1920
gggagtccag cgcgtgaccg ttactgacgc cagacgccgc acctgtccct acgtttacaa 1980
ggccctgggc atagtcgcgc cgcgcgttct ttcaagccgc actttctgat aagcttcgc 2039
<210> 3
<211> 646
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 3
Arg Gly Ser Ala Met Arg Arg Arg Ala Val Leu Gly Gly Ala Val Val
1 5 10 15
Tyr Pro Glu Gly Pro Pro Pro Ser Tyr Glu Ser Val Met Gln Gln Gln
20 25 30
Ala Ala Met Ile Gln Pro Pro Leu Glu Ala Pro Phe Val Pro Pro Arg
35 40 45
Tyr Leu Ala Pro Thr Glu Gly Arg Asn Ser Ile Arg Tyr Ser Glu Leu
50 55 60
Ser Pro Leu Tyr Asp Thr Thr Lys Leu Tyr Leu Val Asp Asn Lys Ser
65 70 75 80
Ala Asp Ile Ala Ser Leu Asn Tyr Gln Asn Asp His Ser Asn Phe Leu
85 90 95
Thr Thr Val Val Gln Asn Asn Asp Phe Thr Pro Thr Glu Ala Ser Thr
100 105 110
Gln Thr Ile Asn Phe Asp Glu Arg Ser Arg Trp Gly Gly Gln Leu Lys
115 120 125
Thr Ile Met His Thr Asn Met Pro Asn Val Asn Glu Tyr Met Phe Ser
130 135 140
Asn Lys Phe Lys Ala Arg Val Met Val Ser Arg Lys Ala Pro Glu Gly
145 150 155 160
Leu Lys Asn Thr Thr Leu Lys Val Phe Arg Gln Val Ile Leu Thr Leu
165 170 175
Gln Thr His His Arg Gly Asp Leu Asp Thr Glu Arg Trp His Glu Arg
180 185 190
Arg Ser Val Ala Gly Ala Thr Glu Asp Ser Val Val Asn Leu Arg Arg
195 200 205
Val Gln Pro Val Thr Arg Gly Pro His Lys Arg Gln Gly Gln His Leu
210 215 220
Val Ala Trp Arg Gln His Leu Ser Arg Gly Tyr Leu Glu Val Val Val
225 230 235 240
Val His Phe Thr Arg Arg Gln Leu Phe Ser His His Asp Asp Arg Pro
245 250 255
Asp Glu Gln Cys His His Gln Leu Pro Gly Asn Trp Gln Thr Glu Trp
260 265 270
Ser Ala Gly Lys His Trp Cys Val His Lys Phe Gln Ala Arg Val Gly
275 280 285
Pro Arg Asn Val Asp Tyr Ala Arg Cys Leu His Leu Gly Ile Pro Ser
290 295 300
His Cys Ile Ala Ala Trp Leu Arg Gly Arg Leu Tyr Lys Pro Thr Gln
305 310 315 320
Leu Ala Trp His Gln Glu Glu Thr Ser Ile Pro Gly Gly Phe Gln Asn
325 330 335
His Val Arg Ser Arg Gly Tyr Ser Cys Pro Phe Gly Cys His Cys Leu
340 345 350
Gly Lys Gln Lys Gly Tyr His Tyr Cys Ala His Gly Glu Leu Arg Trp
355 360 365
Arg Asp Thr Gly Pro Glu Ala Pro Pro His Arg Arg Leu Ile His Ile
370 375 380
Gly Gln Ile Cys Glu Ser His Thr Ile Ser Arg Arg Tyr Asn Arg Arg
385 390 395 400
Tyr Leu Tyr Asn Ala His Gly Glu Leu Arg Trp Arg Asp Thr Gly Pro
405 410 415
Glu Ala Pro Pro His Arg Arg Leu Ile His Ile Gly Gln Ile Cys Glu
420 425 430
Ser His Thr Arg Arg Gln Thr Gln Thr Glu Asn Ser Gly Ala Cys Lys
435 440 445
Ala Val Leu Val Ile Lys Lys Arg Val Lys Asp Pro Thr Ser Gly Lys
450 455 460
Arg Gln Gln Glu Lys Leu Gln Cys Leu Gly Arg Gln Asn Gln His Ser
465 470 475 480
Leu Pro Gln Leu Val Ser Val Leu Gln Leu Arg Pro Glu Arg Asn Lys
485 490 495
Val Leu Asp Thr Ala His His Phe Arg Cys His Leu Trp Gly Arg Ala
500 505 510
Gly Leu Leu Val Ala Pro His Asp Ala Arg Pro Ser His Leu Pro Leu
515 520 525
His Lys Thr Ser Gln Gln Leu Pro Ser Gly Gly Cys Arg Ala Tyr Ala
530 535 540
Arg Leu Leu Lys Glu Phe Leu Gln Ala Ser Arg Val Leu Ser Ala Ala
545 550 555 560
Pro Thr Gly His Phe Ala His Ala Arg Leu Gln Pro Leu Pro Glu Pro
565 570 575
Asp Pro His Pro Pro Ala Gly Thr His Asn Tyr His Arg Gln Lys Arg
580 585 590
Ser Cys Ser His Arg Ser Arg Asp Pro Ala Val Thr Gln Gln Tyr Pro
595 600 605
Gly Ser Pro Ala Arg Asp Arg Tyr Arg Gln Thr Pro His Leu Ser Leu
610 615 620
Arg Leu Gln Gly Pro Gly His Ser Arg Ala Ala Arg Ser Phe Lys Pro
625 630 635 640
His Phe Leu Ile Ser Phe
645
<210> 4
<211> 29
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 4
Glu Asn Tyr Gly Gly Glu Thr Gln Val Gln Arg Arg His His Thr Asp
1 5 10 15
Val Ser Phe Ile Leu Asp Arg Phe Val Lys Val Thr Pro
20 25
<210> 5
<211> 32
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 5
Val Tyr Asn Gly Asn Cys Lys Tyr Ala Gly Gly Ser Leu Pro Asn Val
1 5 10 15
Arg Gly Asp Leu Gln Val Leu Ala Gln Lys Ala Ala Arg Pro Leu Pro
20 25 30
<210> 6
<211> 14
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 6
Arg His Lys Gln Lys Ile Val Ala Pro Val Lys Gln Ser Leu
1 5 10
<210> 7
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<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 7
Val Tyr Asn Gly Asn Cys Lys Tyr Ala Gly Gly Ser Leu Pro Asn Val
1 5 10 15
Arg Gly Asp Leu Gln Val Leu Ala Gln Lys Ala Ala Arg Pro Leu Pro
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Gly Gly Glu Asn Tyr Gly Gly Glu Thr Gln Val Gln Arg Arg His His
35 40 45
Thr Asp Val Ser Phe Ile Leu Asp Arg Phe Val Lys Val Thr Pro
50 55 60
<210> 8
<211> 46
<212> PRT
<213> 2 Ambystoma laterale x Ambystoma jeffersonianum
<400> 8
Glu Asn Tyr Gly Gly Glu Thr Gln Val Gln Arg Arg His His Thr Asp
1 5 10 15
Val Ser Phe Ile Leu Asp Arg Phe Val Lys Val Thr Pro Gly Gly Glu
20 25 30
Arg His Lys Gln Lys Ile Val Ala Pro Val Lys Gln Ser Leu
35 40 45
Claims (8)
1.一种基于ADDomer嵌合猪O型口蹄疫病毒抗原表位的重组蛋白，其特征在于，所述重组蛋白是在ADDomer载体VL区、RGD1区和RGD2区中插入以下猪O型口蹄疫病毒抗原表位的任一或串联的任意组合：所述猪O型口蹄疫病毒抗原表位为T细胞表位第16～44位氨基酸、B细胞表位第129～160位氨基酸和B细胞表位第200～213位氨基酸；所述重组蛋白的氨基酸序列如SEQ ID No.3所示。
2.一种权利要求1所述重组蛋白的编码基因，其特征在于，所述编码基因的核苷酸序列如SEQ ID No.2所示。
3.一种含有权利要求2所述编码基因的转移载体，其特征在于，所述转移载体是将权利要求2所述重组蛋白的编码基因克隆至表达载体所得，所述表达载体为pFBDM。
4.一种含有权利要求2所述编码基因的病毒，其特征在于，是将权利要求3所述转移载体转化感受态细胞，转座重组，获得重组病毒质粒，再将重组病毒质粒转染昆虫细胞，培养后获得重组病毒。
5.一种基于ADDomer嵌合猪O型口蹄疫病毒抗原表位的VLPs，其特征在于，由权利要求1所述重组蛋白组装而成。
6.权利要求5所述的一种基于ADDomer嵌合猪O型口蹄疫病毒抗原表位的VLPs的制备方法，其特征在于，包括以下步骤：扩增权利要求4所述重组病毒，将扩增后的重组病毒感染到昆虫细胞表达重组蛋白，收集，采用蔗糖梯度离心纯化目的蛋白，即得所述VLPs。
7.一种FMDV亚单位疫苗，其特征在于，所述疫苗的活性成分为权利要求5所述的VLPs。
8.权利要求7所述的FMDV亚单位疫苗疫苗在制备用于治疗和预防FMDV感染的药物中的应用。
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| CN109415739A (zh) * | 2016-03-31 | 2019-03-01 | 欧洲分子生物学实验室 | 腺病毒外壳蛋白衍生递送载体 |
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| WO2020025724A1 (en) * | 2018-07-31 | 2020-02-06 | Imophoron Ltd. | Multimerizing polypeptides derived from jelly roll fold domain of adenovirus penton base |
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| WO2020025724A1 (en) * | 2018-07-31 | 2020-02-06 | Imophoron Ltd. | Multimerizing polypeptides derived from jelly roll fold domain of adenovirus penton base |
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