CN113896773A - 重组fcv抗原及猫嵌杯病毒基因工程亚单位疫苗 - Google Patents
重组fcv抗原及猫嵌杯病毒基因工程亚单位疫苗 Download PDFInfo
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Abstract
本发明公开了一种重组FCV抗原及猫嵌杯病毒基因工程亚单位疫苗。所述重组FCV抗原包括分别具有SEQ ID NO：2、SEQ ID NO：3所示序列的两种蛋白。所述疫苗包含所述重组FCV抗原以及医药学上可接受的载剂。本发明提供的重组FCV抗原易于自组装形成结构稳定、免疫原性优良的病毒样颗粒(VLP)，该VLP的形态、稳定性、免疫原性接近野生病毒粒子，同时其能通过昆虫细胞表达系统以生物反应器大规模无血清悬浮培养制备，表达水平高，蛋白免疫原性好，由此制备的疫苗质控容易、批次间稳定、生产成本低。
Description
技术领域
本发明涉及一种重组蛋白，具体涉及一种重组FCV抗原及其应用，例如在制备猫嵌杯病毒重组亚单位疫苗中的应用，属于动物免疫药物技术领域。
背景技术
猫嵌杯病毒(Feline Calicivirus，FCV)是一种引起猫科动物口腔与呼吸道疾病的重要病原。FCV感染动物后临床症状主要表现为鼻炎、结膜炎、气管炎、肺炎和包括舌在内的口腔上皮细胞的水疱和溃疡。该病毒呈世界性分布，所有的猫科动物对FCV都具有易感性，一岁以下的幼猫最易感。该病毒感染后动物发病率较高，死亡率低，但FCV也常常会与细菌、衣原体、支原体、寄生虫以及其他猫科动物上呼吸道病毒发生混合感染导致死亡率增加。FCV主要通过污染物在自然界传播，在猫之间可直接接触传播，病毒粒子也可通过人手触摸间接传播给易感猫。近年来越来越多的FCV变异株在国内外被分离，FCV逐渐发生变异，这进一步给FCV的防控造成极大困难。
FCV是嵌杯病毒科、水疱疹病毒属的一种单股正链RNA病毒，无囊膜，呈二十面体对称。病毒的基因组包含三个开放阅读框，分别为ORF1、ORF2和ORF3。ORF1位于基因组的5’端，编码6种非结构蛋白p5.6、p32、p39(NTPase)、p30、p13(VPg)和p76(Pro-Pol)(SosnovtsevSV，Garfield M，Green KY.Processing map and essential cleavage sites of thenonstructural polyprotein encoded by ORF1 of the feline calicivirusgenome.Journal of virology，2002，76(14)：7060-7072)。ORF2编码囊膜蛋白(VP1)前体。ORF3编码一个小的结构蛋白VP2(Sosnovtsev SV，Green KY.Identification and genomicmapping of the ORF3 and VPg proteins in feline calicivirus virions.Virology，2000，277：193-203.)。VP1在病毒-宿主细胞的相互作用及抗原决定方面具有关键作用。单独的VP1可以自动组装形成病毒样颗粒(Virus-like particles，VLPs)，但是这种VLP在形态和大小上与真实的病毒粒子存在一些差异。VP2在RNA复制和感染性病毒颗粒的成熟中起作用，是感染病毒粒子必不可少的，与VP1自组装成病毒样颗粒有关。大量成熟的VPl加上少量VP2能够与结合VPg的病毒基因组共同组装得到完整的具有感染性的病毒粒子。
当前市面上用于预防和控制FCV的商品化疫苗主要是灭活疫苗和弱毒疫苗，这两类疫苗虽能起到预防效果，但对病毒携带者起不到保护作用，很多免疫猫出现亚临床感染和持续感染症状，并能够持续地向外界排出病毒，成为潜在的传染源。国内关于FCV新型疫苗的研制和商品化几乎是空白，CN108371710A提出一种利用杆状病毒表达系统表达制备猫传染性鼻结膜炎和猫泛白细胞减少症二联疫苗的办法，但该办法只表达FCV的VP1蛋白，其形成的VLP与野生病毒的形态、稳定性、免疫原性等均存在较为明显的差异。
发明内容
本发明的主要目的在于提供一种重组FCV抗原及其应用，以克服现有技术中的不足。
为实现前述发明目的，本发明采用的技术方案包括：
本发明实施例提供了一种重组FCV抗原，其包括第一蛋白和第二蛋白。其中，所述第一蛋白具有SEQ ID NO：2所示序列或其增加或截短的序列，特别是与SEQ ID NO：2的全长序列95％以上相同的序列。所述第二蛋白具有SEQ ID NO：3所示序列或其增加或截短的序列，特别是与SEQ ID NO：3的全长序列95％以上相同的序列。
进一步的，所述第一蛋白与第二蛋白的表达量之比为10∶1。
本发明实施例还提供了用于编码所述重组FCV抗原的基因。
在一些实施方式中，所述基因具有SEQ ID NO：1所示序列或其增加或截短的序列，特别是与SEQ ID NO：1的全长序列95％以上相同的序列。
本发明中通过对FCV的VP1、VP2的分子结构进行优化，之后将两者有机组合，实现了特殊的分子设计，包括：
(1)使重组VP1和重组VP2蛋白的表达量与组装的VLP中VP1和VP2蛋白的质量比例大致呈现10∶1，与野生病毒中二者比例类似，形成的VLP(下文命名为FCV-VLP)形态、稳定性、免疫原性接近野生病毒粒子；
(2)使用多顺反子的设计，即，启动子+VP1+IRES+VP2+polyA，其中重组VP2的起始密码子ATG突变成弱起始密码子ACG，在ACG前面加上KOZAK序列GCCACC，另外重组VP2中出现最多的L，使用稀有密码子CTA(其中的7-14个L使用稀有密码子，优选为14个)，其可以协同减少重组VP2的表达，以将重组VP2与重组VP1的表达比例控制为约1∶10。
概言之，本发明中采用前述的特殊分子设计，既可实现重组VP2蛋白的表达，有利于重组VP1结构蛋白的组装，能显著提高其组装效率，并且所表达的重组VP1蛋白与重组VP2蛋白的比例合适，使重组VP2蛋白的表达不会影响重组VP1蛋白的表达，因此共同组装形成的VLP结构稳定性明显提高，免疫原性大幅增强。
本发明实施例还提供了一种病毒样颗粒，它由第一蛋白与第二蛋白组装形成。优选的，所述第一蛋白与第二蛋白的表达量之比为10∶1。
本发明实施例还提供了包含所述重组FCV抗原的编码基因的重组载体。
在一些实施方式中，所述重组载体包括但不限于pVL1393、pFastBac 1、pFastBacDual等，并优选采用pVL1393。
本发明实施例还提供了包含所述重组FCV抗原的编码基因的宿主细胞。
在一些实施方式中，所述宿主细胞选自昆虫细胞，包括但不限于Sf9、High Five或Sf2l细胞等，优选为Sf9细胞。
本发明实施例还提供了一种免疫组合物，其包括所述的重组FCV抗原或者所述的病毒样颗粒以及医药学上可接受的载剂。
在一些实施方式中，所述医药学上可接受的载剂包括但不限于MONTANIDE ISA206、MONTANIDE ISA 201、MONTANIDE GEL 01 ST、氢氧化铝胶佐剂、明矾、弗氏佐剂、脂多糖、胆固醇、植物油、细胞因子之中的任意一种或者两种以上的组合之中的一种或者两种以上的组合，并优选使用氢氧化铝胶佐剂。
本发明实施例还提供了所述重组FCV抗原的一种制备方法，其包括：在合适条件下培养所述的宿主细胞，之后分离获得所述的重组FCV抗原。
在一些实施方式中，所述的制备方法具体包括：
将所述重组FCV抗原的编码基因克隆到杆状病毒表达系统转移载体上，获得重组表达载体；
将所述重组表达载体与杆状病毒基因组质粒共转染昆虫细胞，再筛选获得重组杆状病毒；
将所述重组杆状病毒接种昆虫细胞，进而表达所述重组FCV抗原。
所述制备方法还包括分离、纯化所述重组FCV抗原的步骤，其中可选的分离、纯化方法包括但不限于层析、透析等方法或本领域已知的其它方法。
在一些实施方式中，所述杆状病毒表达系统转移载体包括但不限于pVL1393、pFastBac 1、pFastBac Dual等，例如可以优选采用pVL1393。
在一些实施方式中，所述昆虫细胞可以选自Sf9细胞系，例如Sf9、High Five或Sf21细胞等，优选为Sf9。
本发明实施例还提供了一种猫嵌杯病毒重组亚单位疫苗的制备方法，其包括：采用前述的任一种方法制备重组FCV抗原，并将所述重组FCV抗原与医药学上可接受的载剂混合。
本发明实施例将设计好的优化后重组FCV抗原编码基因克隆到杆状病毒表达系统转移载体上，利用杆状病毒昆虫细胞表达系统，并使用悬浮培养的Sf9细胞进行表达，表达水平较高，蛋白免疫原性好。
本发明实施例还提供了所述重组FCV抗原、所述病毒样颗粒或所述免疫组合物在制备猫嵌杯病毒检测试剂，在生产用于在受试动物中诱导针对猫嵌杯病毒抗原的免疫反应的药剂，或者，在生产用于预防动物受猫嵌杯病毒感染的药剂中的用途。
本发明实施例还提供了所述重组FCV抗原、所述病毒样颗粒或所述免疫组合物在制备猫嵌杯病毒重组亚单位疫苗中的用途。
本发明实施例提供了一种猫嵌杯病毒重组亚单位疫苗，其包含前述的任一种免疫组合物。进一步的，所述疫苗还可包含医药学上可接受的载剂。
本发明实施例提供的猫嵌杯病毒重组亚单位疫苗在应用时，只需将有效量接种于动物即可。所述“有效量”是指足以获得或至少部分获得期望的效果的量。
本发明实施例还提供了包含所述重组FCV抗原的编码基因的重组载体或宿主细胞在生产用于检测动物受猫嵌杯病毒感染的试剂、用于在受试动物中诱导针对猫嵌杯病毒抗原的免疫反应的药剂、用于预防动物受猫嵌杯病毒感染的药剂中任一者中的用途。
本发明实施例还提供了一种诱导针对猫嵌杯病毒抗原的免疫反应或保护受试动物免受猫嵌杯病毒感染的方法，所述方法包括向受试动物施用所述猫嵌杯病毒重组亚单位疫苗。
本发明实施例还提供了一种适用于在受试动物中产生针对猫嵌杯病毒感染的免疫反应的疫苗，所述疫苗包含：本发明的重组FCV抗原以及佐剂。如本说明书所述的“佐剂”意指被添加到本说明书所述的疫苗中来增强由所述基因所编码的抗原的免疫原性的任何分子。优选的，所述佐剂可以采用苏州世诺生物技术有限公司生产的相关佐剂，以提高疫苗效果。
本发明实施例还提供了一种试剂盒，其包括所述的重组FCV抗原、所述病毒样颗粒、所述重组FCV抗原的编码基因、所述的重组载体、所述的宿主细胞或所述的免疫组合物。进一步的，所述试剂盒还可以包括用于包装或向动物施用所述重组FCV抗原、所述重组FCV抗原的基因、所述重组载体、所述宿主细胞或所述免疫组合物的容器或器械，如注射器等。
较之现有技术，本发明实施例提供的技术方案至少有如下优点：
(1)通过对重组FCV抗原进行特殊的分子设计，使得其中VP1和VP2蛋白的比例合适，更易于自组装形成VLP，且形成的VLP形态、稳定性、免疫原性接近野生病毒粒子，具有结构稳定、免疫原性优良等特点；
(2)通过采用昆虫细胞表达系统，可以使用生物反应器大规模无血清悬浮培养制备重组FCV抗原产品，且所获产品的抗原性、免疫原性和功能与天然蛋白质相似，表达水平较高，免疫原性强，只需要很少量就能提供很好的免疫效果，对动物没有致病性，适于作为低成本且高效的猫嵌杯病毒重组亚单位疫苗广泛应用。
附图说明
图1是实施例1中密码子优化的FCV-VLP基因PCR扩增产物的凝胶电泳图，其中在2.9kbp位置出现目的条带。
图2是实施例1中菌落PCR扩增产物的凝胶电泳图，其中在2.9kbp位置出现目的条带。
图3是实施例1中转移载体pVL-FCV-VLP的示意图。
图4是实施例3中rBac-FCV-VLP细胞培养物以及各对照组细胞培养物的SDS-PAGE检测图谱。
图5是实施例4中各SDS-PAGE电泳后产物的化学发光成像图。
图6是实施例5中接种重组杆状病毒和空杆状病毒的Sf9细胞的间接免疫荧光检测图。
图7是实施例6中纯化后FCV-VLP蛋白的灰度扫描图。
图8是实施例6中rBac-FCV-VLP组样品的电镜图。
图9a-图9d分别是实施例7中rBac-FCV-VLP组及各对照组样品的电镜图。
具体实施方式
下面通过实施例的方式进一步说明本发明。如下实施例中所用试剂和原料均市售可得，而其中未注明具体条件的试验方法，通常按照常规条件，或者按照各制造商所建议的条件。又及，除非另外说明，本发明中所公开的实验方法、检测方法、制备方法均采用本技术领域常规的分子生物学、生物化学、染色质结构和分析、分析化学、细胞培养、重组DNA技术及相关领域的常规技术。这些技术在现有文献中已有完善说明。
实施例1转移载体pVL-FCV-VLP构建与鉴定
1.FCV-VLP基因扩增与纯化
在南京金斯瑞生物科技有限公司合成了密码子优化后的FCV-VLP基因(SEQ IDNO：1)并克隆到pMD19-T载体上，得到pMD-FCV-VLP质粒载体。以pMD-FCV-VLP质粒作为模板，FCV-VLP-F、FCV-VLP-R作为上下游引物进行PCR扩增(FCV-VLP-F、FCV-VLP-R的基因序列如SEQ ID NO：4、5所示)，扩增体系见表1。
表1 FCV-VLP基因扩增体系
反应条件为：94℃预变性5分钟；95℃变性45秒，54℃退火45秒，72℃延伸1分钟，35个循环；72℃延伸10分钟。
将PCR产物进行凝胶电泳验证目的基因大小，如图1所示，在2.9kbp的位置出现目的条带，目的基因扩增成功，用凝胶回收纯化试剂盒进行回收纯化。
2.酶切与纯化
将pVL1393质粒和FCV-VLP基因PCR扩增产物使用BamH I、Xba I 37℃双酶切3小时，具体酶切反应体系见表2、表3。
将酶切产物进行凝胶电泳，分别使用凝胶回收纯化试剂盒纯化酶切的pVL1393质粒以及FCV-VLP基因片段。
表2 FCV-VLP基因酶切反应体系
表3 pVL1393质粒酶切反应体系
3.连接
将酶切过的pVL1393质粒和FCV-VLP基因酶切产物使用T4 DNA连接酶16℃水浴连接过夜，连接体系见表4。
表4 FCV-VLP基因与pVL1393质粒连接体系
4.转化
将10μl连接产物加入100μl的DH5α感受态细胞中混匀，冰浴30分钟，42℃水浴热休克90秒，再冰浴2分钟，加入900μl不含Amp的LB液体培养基，37℃培养1小时。取1.0ml菌液浓缩成100μl涂布于含有Amp的LB固体培养基上，37℃培养16小时。
5.菌落PCR和测序鉴定
挑取平板上的单菌落分别接种LB液体培养基，37℃培养2小时，以菌液作为模板，FCV-VLP-F和FCV-VLP-R作为引物进行菌落PCR。将PCR产物进行凝胶电泳验证目的基因大小，如图2所示，在2.9kbp附近出现目的条带的样品为阳性样品。将菌落PCR鉴定阳性的菌液送测序公司测序，选择测序正确的菌液进行保存。构建的含有目的基因的转移载体pVL-FCV-VLP其示意图如图3。
实施例2重组杆状病毒rBac-FCV-VLP转染与纯化
1.转染
在直径6cm的组织培养皿中接种2×106个Sf9细胞，细胞汇合度为50～70％。待细胞贴壁后弃除培养基，添加1.0ml转染缓冲液A。将BD BaculoGold LinearizedBaculovirus DNA 0.5μg和pVL-FCV-VLP质粒2μg在无菌EP管中混匀放置5分钟，添加1.0ml转染试剂B，混合均匀。吸取混合物滴加到组织培养皿，27℃孵育4小时，移除转染复合物，细胞培养基洗三次，添加3.0ml新鲜培养基，培养皿用封口膜封口，27℃培养4～5日收获上清，即共转染病毒上清。
2.蚀斑纯化
在直径为6cm的组织培养皿中接种2.0×106～2.5×106个Sf9细胞，室温下放置5分钟。梯度稀释(稀释比例：10-4～10-7)上述共转染病毒上清，每个培养皿添加1.0ml稀释的病毒液，室温放置1小时，每15分钟适当震荡，使病毒充分感染。
无菌水配置2％的低浓度琼脂糖，微波加热到60℃，充分融化。放置42℃水浴恒温，添加1体积的2×Grace培养基，混合均匀。弃细胞培养皿中培养液上清，在细胞表面覆盖4.0ml 1％琼脂糖，同时吸走所有气泡，琼脂糖凝固后将培养皿放置于湿润环境中，27℃培养7日，观察蚀斑。挑取培养皿上单个蚀斑，接种SF-SFM培养基，4℃摇晃过夜。收获培养物，接种Sf9细胞，27℃培养72小时，收获细胞培养物，定为重组杆状病毒F0代，可用于病毒的扩增以及生产。
另外按照上面的实施例方法构建表达如下(表5)各个对照组的重组杆状病毒：
表5对照组设置分组
注：对照组2为VP1和VP2基因分别克隆到不同载体后共转染Sf9细胞；对照组3为VP1和VP2基因分别克隆到一个双启动子载体后转染Sf9细胞。
实施例3 SDS-PAGE检测
将实施例2中收获的F0代重组杆状病毒rBac-FCV-VLP细胞培养物以及各对照组的细胞培养物进行SDS-PAGE检测，同时使用感染空杆状病毒的Sf9细胞作为阴性对照。具体操作如下：取40μl收获的细胞培养物，加入10μl的5×loading buffer，沸水浴5分钟，12000r/min离心1分钟，取上清进行SDS-PAGE凝胶(12％浓度凝胶)电泳，电泳后取凝胶经染色、脱色后观察目的条带。如图4所示，rBac-FCV-VLP细胞培养物在分子量约73kDa和12kDa处出现目的条带，且VP1和VP2蛋白表达量呈现约10∶1的比例，与野生病毒相似；对照组1在分子量约73kDa处出现目的条带；对照组2、对照组3均在分子量约73kDa和12kDa处出现目的条带，其中VP2蛋白大量表达，影响VP1蛋白的表达，形成的比例与野生病毒存在差异，影响了VLP的稳定性及最终产量；阴性对照没有出现目的条带。
实施例4 Western Blot检测
将实施例3中SDS-PAGE电泳后的产物转印到NC(硝酸纤维素)膜上，用5％脱脂奶粉封闭2小时，猫源抗FCV阳性血清作为一抗孵育2小时，漂洗，以HRP标记的羊抗猫多克隆抗体作为二抗孵育2小时，漂洗，然后滴加增强型化学发光荧光底物，使用化学发光成像仪拍照。结果如图5所示，重组杆状病毒表达样品有目的条带，阴性对照没有目的条带，说明目的蛋白在Sf9细胞中得到正确表达。
实施例5间接免疫荧光检测
向96孔细胞培养板中加入Sf9细胞悬液，100μl/孔(细胞浓度为2.5×105～4.0×105个/ml)，接种4孔，27℃静置15分钟，使Sf9细胞贴于培养板底壁，然后每孔加入10μl稀释10倍的F0代重组杆状病毒。同时设接种空杆状病毒Sf9细胞对照。接种后细胞置27℃恒温培养箱中培养72～96小时，弃培养液，冷甲醇/丙酮(1∶1)固定。首先与猫源抗FCV阳性血清反应，然后与FITC标记的羊抗猫IgG反应，荧光显微镜下观察。结果如图6所示，接种空杆状病毒Sf9细胞不能观察到荧光，而接种了重组杆状病毒Sf9细胞能够观察到荧光，说明目的抗原蛋白在Sf9细胞中得到正确表达，重组杆状病毒构建正确。
实施例6蛋白纯化以及电镜观察
1.蔗糖密度梯度离心
将rBac-FCV-VLP细胞培养物超声破碎，12000r/min离心30分钟，取上清，0.22μm滤膜过滤，去除杂质，使用截留分子量为10kDa的超滤管浓缩10倍。然后将浓缩样品经过浓度分别为20％、40％、60％的梯度浓度蔗糖离心，29000r/min超速离心2小时，离心后，rBac-FCV-VLP样品组可在20％～40％浓度交界处观察到白色条带。收集20％～40％浓度处的蛋白溶液使用BCA总蛋白定量法进行蛋白含量测定，用SDS-PAGE电泳的方法结合灰度扫描确定其浓度，结果如图7所示，FCV-VLP蛋白浓度为2.1g/L，纯度为99.5％。
2.电镜观察
将上述收集的蛋白溶液滴加到有碳膜的铜网上，自然风干后，滴加2％磷钨酸钠溶液进行负染，负染后进行电镜观察。电镜下rBac-FCV-VLP组样品观察到与FCV病毒粒子大小相当、形态学相近的病毒样颗粒。具体结果见图8。
3.稳定性检验
将rBac-FCV-VLP组及三个对照组样品于37℃放置三周，再按上述方法进行蔗糖密度梯度离心后，收集20％～40％浓度处的蛋白溶液进行电镜观察。电镜下rBac-FCV-VLP组(即图9a中的“专利组”)样品观察到大量病毒样颗粒，其表面粗糙，有野生病毒“刺突”类结构；对照组1存在少量病毒样颗粒，且表面光滑，无“刺突”类结构；对照组2和对照组3未观察到病毒样颗粒。具体结果见图9a-图9d。
实施例7疫苗制备
取实施例6中纯化后的FCV-VLP蛋白稀释后加入到氢氧化铝胶佐剂中，使得最终蛋白总浓度为100μg/ml。乳化后取样进行检验，合格后分装置于4℃保存。同时以同样的方法将对照组1、对照组2、对照组3样品分别制备成同等浓度的疫苗。
实施例8免疫试验
1.中和抗体检测：
将25只8～9周龄的健康中华田园猫(体质量0.5kg-1kg)随机分为5组，每组5只，分别为免疫A组、免疫B组、免疫C组、免疫D组和对照组。免疫A组试验猫以颈背部皮下注射的方式免疫FCV-VLP疫苗，1.0ml/只；免疫B、C、D组试验猫分别以颈背部皮下注射的方式免疫对照组1、2、3疫苗，2.0ml/只；空白对照组试验猫以同种方式注射生理盐水，2.0ml/只。并于首免后第4周进行二免。免疫后每2周对试验猫进行采血并分离血清，测定每组FCV中和抗体效价。结果如表6所示，免疫A组试验猫产生的中和抗体效价较其他组更高，且免疫后第8周仍能维持较高的抗体水平，免疫效果好。
表6 FCV中和抗体效价(1：n)
2.攻毒试验：
对上述免疫后8周的各试验组猫以滴鼻方式接种1ml病毒滴度为109.0TCID50/mL的FCV CH-SH株强毒。攻毒后每日观察试验猫的临床状态，测量体温，并采集咽拭子进行FCV检测，第14天进行保护率统计(至第14天精神状态良好，未出现典型临床症状，未出现持续排毒情况，确定为免疫保护)。结果如表7所示，免疫A组试验猫无发病情况，精神状态良好，行为活动正常，未出现眼鼻分泌物等临床症状，FCV检测结果为阴性，保护率达100％；免疫B、C、D组试验猫均部分发病，出现体温异常、溃疡、眼鼻分泌物等临床症状，FCV检测结果为阳性；空白对照组猫全部发病，保护率0％。
表7感染情况及保护率统计
应当理解，上述实施例为本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化，均应为等效的置换方式，都包含在本发明的保护范围之内。
序列表
<110> 苏州世诺生物技术有限公司
<120> 重组FCV抗原及猫嵌杯病毒基因工程亚单位疫苗
<130> 202109
<160> 7
<170> SIPOSequenceListing 1.0
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<213> 人工序列(人工序列)
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atgtgctcaa cctgcgctaa cgtgcttaaa tattacaatt gggaccccca ttttaagatg 60
gtgataaacc cgaataaatt tctttctgtg ggcttctgtg ataaacctct tttatgttgc 120
tatcctgatc tgcttcctga attcggaact gtatgggact gtaaccaatc cccacttgaa 180
atttatcttg aatccatcct tggtgatgat gagtggtcct ctacctttga agcaattgac 240
cctgttgtcc caccgatgca ttgggatgat gctggaaaga tcttccagcc acacccaggc 300
attctcatgc atcacctcat ctctgaggtt gcaaaagggt gggaccccaa cctgcctctt 360
ttccgtttag aggcggatga tggttccgtt acaacaccag agcagggaac cactgtgggt 420
ggagtcattg ctgaacccag tgcacagatg tcaacggccg ccgatatggc atctggcaag 480
accgttgact ctgagtggga ggctttcttt tccttccaca ctagcgtcaa ctggagtacc 540
tcggaaacgc aaggaaagat tctattcaaa caatccttgg gacctctaat taacccctac 600
ctggaacacc tgtccaaact ttacgtcgct tggtctggat ctgttgaggt tagattttct 660
atttctggtt cgggagtttt cggagggaag cttgcagcca ttgtggtgcc gccaggagtc 720
gatcctgtac aaagtacctc aatgcttcaa taccctcatg tcttatttga tgctcgtcaa 780
gtagaacctg tgattttcac tatccctgat ctaaggaatt ctctctacca tctcatgtct 840
gacactgaca ctacatcctt ggttattatg atatacaatg atctcataaa tccttacgcc 900
aatgactcta attcttctgg gtgtgtggtc actgtggaaa caaaacccgg acctgatttt 960
aaattccatc ttctaaagcc tcctggttca atgcttaccc atggatcggt cccttccgac 1020
ctgatcccaa aatcttcttc gctttggatt ggaaatcgct tctggagcga catcactgat 1080
tttgtagtta ggccttttgt ctttcaagcg aatcggcact ttgacttcaa tcaggaaact 1140
gctggatgga gcactcctag gtttcgaccc atcacaatta ctatcagtga aagtaaaggg 1200
gagaagcttg gaattggtgt ggccactgat tacattgtgc ctggcatacc tgatggatgg 1260
ccggacacaa caattgatgg aacgttgacg ccagctggtg attacgcaat cgtaaatggc 1320
agtggcaatg atattgccac cccacaagcg tatgatactg ctgacgttat cgtgaacaac 1380
accaacttca aaggtatgta catctgtggt tcacttcaga gagcttgggg tgataaaaag 1440
atatccaaca ctgctttcat cactaccggc actgtctctc aaggcgaaat tacacctagc 1500
aatgtgattg accctacaaa acttgcagtc tttcaggaca accacgccgg cgcggatgta 1560
caaacatctg atgacacact ggcactcctc ggatacactg gtataggaga acatgctatt 1620
ggatctagta gagacagagt tgtgcgcatt agcaccttac ctgaaaccgg ggcgcgtggt 1680
ggcaaccatc caatttttta caaaaattcc attaaacttg gctatgtgat taggtctatt 1740
gatgttttca attctcaaat cctgcacaca tccagacaac tatccttaaa ccactatctc 1800
ttgccacctg actcatttgc tgtctatagg attattgact ctaatggttc ttggtttgat 1860
ataggtattg attctgatgg tttctctttt gtcggtgtct ctaacattgg cagacttgag 1920
tttcctttat ctgcctccta catgggaatt caattggcaa agattcgact tgcctctaac 1980
attaggagca gtatgattaa attatgacgc ccctctccct cccccccccc taacgttact 2040
ggccgaagcc gcttggaata aggccggtgt gcgtttgtct atatgttatt ttccaccata 2100
ttgccgtctt ttggcaatgt gagggcccgg aaacctggcc ctgtcttctt gacgagcatt 2160
cctaggggtc tttcccctct cgccaaagga atgcaaggtc tgttgaatgt cgtgaaggaa 2220
gcagttcctc tggaagcttc ttgaagacaa acaacgtctg tagcgaccct ttgcaggcag 2280
cggaaccccc cacctggcga caggtgcctc tgcggccaaa agccacgtgt ataagataca 2340
cctgcaaagg cggcacaacc ccagtgccac gttgtgagtt ggatagttgt ggaaagagtc 2400
aaatggctct cctcaagcgt attcaacaag gggctgaagg atgcccagaa ggtaccccat 2460
tgtatgggat ctgatctggg gcctcggtgc acatgcttta catgtgttta gtcgaggtta 2520
aaaaaacgtc taggcccccc gaaccacggg gacgtggttt tcctttgaaa aacacgatga 2580
taagccacca cgaattcaat actaggactt attgacactg tcaccaatac agttggaaaa 2640
gctcaacaga ttgaactaga taaggccgca ctaggtcaaa atcgtgaact agctctaaaa 2700
cgcataaatc tagaccaaca ggcactaaac aatcaagttg agcagtttaa caaaattcta 2760
gagcagaggg tacacggccc aatccagtct gtgcgactag ctcgtgcggc tggattcagg 2820
gttgaccctt actcatacac aaaccaaaat ttttatgacg accaactaaa tgcaatcagg 2880
ctatcctaca gaaatctatt taagaatcta tga 2913
<210> 2
<211> 668
<212> PRT
<213> 人工序列(人工序列)
<400> 2
Met Cys Ser Thr Cys Ala Asn Val Leu Lys Tyr Tyr Asn Trp Asp Pro
1 5 10 15
His Phe Lys Met Val Ile Asn Pro Asn Lys Phe Leu Ser Val Gly Phe
20 25 30
Cys Asp Lys Pro Leu Leu Cys Cys Tyr Pro Asp Leu Leu Pro Glu Phe
35 40 45
Gly Thr Val Trp Asp Cys Asn Gln Ser Pro Leu Glu Ile Tyr Leu Glu
50 55 60
Ser Ile Leu Gly Asp Asp Glu Trp Ser Ser Thr Phe Glu Ala Ile Asp
65 70 75 80
Pro Val Val Pro Pro Met His Trp Asp Asp Ala Gly Lys Ile Phe Gln
85 90 95
Pro His Pro Gly Ile Leu Met His His Leu Ile Ser Glu Val Ala Lys
100 105 110
Gly Trp Asp Pro Asn Leu Pro Leu Phe Arg Leu Glu Ala Asp Asp Gly
115 120 125
Ser Val Thr Thr Pro Glu Gln Gly Thr Thr Val Gly Gly Val Ile Ala
130 135 140
Glu Pro Ser Ala Gln Met Ser Thr Ala Ala Asp Met Ala Ser Gly Lys
145 150 155 160
Thr Val Asp Ser Glu Trp Glu Ala Phe Phe Ser Phe His Thr Ser Val
165 170 175
Asn Trp Ser Thr Ser Glu Thr Gln Gly Lys Ile Leu Phe Lys Gln Ser
180 185 190
Leu Gly Pro Leu Ile Asn Pro Tyr Leu Glu His Leu Ser Lys Leu Tyr
195 200 205
Val Ala Trp Ser Gly Ser Val Glu Val Arg Phe Ser Ile Ser Gly Ser
210 215 220
Gly Val Phe Gly Gly Lys Leu Ala Ala Ile Val Val Pro Pro Gly Val
225 230 235 240
Asp Pro Val Gln Ser Thr Ser Met Leu Gln Tyr Pro His Val Leu Phe
245 250 255
Asp Ala Arg Gln Val Glu Pro Val Ile Phe Thr Ile Pro Asp Leu Arg
260 265 270
Asn Ser Leu Tyr His Leu Met Ser Asp Thr Asp Thr Thr Ser Leu Val
275 280 285
Ile Met Ile Tyr Asn Asp Leu Ile Asn Pro Tyr Ala Asn Asp Ser Asn
290 295 300
Ser Ser Gly Cys Val Val Thr Val Glu Thr Lys Pro Gly Pro Asp Phe
305 310 315 320
Lys Phe His Leu Leu Lys Pro Pro Gly Ser Met Leu Thr His Gly Ser
325 330 335
Val Pro Ser Asp Leu Ile Pro Lys Ser Ser Ser Leu Trp Ile Gly Asn
340 345 350
Arg Phe Trp Ser Asp Ile Thr Asp Phe Val Val Arg Pro Phe Val Phe
355 360 365
Gln Ala Asn Arg His Phe Asp Phe Asn Gln Glu Thr Ala Gly Trp Ser
370 375 380
Thr Pro Arg Phe Arg Pro Ile Thr Ile Thr Ile Ser Glu Ser Lys Gly
385 390 395 400
Glu Lys Leu Gly Ile Gly Val Ala Thr Asp Tyr Ile Val Pro Gly Ile
405 410 415
Pro Asp Gly Trp Pro Asp Thr Thr Ile Asp Gly Thr Leu Thr Pro Ala
420 425 430
Gly Asp Tyr Ala Ile Val Asn Gly Ser Gly Asn Asp Ile Ala Thr Pro
435 440 445
Gln Ala Tyr Asp Thr Ala Asp Val Ile Val Asn Asn Thr Asn Phe Lys
450 455 460
Gly Met Tyr Ile Cys Gly Ser Leu Gln Arg Ala Trp Gly Asp Lys Lys
465 470 475 480
Ile Ser Asn Thr Ala Phe Ile Thr Thr Gly Thr Val Ser Gln Gly Glu
485 490 495
Ile Thr Pro Ser Asn Val Ile Asp Pro Thr Lys Leu Ala Val Phe Gln
500 505 510
Asp Asn His Ala Gly Ala Asp Val Gln Thr Ser Asp Asp Thr Leu Ala
515 520 525
Leu Leu Gly Tyr Thr Gly Ile Gly Glu His Ala Ile Gly Ser Ser Arg
530 535 540
Asp Arg Val Val Arg Ile Ser Thr Leu Pro Glu Thr Gly Ala Arg Gly
545 550 555 560
Gly Asn His Pro Ile Phe Tyr Lys Asn Ser Ile Lys Leu Gly Tyr Val
565 570 575
Ile Arg Ser Ile Asp Val Phe Asn Ser Gln Ile Leu His Thr Ser Arg
580 585 590
Gln Leu Ser Leu Asn His Tyr Leu Leu Pro Pro Asp Ser Phe Ala Val
595 600 605
Tyr Arg Ile Ile Asp Ser Asn Gly Ser Trp Phe Asp Ile Gly Ile Asp
610 615 620
Ser Asp Gly Phe Ser Phe Val Gly Val Ser Asn Ile Gly Arg Leu Glu
625 630 635 640
Phe Pro Leu Ser Ala Ser Tyr Met Gly Ile Gln Leu Ala Lys Ile Arg
645 650 655
Leu Ala Ser Asn Ile Arg Ser Ser Met Ile Lys Leu
660 665
<210> 3
<211> 107
<212> PRT
<213> 人工序列(人工序列)
<400> 3
Thr Asn Ser Ile Leu Gly Leu Ile Asp Thr Val Thr Asn Thr Val Gly
1 5 10 15
Lys Ala Gln Gln Ile Glu Leu Asp Lys Ala Ala Leu Gly Gln Asn Arg
20 25 30
Glu Leu Ala Leu Lys Arg Ile Asn Leu Asp Gln Gln Ala Leu Asn Asn
35 40 45
Gln Val Glu Gln Phe Asn Lys Ile Leu Glu Gln Arg Val His Gly Pro
50 55 60
Ile Gln Ser Val Arg Leu Ala Arg Ala Ala Gly Phe Arg Val Asp Pro
65 70 75 80
Tyr Ser Tyr Thr Asn Gln Asn Phe Tyr Asp Asp Gln Leu Asn Ala Ile
85 90 95
Arg Leu Ser Tyr Arg Asn Leu Phe Lys Asn Leu
100 105
<210> 4
<211> 39
<212> DNA
<213> 人工序列(人工序列)
<400> 4
ataggatcca tgtgctcaac ctgcgctaac gtgcttaaa 39
<210> 5
<211> 39
<212> DNA
<213> 人工序列(人工序列)
<400> 5
atatctagat catagattct taaatagatt tctgtagga 39
<210> 6
<211> 2007
<212> DNA
<213> 人工序列(人工序列)
<400> 6
atgtgctcaa cctgcgctaa cgtgcttaaa tattacaatt gggaccccca ttttaagatg 60
gtgataaacc cgaataaatt tctttctgtg ggcttctgtg ataaacctct tttatgttgc 120
tatcctgatc tgcttcctga attcggaact gtatgggact gtaaccaatc cccacttgaa 180
atttatcttg aatccatcct tggtgatgat gagtggtcct ctacctttga agcaattgac 240
cctgttgtcc caccgatgca ttgggatgat gctggaaaga tcttccagcc acacccaggc 300
attctcatgc atcacctcat ctctgaggtt gcaaaagggt gggaccccaa cctgcctctt 360
ttccgtttag aggcggatga tggttccgtt acaacaccag agcagggaac cactgtgggt 420
ggagtcattg ctgaacccag tgcacagatg tcaacggccg ccgatatggc atctggcaag 480
accgttgact ctgagtggga ggctttcttt tccttccaca ctagcgtcaa ctggagtacc 540
tcggaaacgc aaggaaagat tctattcaaa caatccttgg gacctctaat taacccctac 600
ctggaacacc tgtccaaact ttacgtcgct tggtctggat ctgttgaggt tagattttct 660
atttctggtt cgggagtttt cggagggaag cttgcagcca ttgtggtgcc gccaggagtc 720
gatcctgtac aaagtacctc aatgcttcaa taccctcatg tcttatttga tgctcgtcaa 780
gtagaacctg tgattttcac tatccctgat ctaaggaatt ctctctacca tctcatgtct 840
gacactgaca ctacatcctt ggttattatg atatacaatg atctcataaa tccttacgcc 900
aatgactcta attcttctgg gtgtgtggtc actgtggaaa caaaacccgg acctgatttt 960
aaattccatc ttctaaagcc tcctggttca atgcttaccc atggatcggt cccttccgac 1020
ctgatcccaa aatcttcttc gctttggatt ggaaatcgct tctggagcga catcactgat 1080
tttgtagtta ggccttttgt ctttcaagcg aatcggcact ttgacttcaa tcaggaaact 1140
gctggatgga gcactcctag gtttcgaccc atcacaatta ctatcagtga aagtaaaggg 1200
gagaagcttg gaattggtgt ggccactgat tacattgtgc ctggcatacc tgatggatgg 1260
ccggacacaa caattgatgg aacgttgacg ccagctggtg attacgcaat cgtaaatggc 1320
agtggcaatg atattgccac cccacaagcg tatgatactg ctgacgttat cgtgaacaac 1380
accaacttca aaggtatgta catctgtggt tcacttcaga gagcttgggg tgataaaaag 1440
atatccaaca ctgctttcat cactaccggc actgtctctc aaggcgaaat tacacctagc 1500
aatgtgattg accctacaaa acttgcagtc tttcaggaca accacgccgg cgcggatgta 1560
caaacatctg atgacacact ggcactcctc ggatacactg gtataggaga acatgctatt 1620
ggatctagta gagacagagt tgtgcgcatt agcaccttac ctgaaaccgg ggcgcgtggt 1680
ggcaaccatc caatttttta caaaaattcc attaaacttg gctatgtgat taggtctatt 1740
gatgttttca attctcaaat cctgcacaca tccagacaac tatccttaaa ccactatctc 1800
ttgccacctg actcatttgc tgtctatagg attattgact ctaatggttc ttggtttgat 1860
ataggtattg attctgatgg tttctctttt gtcggtgtct ctaacattgg cagacttgag 1920
tttcctttat ctgcctccta catgggaatt caattggcaa agattcgact tgcctctaac 1980
attaggagca gtatgattaa attatga 2007
<210> 7
<211> 324
<212> DNA
<213> 人工序列(人工序列)
<400> 7
atgaattcaa tactgggact tattgacact gtcaccaata cagttggaaa agctcaacag 60
attgaactgg ataaggccgc actgggtcaa aatcgtgaac tggctctgaa acgcataaat 120
ctggaccaac aggcactaaa caatcaagtt gagcagttta acaaaattct ggagcagagg 180
gtacacggcc caatccagtc tgtgcgacta gctcgtgcgg ctggattcag ggttgaccct 240
tactcataca caaaccaaaa tttttatgac gaccaactga atgcaatcag gctgtcctac 300
agaaatctgt ttaagaatct gtga 324
Claims (12)
1.一种重组FCV抗原，其特征在于，它包括：
第一蛋白，具有SEQ ID NO：2所示序列或其增加或截短的序列；以及
第二蛋白，具有SEQ ID NO：3所示序列或其增加或截短的序列；
优选的，所述第一蛋白与第二蛋白的表达量之比为10∶1。
2.一种病毒样颗粒，其特征在于：它由第一蛋白与第二蛋白组装形成；其中，所述第一蛋白具有SEQ ID NO：2所示序列或其增加或截短的序列，所述第二蛋白具有SEQ ID NO：3所示序列或其增加或截短的序列；
优选的，所述第一蛋白与第二蛋白的表达量之比为10∶1。
3.用于编码权利要求1所述重组FCV抗原的基因；
优选的，所述基因具有SEQ ID NO：1所示序列或其增加或截短的序列。
4.包含权利要求3所述基因的重组载体；优选的，所述重组载体包括pVL1393、pFastBac1或pFastBac Dual，更优选为pVL1393。
5.包含权利要求3所述基因的宿主细胞；优选的，所述宿主细胞选自昆虫细胞，所述昆虫细胞包括Sf9、High Five或Sf21细胞，更优选为Sf9细胞。
6.一种免疫组合物，其特征在于包括：权利要求1所述的重组FCV抗原或者权利要求2所述的病毒样颗粒；以及，医药学上可接受的载剂。
7.如权利要求6所述的免疫组合物，其特征在于：所述医药学上可接受的载剂包括MONTANIDE ISA 206、MONTANIDE ISA 201、MONTANIDE GEL 01 ST、氢氧化铝胶佐剂、明矾、弗氏佐剂、脂多糖、胆固醇、植物油、细胞因子之中的任意一种或者两种以上的组合；优选为氢氧化铝胶佐剂。
8.一种重组FCV抗原的制备方法，其特征在于包括：在合适条件下培养权利要求5所述的宿主细胞，之后分离获得权利要求1所述重组FCV抗原。
9.如权利要求8所述的制备方法，其特征在于具体包括：
将所述重组FCV抗原的编码基因克隆到杆状病毒表达系统转移载体上，获得重组表达载体；
将所述重组表达载体与杆状病毒基因组质粒共转染昆虫细胞，再筛选获得重组杆状病毒；
将所述重组杆状病毒接种昆虫细胞，进而表达所述重组FCV抗原。
10.如权利要求1所述重组FCV抗原、权利要求2所述的病毒样颗粒或权利要求6或7所述免疫组合物在制备猫嵌杯病毒检测试剂，在生产用于在受试动物中诱导针对猫嵌杯病毒抗原的免疫反应的药剂，或者，在生产用于预防动物受猫嵌杯病毒感染的药剂中的用途。
11.如权利要求1所述重组FCV抗原、权利要求2所述的病毒样颗粒或权利要求6或7所述免疫组合物在制备猫嵌杯病毒重组亚单位疫苗中的用途。
12.试剂盒，其特征在于包括权利要求1所述重组FCV抗原、权利要求2所述的病毒样颗粒、权利要求3所述基因、权利要求4所述重组载体、权利要求5所述宿主细胞或权利要求6或7所述免疫组合物。
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| CN117886900A (zh) * | 2024-03-15 | 2024-04-16 | 北京纳百生物科技有限公司 | 一种猫杯状病毒vp1蛋白截短体及其应用 |
| CN117886900B (zh) * | 2024-03-15 | 2024-05-31 | 北京纳百生物科技有限公司 | 一种猫杯状病毒vp1蛋白截短体及其应用 |
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