CN116143949A - 一种水痘-带状疱疹病毒疫苗及其制备方法 - Google Patents
一种水痘-带状疱疹病毒疫苗及其制备方法 Download PDFInfo
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Abstract
本发明公开了一种水痘‑带状疱疹病毒疫苗及其制备方法。本发明的水痘‑带状疱疹病毒纳米颗粒包含由组分1和组分2,组分1和组分2利用自组装系统相连接;其中,组分1为水痘—带状疱疹病毒糖蛋白E,组分2为幽门螺旋杆菌铁蛋白。本发明构建了一种基于细菌复合物的水痘‑带状疱疹病毒gE蛋白亚单位纳米颗粒疫苗,是一款基于HPF纳米颗粒内核的gE亚单位(水痘‑带状疱疹病毒纳米颗粒)的疫苗,该疫苗可以使小鼠产生水痘‑带状疱疹病毒gE蛋白特异性抗体,并且制备方法简单,安全性高,相比较于Shingrix,相同质量的VZV gE‑HPF疫苗使用更少的gE抗原量和铝佐剂即达到相应的体液免疫水平,也用于诱导较强的细胞免疫。
Description
技术领域
本发明涉及免疫技术领域,具体地,涉及一种水痘-带状疱疹病毒疫苗及其制备方法。
背景技术
水痘-带状疱疹病毒(Varicella-zoster virus,VZV)是一种双链DNA病毒,是疱疹病毒科的一员。人类是这种病毒的唯一已知的宿主。VZV病毒主要通过空气传播,气溶胶传播及接触疱疹液体直接传播也有报道。首次感染VZV会导致水痘及弥漫性疱疹,潜伏期在7~23天,在此期间病毒会扩散到扁桃体及其他淋巴组织,VZV病毒可以在初次感染后潜伏在交感神经节内,其病毒复制重新激活后,病毒通过神经节到达皮肤,引发带状疱疹(Herpeszoster,HZ)。老年人和免疫力低下人群更容易重新激活VZV病毒,60岁以上老年人HZ的发病率比60岁以下的老年人高8~10倍。HZ最常见的并发症为带状疱疹神经痛,这是一种在HZ皮肤病变区域出现的持续的神经痛,严重影响患者的生活质量。近年来HZ的发病率在成年人中不断上升。
已经上市的VZV的疫苗有两款,分别是默克公司的Zostavax和史葛兰素公司的Shingrix。Zostavax以Oka株为主的减毒活疫苗,在60~69岁的人群中,以单次皮下注射的方式使用Zostavax,疫苗的预防效力达到64%。随着年龄的增长,Zostavax疗效下降,在60岁以上的成年人中,疫苗对PHN的有效率仅为39%。
另一款疫苗是史葛兰素的重组蛋白亚单位疫苗Shingrix。Shingrix是一种VZV gE亚基蛋白疫苗,辅以AS01B(一种QS-21,单磷酰脂质A和脂质体的组合)。通过肌肉注射的方式免疫,显示在60~69岁个体中预防HZ的疫苗效力为97.4%,在所有测试年龄组中均>90%,包括≥80岁的个体。但是其价格昂贵且其独有的佐剂AS01B的产量少,导致Shingrix的产量少,国内供应量严重不足。
发明内容
本发明的目的是为了克服现有技术的上述不足,提供一种水痘-带状疱疹病毒疫苗及其制备方法。
本发明的第一个目的是提供一种水痘-带状疱疹病毒纳米颗粒。
本发明的第二个目的是提供所述水痘-带状疱疹病毒纳米颗粒在制备水痘-带状疱疹病毒疫苗中的应用。
本发明的第三个目的是提供一种水痘-带状疱疹病毒纳米颗粒的制备方法。
本发明的第四个目的是提供所述制备方法制备得到的水痘-带状疱疹病毒纳米颗粒。
本发明的第五个目的是提供所述水痘-带状疱疹病毒纳米颗粒在制备水痘-带状疱疹病毒疫苗中的应用。
为了实现上述目的,本发明是通过以下方案予以实现的:
重组亚单位疫苗是活疫苗的替代品,具有强免疫原性、对免疫功能低下的个体安全性和易于生产的潜在优势。VZV糖蛋白E(gE)是VZV疫苗中最有吸引力的抗原。gE是VZV病毒粒子和VZV感染细胞中最丰富的糖蛋白,是VZV特异性CD4+T细胞应答的主要靶标。
为了提高疫苗的免疫原性,本发明在纳米颗粒上集中展示抗原。来自幽门螺旋杆菌(H.pylori)的铁蛋白纳米颗粒已成功应用于纳米颗粒疫苗中,可引发广泛的中和抗体反应。此外,幽门螺旋杆菌铁蛋白(Ferritin,HPF)与人类Ferritin有显著差异,不太可能诱导自身抗体。因此,选择H.pylori(幽门螺旋杆菌)的Ferritin(HPF)作为我们纳米颗粒疫苗的核心。为了增加呈现两个或更多不同蛋白质亚基的能力,并增加亚基的产量,引入了自主研发优化的GvoTagOpti/SdCatcher系统(如图1所示),将GvoTagOpti(Gvo)融合在gE蛋白的C端,分泌信号肽(SP)位于融合蛋白的N端,促进蛋白质的分泌,在细胞表达融合蛋白过程中被清除;而SdCatcher(Sd)与HPF的N末端融合。
Sd-HPF在大肠杆菌中表达纯化;gE蛋白在真核系统细胞中表达纯化来保留蛋白上的糖基化修饰,而糖基化修饰对疫苗的免疫原性和识别至关重要。纯化得到的Sd-HPF和gE蛋白在没有任何酶的常规缓冲液中孵育。Sd和Gvo形成分子间异肽键,不可逆地结合HPF和抗原亚基,形成VZV gE-HPF纳米颗粒蛋白聚体(VZV gE-NP)。
因此,本发明以水痘-带状疱疹病毒gE糖蛋白(glycoprotein)作为抗原片段,并基于幽门螺旋杆菌铁蛋白实现抗原多聚化,构建了一种gE抗原多聚体复合物。具体的是将gE蛋白通过自主研发优化的连接元件肽段完成抗原与幽门螺旋杆菌铁蛋白的偶联,将抗原展示在纳米颗粒的表面,最后将疫苗接种到小鼠体内,能有效地引起免疫保护反应。
一种水痘-带状疱疹病毒纳米颗粒,包含由组分1和组分2,组分1和组分2利用自组装系统相连接;
其中,组分1为水痘—带状疱疹病毒糖蛋白E,其氨基酸序列如SEQ ID NO:1;
组分2为幽门螺旋杆菌铁蛋白,其氨基酸序列如SEQ ID NO:8。
优选地,所述自组装系统为GvoTagOpti/SdCatcher系统,其中GvoTagOpti的氨基酸序列如SEQ ID NO:2所示;SdCatcher的氨基酸序列SEQ ID NO:7。
优选地,GvoTagOpti设置于水痘—带状疱疹病毒糖蛋白E的C端。
优选地,SdCatcher设置于幽门螺旋杆菌铁蛋白的N端。
更优选地,水痘—带状疱疹病毒糖蛋白E的N端还设置有分泌信号肽(SP)。
更优选地,所述分泌信号肽的氨基酸序列SEQ ID NO:4。
所述水痘-带状疱疹病毒纳米颗粒在制备水痘-带状疱疹病毒疫苗中的应用。
一种水痘-带状疱疹病毒纳米颗粒的制备方法,氨基酸序列如SEQ ID NO:5和6所示的重组蛋白,孵育后纯化即得。
优选地,所述氨基酸序列如SEQ ID NO:5和6所示的重组蛋白的物质的量为1:1。
优选地,孵育的条件为24~26℃孵育3.9~4.1h。
更选地,孵育的条件为25℃孵育4h。
优选地,所述纯化方法为通过Siperose6 Increase10/300GL柱子(GE)进行分子筛层析,其中,分子筛层析缓冲液为:PBS,pH 7.4。
所述制备方法制备得到的水痘-带状疱疹病毒纳米颗粒。
所述水痘-带状疱疹病毒纳米颗粒在制备水痘-带状疱疹病毒疫苗中的应用。
与现有技术相比,本发明具有以下有益效果:
本发明构建了一种基于细菌复合物的水痘-带状疱疹病毒gE蛋白亚单位纳米颗粒疫苗,是一款基于HPF纳米颗粒内核的gE亚单位疫苗(水痘-带状疱疹病毒纳米颗粒),该疫苗可以使小鼠产生水痘-带状疱疹病毒gE蛋白特异性抗体,并且制备方法简单,安全性高,相比较于Shingrix,相同质量的VZV gE-HPF疫苗使用更少的gE抗原量和铝佐剂即达到相应的体液免疫水平,同时也用于诱导较强的细胞免疫。
附图说明
图1为抗原gE蛋白结构示意图。
图2为抗原gE蛋白以幽门螺旋杆菌铁蛋白为核心组装成纳米颗粒疫苗示意图。
图3为VZV gE-Gvo-His蛋白与Sd-HPF形成VZV gE-HPF的自组装过程。
图4为gE蛋白纯化分子筛图。
图5VZV gE-Gvo-His蛋白单体(VZV gE)和VZV gE-HPF纳米颗粒蛋白聚体(VZV gE-NP)的SDS-PAGE检测结果。
图6纳米颗粒疫苗BALB/c小鼠免疫策略图。
图7为疫苗免疫后小鼠血清中gE抗原特异性IgG抗体的滴度。其中A为第2周,B为第4周,C为第6周。
图8为gE(全长)-GVO、gE(截短)-GVO和SP-gE(截短)-GVO融合蛋白表达纯化SDS-PAGE图。
图9为gE(截短)-GVO和SP-gE(截短)-GVO融合蛋白表达纯化Westernblot图。
具体实施方式
下面结合说明书附图及具体实施例对本发明作出进一步地详细阐述,所述实施例只用于解释本发明,并非用于限定本发明的范围。下述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,为可从商业途径得到的试剂和材料。
实施例1VZV gE-HPF纳米颗粒疫苗的构建与纯化
构建水痘-带状疱疹病毒的SP-VZVgE-Gvo-His融合蛋白和Sd-HPF融合蛋白的结构示意图如图1所示。由图1可知,去除水痘-带状疱疹病毒的gE的跨膜域和胞内段后,得到gE蛋白可溶的胞外段(即gE抗原),其氨基酸序列如SEQ ID NO:1所示;上述gE抗原与SEQ IDNO:2所示Gvo通过GSG连接后的氨基酸序列如SEQ ID NO:3所示;为了有更好的蛋白产量,把蛋白原始的信号肽段更换成另一信号肽(SP),SP的氨基酸序列如SEQ ID NO:4所示,把上述抗原与分泌型信号肽(SP)、Gvo和His纯化标签连接后的氨基酸序列如SEQ ID NO:5所示(SP-VZV gE-Gvo-His融合蛋白)。
构建的pcDNA3.1-intron-SP-gE-Gvo-His-WPRE的质粒图谱如图2所示。VZV gE纳米抗原的组装过程如图3所示,将制备得到的SP-VZV gE-Gvo-His融合蛋白与Sd-HPF融合蛋白(Sd-HPF融合蛋白的氨基酸序列如SEQ ID NO:6所示)于25℃条件下孵育过夜,SP-VZVgE-Gvo-His融合蛋白与Sd-HPF融合蛋白通过共价结合GvTagOpti/Sdcatcher(Gvo/Sd)系统,通过Gvo-Sd的自发化学键结合,自组装得到VZV gE-HPF二十四聚体,即纳米抗原。
其中,GvTagOpti/Sdcatcher(Gv/Sd)系统参见公开号为CN113621031A的中国专利。Sd的氨基酸序列如SEQ ID NO:7所示,HPF的氨基酸序列如SEQ ID NO:8所示,Sd(Sdcatcher)通过GSG与幽门螺旋杆菌铁蛋白(HPF)的N端连接得到Sd-HPF融合蛋白。
具体地,SP-VZV gE-Gvo-His融合蛋白(氨基酸序列SEQ ID NO:5所示)的制备方法如下:
1、pcDNA3.1-intron-SP-gE-Gvo-His-WPRE质粒的构建
pcDNA3.1-intron-SP-gE-Gvo-His-WPRE质粒由pcDNA3.1-Intron-WPRE质粒载体(核苷酸序列SEQ ID NO:9)和编码蛋白SP-VZV gE-Gvo-His的核苷酸序列组成,SP-VZV gE-Gvo-His核苷酸序列如SEQ ID NO:10所示,SP-VZV gE-Gvo-His核苷酸序列的5’端为EcoRI酶切位点,3’端为XbaI酶切位点。
(1)EcoRI限制性核酸内切酶和XbaI限制性核酸内切酶在37℃酶切SP-VZV gE-Gvo-His的核苷酸片段(SEQ ID NO:10)以及pcDNA3.1-Intron-WPRE质粒载体(SEQ ID NO:9),酶切2h后通过胶回收试剂盒(OMEGA D2500-02)回收SP-VZV gE-Gvo-His的核苷酸片段以及线性化的pcDNA3.1-Intron-WPRE质粒载体。
通过T4连接酶,将目的片段(SP-VZV gE-Gvo-His)与线性化的pcDNA3.1-Intron-WPRE质粒载体在25℃连接1h,构建得到重组表达载体质粒pcDNA3.1-intron-SP-gE-Gvo-His-WPRE,所述重组表达载体质粒的结构示意图如图2所示。
(2)将构建所得重组表达载体质粒pcDNA3.1-intron-SP-gE-Gvo-His-WPRE加入到DH5α感受态细菌中,在冰盒上静置30min,涂布于氨苄青霉素抗性的LB培养皿上,37℃过夜培养,挑取单菌落并用PCR鉴定出阳性克隆;用质粒提取试剂盒(Omega D6950-02)提取去内毒素的质粒(pcDNA3.1-intron-SP-gE-Gvo-His-WPRE),经测序验证构建得到了正确的pcDNA3.1-intron-SP-gE-Gvo-His-WPRE重组表达载体质粒。
2、VZV gE-Gvo-His蛋白的表达与纯化
(1)准备两个50mL离心管,分别标记为1号管和2号管。这两个50mL离心管中都加入20mL Opti-MEM培养基。在1号管中加入1.25mg上述的pcDNA3.1-intron-SP-gE-Gvo-His-WPRE重组表达载体质粒,混匀后静置5min。在2号管中加入3.75mL的PEI阳离子转染试剂,混匀后静置5min。将上述2号管的混合试剂倒入1号管中,混匀后静置20min。20min后将2号管的混合试剂倒入1L的密度为3×106cells/mL的293F细胞中,5天后离心收获293F细胞上清(即表达SP-VZV gE-Gvo-His的细胞上清)。
(2)用His纯化标签对目的蛋白SP-VZV gE-Gvo-His进行纯化:
将表达SP-VZV gE-Gvo-His的细胞上清通过0.22μm的滤膜过滤,除去细胞碎片,得过滤后的细胞上清液。将过滤后的细胞上清液流穿HisTrap excel镍柱进行粗提纯,目的蛋白通过His标签结合在HisTrap excel镍柱镍填料中,得到富集了目的蛋白的镍填料。用PBS(pH=7.4)缓冲液和低浓度咪唑缓冲液(PBS,20mM Imidazole,pH=7.4)分别洗涤富集了目的蛋白的镍填料,缓冲液的洗涤体积都为50mL。洗涤的目的是去除镍填料上的杂蛋白。其后,用含高咪唑缓冲液(PBS,50或500mM Imidazole,pH=7.4)进行目的蛋白洗脱,得目的蛋白洗脱液。
将洗脱液浓缩至1mL,得到纯化后的SP-VZV gE-Gvo-His蛋白(VZV gE),对其进行SDS-PAGE检测。
3、VZV gE-HPF的纳米颗粒疫苗的制备
将上述纯化后的SP-VZV gE-Gvo-His融合蛋白(氨基酸序列如SEQ ID NO:5所示)与Sd-HPF融合蛋白(氨基酸序列如SEQ ID NO:6所示)按物质的量为1:1的比例于25℃孵育4h,形成VZV gE-HPF(纳米抗原)。其自组装过程如图3所示。
收集孵育后形成的VZV gE-HPF(纳米抗原),通过Siperose6 Increase10/300GL柱子(GE)进行分子筛层析(分子筛层析缓冲液为:PBS,pH 7.4),收集纯化产物,进行SDS-PAGE检测。
将所得VZV gE-HPF蛋白浓缩后分装成小份(500μL/管),用液氮迅速冷冻后于-80℃保存。
VZV gE-HPF纯化分子筛图如图4所示,所得多聚体蛋白VZV gE-HPF的峰单一,表明获得了纯度高于99%的二十四聚体VZV gE-HPF纳米颗粒蛋白聚体(VZV gE-HPF)。
SDS-PAGE检测结果如图5所示,本发明成功表达、纯化、孵育获得了SP-VZV gE-Gvo-His蛋白单体(VZV gE)和VZV gE-HPF纳米颗粒蛋白聚体(VZV gE-NP)。
实施例2gE蛋白特异性IgG滴度的ELISA实验
一、实验方法
将10μg实施例1的VZV gE-HPF纳米颗粒蛋白和HPF蛋白(氨基酸序列如SEQ ID NO:8所示)分别用PBS缓冲液(pH 7.4)稀释到100μL的体积,并与等体积铝佐剂进行乳化,制备得到200μL VZV gE-HPF纳米颗粒疫苗和HPF疫苗。
对6~8周龄体重在18-22g的Balb/C小鼠进行分组免疫。小鼠分组为HPF组和VZVgE-HPF纳米颗粒组,每组6只小鼠,免疫策略如图6所示,即通过皮下注射的方式,所有小鼠均以双针免疫的策略免疫,即在第0周和第4周免疫,首次接种记为第0周,首次接种后的四周,记为第4周,共接种两次,每次200μL的接种体积(实际每只小鼠接种的Sd-HPF或VZV gE-HPF纳米颗粒蛋白的质量都为10μg)。分别在第2、4、6、8和10周,对小鼠进行眼眶取血;小鼠血清静置30min后,在4℃,2800rpm条件下离心10min,获得免疫后的小鼠血清,立刻进行该血清中gE蛋白特异性IgG滴度的ELISA实验。
其中,gE蛋白特异性IgG滴度的ELISA实验过程如下:
1、包板
将SP-VZV gE-Gvo-His用PBS分别稀释到5ng/μL,50μL/孔,分别加入96孔ELISA板(康宁,42592),用贴纸贴紧孔板,防止蒸发,4℃过夜。
2、封闭
甩出并扣干步骤1孔板中液体,用PBS配制5%脱脂奶粉溶液,倒入加样槽,用排枪加入孔板中,100μL/孔,再用贴纸贴紧孔板,25℃下封闭1h。
3、洗板
大力甩出并扣干步骤2封闭后孔板中液体,加入200μL 0.1% PBST溶液,重复此过程3次,彻底洗干净脱脂奶粉溶液。
4、加入稀释后血清
摆好1.5mL EP管,加入450mL PBS,再加入50μL免疫后的小鼠血清进行稀释(10倍稀释);除第一排的板孔外,其余板孔用排枪加入100μL PBS。按浓度梯度3倍稀释,第一孔加150μL稀释后血清,用排枪吸出50μL加入下一排,反复吹吸混匀,重复吸出混匀操作,直至最后一排,混匀后将多余的50μL稀释血清弃去。将最上面的孔板贴上贴纸防止蒸干。
5、孵育一抗
将步骤4加入血清的孔板放入37℃烘箱孵育1h。
6、洗板
大力甩出并扣干步骤5孵育后孔板中液体,加入200μL 0.1% PBST溶液,重复此过程3次,彻底洗干净。
7、孵育二抗
用TBS缓冲液按照1:4000的比例稀释HRP酶标记的鼠二抗(Invitrogen,31430),稀释后用排枪加入100μL/孔,最上面的贴纸贴紧板孔防止蒸发,37℃孵育1h。
8、洗板
大力甩出并扣干步骤7孵育后孔板中液体,加入200μL 0.1% PBST溶液,重复此过程4次。
9、加入显色底物和终止显色
全程避光操作,用排枪向步骤8洗板后的孔板加入显色底物TMB 50μL/孔,显色10min后加入50μL终止液终止显色。
10、酶标仪检测
设定检测波长为450nm进行检测,每个板子作3次重复检测。保存原始数据及导出的Excel表格。
二、实验结果
用VZV gE-HPF纳米颗粒疫苗免疫小鼠第2周,第4周和第6周后所采血清中gE蛋白特异IgG滴度的检测结果如图7所示。将VZV gE-HPF纳米抗原免疫Balb/c小鼠后第2周(图7A),第4周(图7B)和第6周(图7C),从小鼠血清中分别检测对gE蛋白特异性IgG滴度,sidak多重比较检验显示实验组与对照组组间存在差异显著性,在显著性水平为0.0001的情况下,双尾概率水平小于0.0001。结果表明,利用本发明所述VZV gE-HPF纳米颗粒疫苗,在距离第一次免疫后的第2周,第4周和第6周后能激发小鼠产生高水平的针对VZV gE蛋白的IgG滴度。
实施例3gE蛋白片段对纳米颗粒疫苗构建的影响
一、实验方法
将Gvo和His纯化标签分别与水痘-带状疱疹病毒的全长gE蛋白(氨基酸序列如SEQID NO:11所示)、去除水痘-带状疱疹病毒的gE的跨膜域和胞内段后的gE蛋白可溶的胞外段(氨基酸序列如SEQ ID NO:1所示)和把gE蛋白可溶的胞外段的原始的信号肽更换成另一信号肽(SP)的截短后gE蛋白连接,构建得到3种融合蛋白:(1)gE(全长)-GVO(氨基酸序列如SEQ ID NO:12所示)、(2)gE(截短)-GVO(氨基酸序列如SEQ ID NO:3所示)和(3)SP-gE(截短)-GVO(氨基酸序列如SEQ ID NO:5所示)。
将上述gE(全长)-GVO、gE(截短)-GVO和SP-gE(截短)-GVO融合蛋白的编码基因分别均构建在pcDNA3.1-Intron-WPRE质粒载体上,构建方法与实施例1相同,得到重组表达载体质粒。按照实施例1的方法,将3种重组表达载体质粒瞬时分别转染293F细胞,5天后离心收获293F细胞上清,取80μL细胞上清,加入20μL SDS-PAGE loading buffer,95℃煮样10min,得3种待测样品。在12孔4~12% SDS-PAGE蛋白预制胶上样30μL,140V,50min,进行蛋白电泳。蛋白电泳结束后取出SDS-PAGE蛋白胶放入考马氏亮蓝染液,染色30min,后换上清水。
gE(截短)-GVO和SP-gE(截短)-GVO融合蛋白经上述蛋白胶电泳样品后,用金斯瑞的半干转快速转膜仪standard模式将蛋白胶上的蛋白转印到NC膜上,转膜后用TBS溶液配制5%脱脂奶粉摇床上封闭30min,封闭后用TBST溶液清洗3遍,每遍5min。用鼠抗gE的特异性抗体25℃孵育NC膜1h,一抗孵育完成后,用TBST溶液清洗3遍,每遍8min。用山羊抗鼠的二抗25℃孵育NC膜1h,二抗孵育完成后,用TBST溶液清洗3遍,每遍8min。
二、实验结果
如图8所示,泳道1是gE(全长)-GVO,其在293F真核表达系统中无法表达,因此需要对gE进行截短。泳道3是截短后的gE(截短)-GVO融合蛋白,截短后的gE-GVO能够在293F真核表达系统表达,但产量不够高。因此对gE的信号肽进行改进,泳道2是改进信号肽后的SP-gE(截短)-GVO融合蛋白,改进信号肽并且截短的SP-gE(截短)-GVO融合蛋白在293F真核表达系统中表达良好,其蛋白产量较高。
如图9所示,看到改进信号肽后的SP-gE(截短)-GVO融合蛋白产量比gE(截短)-GVO融合蛋白的高。
最后所应当说明的是,以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制,对于本领域的普通技术人员来说,在上述说明及思路的基础上还可以做出其它不同形式的变化或变动,这里无需也无法对所有的实施方式予以穷举。凡在本发明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。
Claims (10)
1.一种水痘-带状疱疹病毒纳米颗粒,其特征在于,包含由组分1和组分2,组分1和组分2利用自组装系统相连接;
其中,组分1为水痘—带状疱疹病毒糖蛋白E,其氨基酸序列如SEQ ID NO:1;
组分2为幽门螺旋杆菌铁蛋白,其氨基酸序列如SEQ ID NO:8。
2.根据权利要求1所述的水痘-带状疱疹病毒纳米颗粒,其特征在于,所述自组装系统为GvoTagOpti/SdCatcher系统,其中GvoTagOpti的氨基酸序列如SEQ ID NO:2所示;SdCatcher的氨基酸序列SEQ ID NO:7。
3.根据权利要求2所述的水痘-带状疱疹病毒纳米颗粒,其特征在于,GvoTagOpti设置于水痘—带状疱疹病毒糖蛋白E的C端。
4.根据权利要求2所述的水痘-带状疱疹病毒纳米颗粒,其特征在于,SdCatcher设置于幽门螺旋杆菌铁蛋白的N端。
5.根据权利要求3所述的水痘-带状疱疹病毒纳米颗粒,其特征在于,水痘—带状疱疹病毒糖蛋白E的N端还设置有分泌信号肽。
6.根据权利要求4所述的水痘-带状疱疹病毒纳米颗粒,其特征在于,所述分泌信号肽的氨基酸序列SEQ ID NO:4。
7.权利要求1到6任一所述水痘-带状疱疹病毒纳米颗粒在制备水痘-带状疱疹病毒疫苗中的应用。
8.一种水痘-带状疱疹病毒纳米颗粒的制备方法,其特征在于,氨基酸序列如SEQIDNO:5和6所示的重组蛋白,孵育后纯化即得。
9.权利要求8所述制备方法制备得到的水痘-带状疱疹病毒纳米颗粒。
10.权利要求9所述水痘-带状疱疹病毒纳米颗粒在制备水痘-带状疱疹病毒疫苗中的应用。
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