CN116444623B - 一种基于纳米颗粒支架的纳米疫苗及其制备方法与应用 - Google Patents
一种基于纳米颗粒支架的纳米疫苗及其制备方法与应用 Download PDFInfo
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Abstract
本发明提供了一种基于纳米颗粒支架的纳米疫苗及其制备方法与应用,属于生物技术领域。本发明提供一种纳米颗粒支架蛋白,所述纳米颗粒支架蛋白的氨基酸序列如SEQ ID No.1~SEQ ID No.3任一项所示。本发明以二氧四氢喋啶合酶多聚物蛋白(lumazinesynthase,LS)作为蛋白支架,利用SpyCatcher‑SpyTag/SnoopCatcher‑SnoopTag正交体系,对LS进行优化和改造,研发SpyCatcher‑LS‑SnoopTag纳米疫苗通用型平台。本发明的纳米颗粒支架蛋白可应用于双联疫苗的研发,为其他病种多价疫苗研发提供崭新的思路。
Description
技术领域
本发明属于生物技术领域,具体涉及一种基于纳米颗粒支架的纳米疫苗及其制备方法与应用。
背景技术
基于纳米颗粒研发的疫苗表现出诸多优良的理化性质,如作为抗原展示支架或免疫佐剂,改善抗原向抗原呈递细胞传递的特异性,在疾病的预防、控制和治疗中具有广泛的应用价值。许多天然的低聚物自组装纳米颗粒,如铁蛋白家族蛋白质、IMX313和C4bp的同源物已被开发用于疫苗支架平台的设计,它们在纳米疫苗的开发中显示出巨大的潜力。来自Aquifex aeolicusx的二氧四氢喋啶合酶多聚物蛋白(lumazine synthase,LS)是一种参与核黄素合成的酶,由60个五聚体蛋白质单体自然组装成的纳米颗粒,具有球形和空心二十面体的衣壳结构,外径和内径分别为15.4nm和9nm。LS的N-端和C-端均暴露在衣壳表面,具有三倍和五倍的对称性,端部与对称轴的接近可以稳定三聚体或五聚体抗原的呈现。鉴于LS较强的免疫刺激能力,且纳米尺寸可控、生物安全性和兼容性较好,已被开发用作纳米疫苗的载体颗粒。
抗原与LS纳米颗粒的连接可以通过基因融合或肽-蛋白质共价偶联体系实现,基因融合的形式限制抗原序列长度。肽-蛋白偶联体系允许抗原以独立形式表达或模块化抗原附着。SpyCatcher-SpyTag和SnoopCatcher-SnoopTag系统分别来源于化脓性链球菌黏连蛋白CnaB2结构域的和肺炎链球菌的黏附素RrgA。该正交体系可形成稳定且特异的酰胺键,赋予蛋白质特定的拓扑结构,使其兼具稳定性与生物功能。另外,该系统将蛋白质工程模块化,通过“即插即用”式组装,开发不同病种的疫苗,大大缩减了疫苗研发周期,减轻了疫苗研发的复杂程度,使该系统所携带的功能区既为整体服务,又发挥了特定靶点研究的作用。作为快速、可靠、不可逆的蛋白质偶联工具,SpyCatcher-SpyTag/SnoopCatcher-SnoopTag系统是在生物大分子研究领域中模块化纳米疫苗平台研发的理想选择。
SpyTag或SnoopTag融合外源性抗原产物与LS纳米颗粒混合,可通过形成异肽键使抗原均匀地装饰、附着在LS表面,获得病毒样颗粒。Nisreen等将MERS受体结合域蛋白RBD通过SpyCatcher-SpyTag连接到LS纳米颗粒的表面,RBD以一种有序的方式呈现多个拷贝,诱发有效、广泛的免疫反应。但是,对于LS偶联体系的研究仅限于单一蛋白的附着,即选择SpyCatcher-SpyTag或者SnoopCatcher-SnoopTag中的一种来用于抗体标记、示踪、药物递送、疫苗研发等。这就意味着复合物向免疫系统展示病毒的一个片段。相较于单体,双抗原的高效合成装配能够保护一种病原体的不同菌株,甚至保护两种不同的病原体。将两个携带单一抗原的病毒样颗粒混合在一起,这种方法增加了生产的复杂性,且双抗原携带的病毒样颗粒通过基因共轭产生,其抗原错误折叠的可能性更大,无法精确保证两种抗原的产量。
基孔肯雅病毒(CHIKV)基因组长度约为11.8kb,编码两个开放阅读框(ORF),即结构性和非结构性ORF。结构ORF编码衣壳(C)、包膜蛋白(E3-E2-6 K-E1),而非结构ORF编码四种非结构蛋白nsp1-4。CHIKV的外包膜表面包含80个由E1和E2糖蛋白的异二聚体形成的三聚体尖峰。E1和E2糖蛋白主要负责细胞膜融合和病毒进入宿主细胞,其中E2与细胞受体相互作用并附着于细胞,E1参与病毒与细胞膜的融合。E2蛋白包含三个不同的结构域:A(16-134aa)、B(173-231aa)和C(269-341aa),它们参与受体结合同时具有免疫原性。CHIKV E2蛋白是一种高度免疫原性的病毒蛋白,参与宿主-细胞相互作用并能够诱导针对CHIKV感染的强烈抗体反应,且E2蛋白已被认为是整个疾病过程中(从恢复期到康复期)抗CHIKV抗体反应的主要目标。寨卡(ZIKV)是一种有包膜的正链单链RNA基因组病毒,长约11kb,具有单个ORF,可被翻译成多蛋白,被病毒和宿主蛋白酶切割产生结构性(C、prM/M和E)和病毒颗粒的非结构蛋白(NS1、NS2A、NS2B、NS3、NS4A、NS4B和NS5)。E蛋白负责病毒与细胞受体结合、进入宿主细胞和膜融合。每个E蛋白单体具有三个结构域:中心结构域I(EDI)、包含融合肽的二聚化结构域(EDII)和受体结合结构域(EDIII)。EDIII具有类似于由反平行β折叠形成的免疫球蛋白结构域的桶状结构。该结构域具有与细胞受体结合的位点以及有效的抗原性和免疫原性表位,刺激免疫系统产生针对ZIKV的特异性抗体。全球持续性贸易和国际旅行增长,使得(CHIKV)和(ZIKA)病毒等其它蚊媒传染病存在输入性病例和输入后发生本地传播的风险。2019年,广州海关技术中心在入境人员中首次截获合并感染CHIKV与ZIKA的个案,这是我国首次检出合并感染CHIKV病毒和ZIKA病毒病例。外域输入引起当地流行的风险依然很高,且目前尚未批准有效的治疗方法和注册疫苗用于预防CHIKV和ZIKA感染。因此,有必要通过疫苗接种或主动免疫策略来预防CHIKV和ZIKA。
发明内容
针对上述问题,本发明的目的在于提供一种基于纳米颗粒支架的纳米疫苗及其制备方法与应用,本发明提供一种纳米颗粒支架蛋白,并通过纳米颗粒支架蛋白自组装策略,将其应用于CHIKV-ZIKA病毒的双联疫苗研发,为其他病种多价疫苗研发提供崭新的思路。
为实现上述目的,本发明采取的技术方案为:一种纳米颗粒支架蛋白,所述纳米颗粒支架蛋白的氨基酸序列如SEQ ID No.1~SEQ ID No.3任一项所示。
本发明以二氧四氢喋啶合酶多聚物蛋白(lumazine synthase,LS)作为蛋白支架,利用SpyCatcher-SpyTag/SnoopCatcher-SnoopTag正交体系,对LS进行优化和改造,研发SpyCatcher-LS-SnoopTag纳米疫苗通用型平台。本发明的SpyCatcher-LS-SnoopTag纳米疫苗通用型平台具有高度的生物相容性,可以均匀地组装,并可以有效地定制以适应任何抗原,提高附着物的生产效率。本发明的SpyCatcher-LS-SnoopTag纳米疫苗通用型平台以双标签复合物形式使抗原附着,这使得平台上的两种抗原可以均匀地装饰,以一种有序的方式呈现多个蛋白拷贝可以诱发有效和广泛的免疫反应。
本发明还提供所述的纳米颗粒支架蛋白的制备方法,包括以下步骤:
(1)获取编码SEQ ID No.1~SEQ ID No.3任一项所示的氨基酸序列的基因序列;
(2)将步骤(1)的基因序列插入到pCold-I表达载体中,得重组质粒;
(3)将重组质粒进行转化、诱导表达和纯化,得所述纳米颗粒支架蛋白。
作为本发明所述纳米颗粒支架蛋白的制备方法的优选实施方式,步骤(2)所述基因序列插入pCold-I表达载体的Nde I和Xba I酶切位点之间。
作为本发明所述纳米颗粒支架蛋白的制备方法的优选实施方式,所述pCold-I表达载体的N端具备His6标签修饰。本发明的纳米颗粒支架蛋白的制备方法制备得到的纳米颗粒支架蛋白纯度>90%。
本发明还提供一种氨基酸序列,所述序列如SEQ ID No.1~SEQ ID No.3任一项所示。
本发明还提供一种纳米疫苗,所述纳米疫苗包括所述的纳米颗粒支架蛋白。
作为本发明所述纳米疫苗的优选实施方式,所述纳米疫苗包括单联疫苗和双联疫苗。
本发明还提供所述的纳米疫苗的制备方法,将所述的纳米颗粒支架蛋白与病毒抗原进行共轭偶联得到。
本发明还提供所述纳米颗粒支架蛋白在制备多价疫苗中的应用。
本发明的有益效果为:本发明基于LS提供一种纳米颗粒支架蛋白,(1)该纳米颗粒支架蛋白以二氧四氢喋啶合酶多聚物蛋白(lumazine synthase,LS)作为蛋白支架,由于LS是含有六十聚体近似对称球形的纳米颗粒,表面展示蛋白要优于IMX313、铁蛋白等传统纳米颗粒支架,且LS的N端与C端均暴露在颗粒表面,易于抗原的展示;(2)本发明采用的LS来源于超嗜热菌,其蛋白质在高温下仍很稳定,基于LS研发的疫苗可显著提升抗原的热稳定性,减少对冷链运输的依赖,进一步降低疫苗储存和运输的成本;(3)采用本发明的纳米颗粒支架蛋白制备得到的疫苗在大肠杆菌中以可扩展的水平生产支架,降低成本、减少开发时间;(4)本发明的纳米颗粒支架蛋白利用SpyCatcher-SpyTag/SnoopCatcher-SnoopTag正交体系,可以实现蛋白质空间结构的精准控制,在操纵抗原密度和方向方面具有绝对优势,为研究多价效应的潜在机制及其优化策略提供了良好的平台。
附图说明
图1为基于LS纳米颗粒蛋白重组载体示意图;
图2为基于原核表达的LS纳米颗粒蛋白SDS-PAGE图和负染透射电镜图;
图3为纳米颗粒支架LS-SUMO热稳定性DSC结果图;
图4为纳米颗粒支架LS-SUMO热稳定性SDS-PAGE结果图;
图5为基于纳米颗粒支架LS-SUMO设计的双联纳米疫苗示意图;
图6为抗原单体、LS-SUMO、LS-SUMO偶联纳米颗粒和未偶联的空载纳米颗粒SDS-PAGE图;
图7为LS-SUMO偶联纳米颗粒的负染透射电镜图和颗粒粒径分布图;
图8为抗原单体、LS-SUMO、LS-SUMO偶联纳米颗粒与抗体亲和检测ELSIA结果图;
图9为BALB/c小鼠免疫方案示意图;
图10为LS-SUMO纳米颗粒疫苗总抗原特异性IgG滴度结果图(AUC=吸光度vs.对数稀释);
图11为LS-SUMO纳米颗粒疫苗IgG分型分布图(AUC=吸光度vs.对数稀释);
图12为免疫后小鼠淋巴细胞CD4+和CD8+细胞流式结果图;
图13为免疫后小鼠淋巴细胞中Th1和Th2介导的细胞因子定量结果图;
图14为免疫后小鼠血清活病毒中和滴度结果图。
具体实施方式
为了更加简洁明了的展示本发明的技术方案、目的和优点,下面结合具体实施例和附图详细说明本发明的技术方案。
实施例1基于SpyCatcher-LS-SnoopCatcher新型自组装纳米颗粒蛋白支架的构建
1、重组质粒构建
按以下排序合成基因序列:
A:ΔN端N1-SpyCatcher(GenBank:AFD50637.1),GGSGS linker,LS((GenBank:WP_010880027.1;C37A和D102Q位氨基酸替换),L9 linker,SnoopCatcher(GenBank:KU500646),N端不添加SUMO标签,命名为LS-ΔSUMO。
B:ΔN端N1-SpyCatcher(GenBank:AFD50637.1),GGSGS linker,LS((GenBank:WP_010880027.1;C37A和D102Q位氨基酸替换),L9 linker,SnoopCatcher(GenBank:KU500646),N端添加SUMO标签修饰,命名为LS-SUMO。
C:ΔN端N1-SpyCatcher(GenBank:AFD50637.1),L9 linker,GGSGS linker,LS(GenBank:WP_010880027.1),N段添加SUMO标签,命名为LL-SUMO。
将上述基因序列克隆至N端His6标签修饰的pCold-I载体,同时在上下游分别设计含Nde I和Xba I酶切位点的引物,构建重组质粒,委托通用生物(安徽)股份有限公司合成该质粒。重组载体示意图如图1所示,其中A为LS-ΔSUMO;B为LS-SUMO;C为LL-SUMO。
2、自组装纳米颗粒支架的表达及纯化
将上述三种重组质粒转化至表达菌株BL21-CodonPlus(DE3)-RIRL,获得重组蛋白表达株。取平板划线、过夜培养的表达株单菌落于10mL氨苄青霉素抗性的LB培养基中,250rpm、37℃下培养5h。按1:100转接到至1L含氨苄青霉素的LB培养基中,250rpm 37℃下培养至菌液OD600=0.6-0.8,加入终浓度0.5mM IPTG,15℃下诱导培养15h。诱导表达后的菌液4000rpm离心30min,弃上清,Lysis Buffer(50mM Tris-HCl,2M NaCl,pH8.0)重悬菌体,冰浴下超声破碎20min(超声条件,300W,超声3s,停止5s),4℃、18000g离心25min。可溶性重组蛋白使用Ni柱亲和层析纯化,经终浓度500mM咪唑洗脱目的蛋白后,使用30kD超滤管4℃、3000rpm下浓缩样品。利用AKTA蛋白纯化系统对样品进行二次洗脱、纯化,将ElutionBuffer(50mM Tris-HCl,200mM NaCl,60mM咪唑,pH8.0)洗脱得到的蛋白峰进行收集、浓缩。通过SDS-PAGE检测,预期蛋白如图2所示,A为LS-ΔSUMO;B为LS-SUMO;C为LL-SUMO。
3、自组装纳米颗粒支架形态学特征
将上述制备的LS-ΔSUMO、LS-SUMO和LL-SUMO纳米颗粒进行负染处理,观察纳米颗粒的大小和形态。取10uL蛋白质溶液滴于300目碳膜铜网,吸附1min;用滤纸从碳膜铜网边缘轻轻吸去多余的溶液,滴加2%磷钨酸孵育染色2min,洗2次,染色1min;用滤纸吸去多余液体,室温晾干;晾干后将样品置于120kV透射电镜中观察,选择样品颗粒分散性和均一性较好的区域进行数据收集。
如图2所示,A为LS-ΔSUMO;B为LS-SUMO;C为LL-SUMO。经2%磷钨酸溶液负染,通过120kV透射电镜观察,LS-SUMO颗粒分散性、均一性良好,虽存在少量细小的小蛋白颗粒,但大部分颗粒近似圆形,直径约为10-15nm,与预期大小基本相符。LS-ΔSUMO和LL-SUMO颗粒凝聚成团,分布不均匀,轮廓不清,结构具有不同程度的缺损或消失。
4、自组装纳米颗粒支架热稳定性研究
采用MicroCal PEAQ-DSC(Malvern)通过差示扫描量热法(DSC)测定熔融温度(Tm)。测量蛋白溶液和参考缓冲液的体积为400μL。仪器参数设置:起始温度10℃,终止温度100℃,升温速率1℃/min。使用MicroCal PEAQ-DSC软件记录和分析样品的热稳定性。如图3所示,LS-SUMO纳米颗粒蛋白的Tm值为86.78℃。为了在较长时间内监测纳米颗粒稳定性,将样品分别置于4℃、25℃、37℃、60℃条件下孵育35天,13,000rpm/min离心处理后,取上清进行SDS-PAGE检测。LS-SUMO纳米颗粒蛋白经不同温度处理后可溶性分析如图4所示。
实施例2基于SpyCatcher-LS-SnoopCatcher自组装支架蛋白制备联合纳米颗粒疫苗
1、CHIKV E2-SpyTag抗原制备
参照GenBank:HQ846359.1序列,合成DNA编码序列,在C端添加SpyTag(GeneBank:WP_129284416.1)和His8标签,添加标签后的融合蛋白命名为CHIKV E2-SpyTag,将其克隆至表达载体pFastBac1中,得到pFastBac1-CHIKV-E2-SpyTag质粒,转化至DH10Bac感受态细胞中,经过蓝白斑筛选出阳性克隆。提取阳性质粒,通过Sinofection试剂转染入密度约为1×106的Sf9细胞中,96h后吹混匀细胞,离心收集细胞上清。以MOI=0.1将P1代重组杆状病毒颗粒接种Sf9细胞中进行病毒扩增,连续扩增两代后得到P3代重组病毒。将P3代病毒以MOI=0.5剂量接种摇瓶培养的悬浮High-five细胞,接种后96h通过低速离心收集上清。利用不同梯度咪唑洗脱目的蛋白,SDS-PAGE检测。
2、ZIKAEDIII-SnoopTag抗原制备
参照GenBank:AMK79468.2序列,合成DNA编码序列,在N端添加SnoopTag(GenBank:KU35687)和和His6标签,添加标签后的融合蛋白命名为ZIKA EDIII-SnoopTag,将其克隆至表达载体pCold-I中,得到pColdI-ZIKA-EDIII-SnoopTag质粒,转化至大肠杆菌TOP10。挑选阳性克隆进行表达,ZIKAEDIII-SnoopTag以包涵体形式表达。收集包涵体并用包涵体溶解液(20mmol/LTris-HCl pH8.0,0.5mol/LNaCl,6mol/L尿素,0.2mmol/LDTT,2%TritonX-100)重悬,室温摇床30-60min以充分溶解,4℃12,000rpm离心15min,弃沉淀取上清。取上述包涵体变性溶解液,利用TALON Metal affinity Resin(含CO2+)纯化纳米抗体,取CO2+柱沉淀树脂,灭菌ddH2O清洗2-3次,含10mM咪唑缓冲液平衡CO2+柱,将制备的超声裂解物加入CO2+柱中,震荡混匀,4℃孵育1h,2倍柱体积的洗脱液液(含20mM咪唑)洗去杂蛋白,最后用等体积的洗脱液(含250mM咪唑)洗脱目的蛋白,并收集洗脱液。取3.5kDa透析袋,将上述纯化的包涵体依次分别在2M尿素溶液和0M尿素溶液中透析8-12h,最后在PBS(pH 7.4)中透析8-12h。将复性后的蛋白进行SDS-PAGE检测。
3、基于SpyCatcher-LS-SnoopCatcher制备联合纳米颗粒疫苗
根据SpyCatcher-LS-SnoopCatcher形态特征,选择LS-SUMO作为支架应用于CHIKV-ZIKA病毒双联疫苗的研发。偶联示意图如图5所示。根据预期目的骨架蛋白,将纯化的LS-SUMO纳米颗粒支架分别与CHIKV E2-SpyTag、ZIKA EDⅢ-SnoopTag抗原以1:2过量摩尔比孵育,制备LS-CHIKV和LS-ZIKA单联疫苗。CHIKV-ZIKA病毒双联疫苗则LS-SUMO和CHIKVE2-SpyTag、ZIKA EDⅢ-SnoopTag按照LS-SUMO:CHIKV E2-SpyTag、ZIKAEDⅢ-SnoopTag=1:2:3过量摩尔比孵育。所有偶联体系均在TBS缓冲液(pH7.4,Leagene)中于4℃下静置连接12h。加入5X SDS-PAGE上样缓冲液并在80℃加热10分钟终止反应。偶联结果如图6所示。
实施例3联合纳米颗粒疫苗表征
1、负染电镜
将偶联后的经超滤管截留后,采用负染电镜法对样品进行处理,形态和大小如图7所示。负染电镜显示三种LS-SUMO共轭纳米疫苗呈现出均匀单分散的球形纳米颗粒,平均直径分别为14.56±2.30nm(LS-CHIKV)、11.29±4.30nm(LS-ZIKA)和25.31±5.63nm(LS-CIKV/ZIKA)。
2、使用NanoSightNS300(Malvern Instruments,Malvern,UK)测定联合纳米颗粒疫苗的大小和浓度分布。纯化的蛋白质在PBS中以1:10的比例稀释,用注射泵以恒定的流速注射到跟踪室。在60秒内记录三个独立的重复,摄像级别设为10,检测阈值设为5。纳米颗粒浓度和大小分布如图7所示。利用NTA分析颗粒密度和粒径分布,LS-CHIKV、LS-ZIKA和LS-CHIKV/ZIKA分别在69nm、56nm和121nm处显示出最高峰值。
3、ELISA
利用纯化的Nb-3C5/mAb-8E6抗体分别通过ELISA检测与CHIKV E2/ZIKA EDIII的结合能力。将抗原单体及LS-SUMO偶联产物在碳酸盐缓冲液(CBS,pH9.6)下稀释至1μg/ml,4℃下包被过夜。3%的BSA 37℃下孵育1h阻断非特异性位点。Nb-3C5从10μg/ml开始1:10连续稀释至8个梯度。鼠抗mAb-8E6从100μg/ml开始1:2连续稀释至8个梯度。用HRP结合的山羊抗人IgG(1:5000)或HRP结合的山羊抗小鼠IgG(1:5000稀释)检测目标抗原。洗涤后,100μLTMB避光显色,100μL 1N HCl停止反应。在450nm处测量吸光度。LS-SUMO空白支架作为阴性对照。ELISA检测结果如图8所示。ELISA分别比较分析单体和LS-SUMO共轭纳米疫苗结合的抗体的反应性,其识别的抗体的亲和力均高于单体形式。
实施例4CHIKV-ZIKA双联纳米颗粒疫苗诱导BALB/c小鼠产生免疫应答
1、BALB/c小鼠免疫方案
6周雌性小鼠给予肌肉注射,ZIKAEDIII单体(5μg,n=8),CHIKV E2单体(5μg,n=8),LS-CHIKV(含有5μg CHIKV-SpyTag,n=8),LS-ZIKA(含有5μgZIKA-SnoopTag,n=8),LS-CHIKV/ZIKA(含有5μg CHIKV-SpyTag和5μgZIKA-SnoopTag,n=8)。注射PBS(100μL,n=8)或LS(5μg,n=8)作为阴性对照。所有组都接受了三次免疫(第0、14和21天),并在第0、7、14、28、35、42和56天收集血清样本。免疫方案如图9所示。
2、CHIKV-ZIKA双联纳米颗粒疫苗诱导BALB/c小鼠产生体液免疫应答
在96板上包被100ng抗原单体(不含SpyTag或SnoopTag)4℃过夜,用含0.05%Tween-20的PBS(PBST)洗孔三次。加入200μLBSA(5%),在37℃下孵育2h。用PBST洗三次后,在不同的孔中加入100μl 10倍连续稀释(从1:10开始)的血清样品,在37℃下孵育1h。用PBST洗5次后,用抗小鼠IgG(1:5000稀释)、同型特异性抗小鼠IgG、IgG1、IgG2a、IgG2b和IgG3(1:2000稀释)在37℃下孵育HRP结合的二级抗体1h。用PBST洗板后,加入TMB底物,用1MHCl停止反应。使用吸光度与对数稀释因子的曲线下面积(AUC)报告血清滴度。免疫后小鼠血清总IgG抗体滴度结果如图10所示,IgG分型结果如图11所示。
对于抗CHIKV IgG反应,LS-CHIKV或LS-CHIKV/ZIKA组以及CHIKV单体组诱导E2免疫原特异性总IgG反应,第42天AUC值为7.16-8.50。在所有抗原接种组中,总IgG应答随着时间的延长而明显增加。LS-CHIKV组在每个时间点诱导的抗体反应显著高于单体CHIKV E2组,平均AUC值平均增加1.11-2.10倍。对于抗ZIKAIgG反应,抗原在第二次接种后,显示出对EDIII抗原的总IgG结合抗体反应。LS-ZIKA和LS-CHIKV/ZIKA组IgG抗体应答水平表现出相同的趋势。此外,与单体ZIKA EDIII组相比,LS-ZIKA和LS-CHIKV/ZIKA组的AUC值分别增加了1.73倍和1.76倍。CHIKV E2特异性IgG应答主要由IgG1亚类组成,并显示出与总IgG抗体水平相同的趋势。在ZIKA EDIII特异性IgG亚类反应中,IgG1是主要的亚类,而血清中IgG2a和IgG2b水平较低,IgG3是检测水平最低的亚类。LS-SUMO共轭纳米疫苗免疫的血清IgG2a/IgG1比率均低于1,表明LS-SUMO共轭纳米疫苗更倾向诱导Th2免疫应答。
3、CHIKV-ZIKA双联纳米颗粒疫苗诱导BALB/c小鼠产生细胞免疫应答
分离小鼠脾脏,单细胞悬浮液通过70μm滤网过滤以去除碎片,以2000rpm离心5min,并用红细胞裂解缓冲液处理以去除红细胞。小鼠富集的T细胞样本用CD45、CD3、CD4和CD8抗体进行染色,流式细胞仪分选T细胞,并使用FlowJo软件分析CD4和CD8数据,结果如图12所示。通过R&D Systems ELISA试剂盒检测细胞因子,分析细IL-2、IL-4、IL-6、IL-10、TNF-α和IFN-γ表达水平,PBS作为阴性对照,结果如图13所示。
与PBS对照组相比,CD3+CD4+T细胞的百分比显著升高,但CD3+CD8+T细胞组之间没有差异。ELISA法测定上清中细胞因子浓度。对于CHIKV抗原产生的应答,LS-CHIKV/ZIKA组中的IL-2、IL-4、IL-6和IFN-γ表达水平明显高于单体组。对于ZIKAEDⅢ抗原产生的应答,LS-CHIKV/ZIKA组中IL-2、IL-4、IL-6、IL-10、TNF-α和IFN-γ的表达水平显著高于单体ZIKA组。与PBS组相比,所有抗原免疫组均能分泌Th1和Th2细胞因子。
实施例5CHIKV-ZIKA双联纳米颗粒疫苗诱导BALB/c小鼠产生病毒中和抗体滴度
具体实验方法如下:2.0×105/个Vero细胞铺板。免疫后血清样品用DMEM培养基1:5稀释,然后对所有样品进行2倍的连续稀释,最终达到1:5至1:2560的血清稀释度。每个样品都包括一个无血清的阴性对照。稀释的血清随后与相同体积的病毒混合,生成含有约100个斑块形成单位(PFU)/100μL病毒的混合物,形成最终1:10至1:5020血清稀释度。在37℃下孵育1h后,将血清-病毒混合物加入到含有80-90%汇合单层Vero细胞的12孔板中。37℃病毒吸附2h后,在病毒维持培养基(含2%FBS和1%青霉素-链霉素的DMEM)中用1%甲基纤维素覆盖细胞。感染后3-4天,用4%多聚甲醛固定细胞,用0.4%亚甲蓝染色以观察斑块。终点滴度被定义为空斑减少50%(NT50)为最高血清稀释度。
结果如图14所示,单体E2、LS-CHIKV和LS-CHIKV/ZIKA组中小鼠血清中和CHIKV毒株(GD134)的几何平均NT50滴度分别为106、490和526。LS-CHIKV/ZIKA诱导的中和抗体水平比CHIKV E2单体组高4.96倍。单体EDIII、LS-ZIKA和LS-CHIKV/ZIKA组中小鼠血清中和ZIKA毒株(GD01)的几何平均NT50滴度分别为63、195和408,比EDIII单体免疫小鼠高3.09-6.48倍。PBS组和LS-SUMO组组中小鼠血清没有观察对CHIKV和ZIKA病毒中和能力。
最后应当说明的是,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,尽管参照较佳实施例对本发明作了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的实质和范围。
Claims (8)
1.一种纳米颗粒支架蛋白,其特征在于,所述纳米颗粒支架蛋白的氨基酸序列如SEQID No.2所示。
2.根据权利要求1所述的纳米颗粒支架蛋白的制备方法,其特征在于,包括以下步骤:
(1)获取编码SEQ ID No.2所示的氨基酸序列的基因序列;
(2)将步骤(1)的基因序列插入到pCold-I表达载体中,得重组质粒;
(3)将重组质粒进行转化、诱导表达和纯化,得所述纳米颗粒支架蛋白。
3.根据权利要求2所述的纳米颗粒支架蛋白的制备方法,其特征在于,步骤(2)所述基因序列插入pCold-I表达载体的Nde I和Xba I酶切位点之间。
4.根据权利要求2所述的纳米颗粒支架蛋白的制备方法,其特征在于,所述pCold-I表达载体的N端具备His6标签修饰。
5.一种纳米疫苗,其特征在于,所述纳米疫苗包括权利要求1所述的纳米颗粒支架蛋白。
6.根据权利要求5所述的纳米疫苗,其特征在于,所述纳米疫苗包括单联疫苗和双联疫苗。
7.根据权利要求5~6任一项所述的纳米疫苗的制备方法,其特征在于,将权利要求1所述的纳米颗粒支架蛋白与病毒抗原进行共轭偶联得到。
8.权利要求1所述纳米颗粒支架蛋白在制备多价疫苗中的应用。
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