CN116334139A - 基于猪圆环病毒2型病毒样颗粒的通用型模块化纳米抗原展示载体及其应用 - Google Patents
基于猪圆环病毒2型病毒样颗粒的通用型模块化纳米抗原展示载体及其应用 Download PDFInfo
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Abstract
本发明提供一种基于猪圆环病毒2型病毒样颗粒的通用型模块化纳米抗原展示载体,所述纳米抗原展示载体由与SpyCatcher003融合表达的猪圆环病毒2型病毒样颗粒自组装而成;SpyCatcher003与猪圆环病毒2型病毒样颗粒的融合蛋白的氨基酸序列如SEQ ID NO:1所示。本发明制备的纳米抗原展示载体,能够快速、高效的展示包括保护性多肽、单体蛋白、二聚体蛋白和三聚体蛋白在内的多种不同猪源病毒亚单位疫苗候选抗原,能够诱导产生高于相应单体抗原5‑8倍的抗体水平,不但有效降低了亚单位疫苗的研发成本,而且为多联疫苗的研制提供了技术支撑。
Description
技术领域
本发明涉及一种基于猪圆环病毒2型病毒样颗粒的通用型模块化纳米抗原展示载体及其应用,属于纳米颗粒载体技术领域。
背景技术
以纳米颗粒为载体多价展示抗原是一种增强亚单位疫苗抗原免疫原性进而诱导高水平中和抗体的有效策略。纳米粒子能够以高密度显示偶联的的抗原阵列,并提供与BCR结合的多种分子场景。成熟的幼稚型B细胞通过其表面分布的B细胞受体识别外界抗原,随后BCR发生簇集,这是机体产生保护性抗体及免疫记忆的关键步骤。多价抗原可诱导BCR形成微簇体,在没有抗原呈递细胞或辅助性T细胞帮助的情况下,有序的抗原阵列就可以引发B细胞的活化应答。例如,水疱性口炎病毒G糖蛋白排列越重复和严格,其免疫原性就越强,越能诱导高水平抗体。
病毒样颗粒(Virus like particles,VLPs)可能是最精确定义可以通过自组装形成的纳米级蛋白质笼结构。具有近似球形结构的正二十面体的VLPs在生物医学和纳米生物技术的发展中表现出显著效用,如药物输送、成像、生物催化剂,特别是在纳米疫苗开发中。VLPs与真正的病毒粒子具有抗原相似性,可作为有效的独立疫苗。更重要的是,VLPs可作为分子支架,显著增加异源抗原的免疫原性。外源抗原可插入病毒的结构蛋白然后形成嵌合VLPs,使得外源抗原展示在所得的嵌合VLPs表面上。传统方法是通过基因融合实现将异源表位插入病毒衣壳蛋白中,然后自组装形成嵌合VLPs或通过化学缀合的方式将异源表位偶联到预组装的VLPs上。小肽(通常是单个表位)的融合后自组装成功率很高。采用融合表达大尺寸抗原蛋白再组装形成纳米颗粒的策略常会由于复杂抗原折叠效率低而陷入困境。化学交联的方法已经广泛用于将目的抗原偶联到VLPs上,但由于化学偶联剂以及氨基酸侧链偶联位点的不确定性,通常会破坏或遮蔽目的抗原的中和表位。因此,目的抗原和VLPs展示平台先单独表达和纯化,随后再组合模块化设计的策略更为合理。
猪圆环病毒2型(Porcine circovirus type 2,PCV2)是最小的哺乳动物病毒之一,会给养猪业带来巨大的经济损失。PCV2是猪最易感染的病原体之一,导致淋巴细胞凋亡和耗竭,造成免疫抑制,增加其他致病微生物的感染风险。PCV2唯一的衣壳(Capsid,Cap)蛋白能够自组装形成VLPs,是PCV2的理想候选疫苗之一。Cap蛋白的C末端延伸到VLPs的表面形成突起,最后四个氨基酸构成构象中和表位,表明C末端容易被免疫系统识别,提示C末端是一个潜在的外源抗原插入位点。本研究团队前期的研究表明Cap蛋白的C末端至少可以容受81个氨基酸(3个拷贝的甲型流感病毒M2e表位)的插入而不影响嵌合VLPs的形成,同时能够诱导产生高水平的PCV2中和抗体和M2e特异性抗体。并且由于载体效应,体内预先存在的PCV2抗体促进了抗原递呈过程,显著提高嵌合VLPs的诱导M2e抗体。猪体内广泛存在PCV2抗体,以PCV2 VLPs作为纳米抗原载体可以利用载体效应进一步提高免疫效果。因此基于PCV2VLPs开发一种模块化、坚固的、可扩展的纳米抗原展示载体是非常有前景的。
SpyTag/SpyCatcher系统来源于化脓性链球菌纤粘蛋白的FbaB结构域,该系统分为两个部分:SpyCatcher(113aa)和SpyTag(13aa)。SpyCatcher上的赖氨酸(Lys)和SpyTag上的天冬氨酸(Asp)在溶液中相互作用形成高度稳定的酰胺键。该过程在不同的pH值、温度和缓冲条件下均可以在几分钟内完成,反应过程不可逆。SpyCatcher和SpyTag分子量较小,将其分别与纳米颗粒平台或抗原融合表达后不影响酰胺键的形成。SpyTag003/SpyCatcher003是这一系统的最新版本,解决了SpyTag/SpyCatcher的中等反应速率限制低浓度蛋白质的时间分辨率和标记效率问题。经改造后反应速率接近扩散极限,有助于共价修饰在细胞和生物体中的更多应用。目前已有多种纳米颗粒与该系统相结合构建出模块化的纳米颗粒展示载体,例如铁蛋白纳米颗粒、AP205 VLPs、mi3正二十面体纳米颗粒等。
发明内容
针对现有技术的不足,本发明的目的是提供一种基于猪圆环病毒2型病毒样颗粒的通用型模块化纳米抗原展示载体,其中SpyCatcher003与猪圆环病毒2型病毒样颗粒融合蛋白的氨基酸序列如SEQ ID NO:1所示。
本发明还有一个目的在于,评估这种基于猪圆环病毒2型病毒样颗粒的纳米抗原展示载体展示猪源病毒保护性多肽、单体蛋白、二聚体蛋白和三聚体蛋白的能力。
在本发明的应用方面,测试了猪繁殖与呼吸综合征病毒GP5与GP3蛋白上的B细胞表位、猪流行性腹泻病毒COE蛋白、猪瘟病毒E2蛋白二聚体、猪流感病毒HA蛋白三聚体等多种抗原,与未连接的相应单体抗原相比,由猪圆环病毒2型病毒样颗粒纳米抗原展示载体展示的抗原诱导产生了高于前者5-8倍的抗体应答水平。
为了实现上述目的,本发明所采用的技术方案是:
基于猪圆环病毒2型病毒样颗粒的通用型模块化纳米抗原展示载体,所述纳米抗原展示载体由与SpyCatcher003融合表达的猪圆环病毒2型病毒样颗粒自组装而成;SpyCatcher003与猪圆环病毒2型病毒样颗粒的融合蛋白的氨基酸序列如SEQ ID NO:1所示。
SpyCatcher003融合表达在猪圆环病毒2型病毒样颗粒的羧基端。
所述猪圆环病毒2型病毒样颗粒为PCV2b亚型。
将SpyCatcher003序列插入到PCV2 Cap蛋白的羧基末端,二者以GSGSGSGSGSGS序列相连,经大肠杆菌偏好性密码子优化,得到氨基酸序列SEQ ID NO:1。
利用所述的纳米抗原展示载体展示的装载抗原,所述装载抗原为:与SpyTag003标签融合表达的猪源病毒保护性多肽,或与SpyTag003标签融合表达的猪源病毒保护性单体蛋白,或与SpyTag003标签融合表达的猪源病毒保护性二聚体蛋白,或与SpyTag003标签融合表达的猪源病毒保护性三聚体蛋白。
与SpyTag003标签融合表达的猪源病毒保护性多肽的氨基酸序列如SEQ ID NO:2所示;
与SpyTag003标签融合表达的猪源病毒保护性单体蛋白的氨基酸序列如SEQ IDNO:3所示;
与SpyTag003标签融合表达的猪源病毒保护性二聚体蛋白的氨基酸序列如SEQ IDNO:4所示;
与SpyTag003标签融合表达的猪源病毒保护性三聚体蛋白的氨基酸序列如SEQ IDNO:5所示。
所述装载抗原与纳米抗原展示载体是通过SpyCatcher003与SpyTag003的相互作用而共价连接。
所述的纳米抗原展示载体在疫苗制备中的应用。
本发明有益效果:
本发明通过基因工程技术将SpyCatcher003(SpyTag003/SpyCatcher003系统)插入到猪圆环病毒2型病毒样颗粒(PCV2 Cap)蛋白的羧基末端,在大肠杆菌中表达并自组装形成Cap-SpyCatcher003纳米颗粒。本发明制备的基于猪圆环病毒2型病毒样颗粒的通用型模块化纳米抗原展示载体,能够快速、高效的展示包括保护性多肽(猪繁殖与呼吸综合征病毒GP5与GP3蛋白上的B细胞表位)、单体蛋白(猪流行性腹泻病毒COE蛋白)、二聚体蛋白(猪瘟病毒E2蛋白)和三聚体蛋白(流感病毒HA蛋白)在内的多种不同猪源病毒亚单位疫苗候选抗原。
本发明纳米抗原展示载体能显著提高机体抗体应答水平,通过装载抗原(与SpyTag003标签融合表达的猪源病毒保护性多肽、单体蛋白、二聚体蛋白和三聚体蛋白)与Cap-SpyCatcher003纳米颗粒的高效偶联,验证了纳米抗原展示载体的装配功能,其能够诱导产生高于相应单体抗原5-8倍的抗体水平。不但有效降低了亚单位疫苗的研发成本,而且为多联疫苗的研制提供了技术支撑。
本发明应用猪圆环病毒2型病毒样颗粒与SpyTag003/SpyCatcher003系统构建的通用型模块化纳米抗原展示载体是对现有猪用亚单位疫苗研制技术的改进,成本低廉,制法简便,装配迅速,效果显著,具有良好的科学和应用价值。
附图说明
图1是猪圆环病毒2型病毒样颗粒通用型模块化纳米抗原展示载体结构示意图;
图2是Cap-SpyCatcher003纯化后的SDS-PAGE图;
其中,M是蛋白质标记,泳道1是纯化后的Cap-SpyCatcher003纳米颗粒;
图3是Cap-SpyCatcher003与SpyTag003标签装载抗原(GP53)在不同摩尔比下偶联的SDS-PAGE图;
图4是Cap-SpyCatcher003与SpyTag003标签装载抗原(COE)在不同摩尔比下偶联的SDS-PAGE图;
图5是Cap-SpyCatcher003与SpyTag003标签装载抗原(E2)在不同摩尔比下偶联的SDS-PAGE图;
图6是Cap-SpyCatcher003与SpyTag003标签装载抗原(HA)在不同摩尔比下偶联的SDS-PAGE图;
图7是VLP-抗原偶联物(Cap-Cat-GP53)免疫小鼠后,第21和42天采集的小鼠血清效价图;
图8是VLP-抗原偶联物(Cap-Cat-COE)免疫小鼠后,第21和42天采集的小鼠血清效价图;
图9是VLP-抗原偶联物(Cap-Cat-E2)免疫小鼠后,第21和42天采集的小鼠血清效价图;
图10是VLP-抗原偶联物(Cap-Cat-HA)免疫小鼠后,第21和42天采集的小鼠血清效价图。
具体实施方式
为了使本领域的技术人员更好地理解本发明的技术方案,下面结合具体实施例对本发明的优选实施方案进行描述。应理解,以下实施例仅用于说明本发明而非用于限定本发明的范围。
下述实施例中所述实验方法,如无特殊说明,均为常规方法;所述试剂如无特殊说明,均从常规商业途径获得,或以常规方法制备。
实施例1:Cap-SpyCatcher003纳米抗原展示载体的构建、表达及纯化
1.Cap-SpyCatcher003纳米抗原展示载体的构建
本发明利用猪圆环病毒2型病毒样颗粒和SpyTag003/SpyCatcher003系统,将SpyCatcher003展示在猪圆环病毒2型Cap蛋白的羧基末端,然后自组装形成嵌合VLPs,进而开发一种基于猪圆环病毒2型病毒样颗粒的通用型模块化纳米抗原展示载体。
具体的,将SpyCatcher003序列(第4-116位氨基酸)插入到PCV2 Cap蛋白(PCV2b亚型,GenBank:ABM54440.1,第15-230位氨基酸)的羧基末端,二者间以GSGSGSGSGSGS序列相连,以增加蛋白质链的灵活性,得到PCV2 Cap VLPs-SpyCatcher003纳米载体,简写为Cap-SpyCatcher003。整个序列经过大肠杆菌偏好性密码子优化,由生工生物工程(上海)股份有限公司合成,并利用NcoI和HindIII酶切位点将合成好的基因插入到pET28a载体中,得到pET28a-Cap-SpyCatcher003质粒。
其中,SpyCatcher003与猪圆环病毒2型病毒样颗粒的融合蛋白Cap-SpyCatcher003的氨基酸序列如SEQ ID NO:1所示。
SEQ ID NO:1:
PRSHLGQILRRRPWLVHPRHRYRWRRKNGIFNTRLSRTFGYTIKRTTVKTPSWAVDMMRFNINDFLPPGGGSNPRSVPFEYYRIRKVKVEFWPCSPITQGDRGVGSSAVILDDNFVTKATALTYDPYVNYSSRHTITQPFSYHSRYFTPKPVLDSTIDYFQPNNKRNQLWLRLQTAGNVDHVGLGTAFENSIYDQEYNIRVTMYVQFREFNLKDPPLNPGSGSGSGSGSGSVTTLSGLSGEQGPSGDMTTEEDSATHIKFSKRDEDGRELAGATMELRDSSGKTISTWISDGHVKDFYLYPGKYTFVETAAPDGYEVATPIEFTVNEDGQVTVDGEATEGDAHT
2.Cap-SpyCatcher003纳米抗原展示载体的表达
将上述构建好的pET28a-Cap-SpyCatcher003质粒转化BL21(DE3)大肠杆菌表达感受态细胞,转化产物涂布至含50μg/mL卡那霉素的LB琼脂平板上,并在37℃孵育过夜,得单克隆菌落。将单克隆菌落挑入含50μg/mL卡那霉素的5mL LB培养基中,在37℃以200r/min振荡孵育16h,得预培养物。
然后将预培养物以1:100(v/v)稀释至500mL LB培养基中,加入50μg/mL卡那霉素,并在37℃、200r/min下振荡培养。当培养至OD600达到0.6左右时,加入终浓度为0.2mM的IPTG,并在16℃200r/min下继续诱导18h,得菌体。
离心收集菌体,将离心后的菌体按10:1的体积比(原始菌液量:破菌缓冲液)加入50mL,pH为8.0的破菌缓冲液(含20mM Tris-HCl、150mM NaCl)使菌体重悬,得菌体重悬液。
3.Cap-SpyCatcher003纳米抗原展示载体的纯化
将上述获得的菌体重悬液进行超声破碎处理,在4℃下以12000r/min离心20min,收集上清液。使用0.22μm过滤器过滤收集到的上清液,接着向上清液中加入5%体积的饱和硫酸铵溶液后放于4℃静置6h。以12000r/min在4℃下离心20min,弃去上清液,将沉淀重新悬浮在20mL pH为8.0的破菌缓冲液中。用过量的破菌缓冲液透析24h,除去残留的硫酸铵,得透析后的Cap-SpyCatcher003。
在4℃下,以6000r/min将透析后的Cap-SpyCatcher003离心20min,再用0.22μm过滤器过滤上清液,以进一步去除任何不溶性物质。最后,将上清液以0.75mL/min的流速通过装填CaptoTM Core 700填料的柱子,收集流穿液,得纯化后的Cap-SpyCatcher003。
使用SDS-PAGE凝胶电泳对纯化后的Cap-SpyCatcher003进行鉴定。结果见图2。
图2是纯化后的SDS-PAGE图,其中M是蛋白质标记,泳道1是纯化后的Cap-SpyCatcher003纳米颗粒。
实施例2:4种SpyTag003标签装载抗原的制备与装配
研究猪圆环病毒2型病毒样颗粒纳米抗原展示载体展示猪源病毒保护性多肽、单体蛋白、二聚体蛋白和三聚体蛋白的潜能,并在小鼠模型中研究其诱导高效免疫应答反应的规律。
1.4种SpyTag003标签装载抗原的制备
与SpyTag003标签装载抗原融合表达的猪源病毒保护性多肽(猪繁殖与呼吸综合征病毒(PRRSV)GP5与GP3蛋白上的B细胞表位),GP53-SpyTag003,简称为GP53,由生工生物工程(上海)股份有限公司合成,其氨基酸序列如SEQ ID NO:2所示。
与SpyTag003标签装载抗原融合表达的猪源病毒保护性单体蛋白(猪流行性腹泻病毒(PEDV)COE蛋白),COE-SpyTag003,简称为COE,其氨基酸序列如SEQ ID NO:3所示;与SpyTag003标签装载抗原融合表达的猪源病毒保护性二聚体蛋白(猪瘟病毒(CSFV)E2蛋白),E2-SpyTag003,简称为E2,其氨基酸序列如SEQ ID NO:4所示;与SpyTag003标签装载抗原融合表达的猪源病毒保护性三聚体蛋白(猪流感病毒(SIV)HA蛋白),HA-SpyTag003,简称为HA,其氨基酸序列如SEQ ID NO:5所示;这三种蛋白均由各自对应的pcDNA3.1载体瞬时转染Expi293F细胞表达,并使用Spy&IAC免疫亲和层析方法通过pure色谱系统纯化得到。
SEQ ID NO:2:
TPLTRVSAERWGRLKKQAAAEILEPGKSGGSRGVPHIVMVDAYKRYK
其中,划线部分“RGVPHIVMVDAYKRYK”为SpyTag003序列。
SEQ ID NO:3:
MRSLIYFWLLLPVLPTLSLPTSFVTLPSFNDHSFVNITVSAAFGGYSGANLIASDTTINGFSSFCVDTRQFTISLFYNVTNSYGYVSKSQDSNCPFTLQSVNDYLSFSKFCVSTSLLASACTIDLFGYPEFGSGVKFTSLYFQFTKGELITGTPKPLEGVGGSGGSGGSRGVPHIVMVDAYKRYK
SEQ ID NO:4:
MYRMQLLSCIALSLALVTNSRLACKEDYRYAISSTNEIGPLGAGGLTTTWKEYSHDLQLYDGTVKAICVAGSFKVTALNVVSRRYLASLHKGALLTSVTFELLFDGTNPSTEEMGDDFGFGLCPFDTSPVVKGKYNTTLLNGSAFYLVCPIGWTGVIECTAVSPTTLRTEVVKTFRREKPFPHRMDCVTTTVENEDLFYCKLGGNWTCVKGEPVVYTGGQVKQCKWCGFDFNEPDGLPHYPIGKCILANETGYRIVDSTDCNRDGVVISAEGSHECLIGNTTVKVHASDERLGPMPCRPKEIVSSAGPVRKTSCTFNYAKTLKNKYYEPRDSYFQQYMLKGEYQYWFDLDVTDRHSDYFAELQRMKQLEDKVEELLSKNYHLENEVARLKKLVGEGGSGGSGGSRGVPHIVMVDAYKRYKSEQ ID NO:5:
MKANLLVLLCALAAADADTICIGYHANNSTDTVDTVLEKNVTVTHSVNLLEDSHNGKLCRLKGIAPLQLGKCNIAGWLLGNPECDPLLPVRSWSYIVETPNSENGICYPGDFIDYEELREQLSSVSSFERFEIFPKESSWPNHNTNGVTAACSHEGKSSFYRNLLWLTEKEGSYPKLKNSYVNKKGKEVLVLWGIHHPPNSKEQQNLYQNENAYVSVVTSNYNRRFTPEIAERPKVRDQAGRMNYYWTLLKPGDTIIFEANGNLIAPMYAFALSRGFGSGIITSNASMHECNTKCQTPLGAINSSLPYQNIHPVTIGECPKYVRSAKLRMVTGLRNNPSIQSRGLFGAIAGFIEGGWTGMIDGWYGYHHQNEQGSGYAADQKSTQNAINGITNKVNTVIEKMNIQFTAVGKEFNKLEKRMENLNKKVDDGFLDIWTYNAELLVLLENERTLDFHDSNVKNLYEKVKSQLKNNAKEIGNGCFEFYHKCDNECMESVRNGTYDYPKYSEESKLNREKVDGVKLESMGIYQGSGGSGYIPEAPRDGQAYVRKDGEWVLLSTFLGGGSGGSGGSRGVPHIVMVDAYKRYK
2.用Cap-SpyCatcher003纳米抗原展示载体展示4种SpyTag003标签装载抗原
将上述得到的4种SpyTag003标签装载抗原(分别简称为GP53、COE、E2、HA),在4℃条件下,以不同摩尔比与Cap-SpyCatcher003(简称为Cap-Cat)结合2h(如Cap-Cat:GP53的摩尔比为1:1、1:2或1:3),并通过离心去除聚集体,分别得到不同偶联比的VLP-抗原偶联物(Cap-Cat-GP53、Cap-Cat-COE、Cap-Cat-E2、Cap-Cat-HA)。
使用SDS-PAGE,分别对SpyTag003标签装载抗原(GP53、COE、E2、HA)、纯化后的Cap-SpyCatcher003(Cap-Cat)、VLP-抗原偶联物(Cap-Cat-GP53、Cap-Cat-COE、Cap-Cat-E2、Cap-Cat-HA)进行鉴定,显示每种偶联比率下形成的共价VLP-抗原偶联物的量。
如图3-图6所示,SDS-PAGE证实了有效的共价反应,所有SpyTag标签抗原都在Cap-SpyCatcher003纳米抗原展示载体上高效展示。其中,在VLP:抗原1:3(摩尔比)的条件下,Cap-SpyCatcher003载体与装载抗原GP53和E2的缀合显示出最少的残留非结合载体;在VLP:抗原为1:2(摩尔比)的条件下,Cap-SpyCatcher003载体与装载抗原COE和HA的缀合显示出较少的残留非结合载体,且相应VLP-抗原偶联物的装配能力明显最优。
通过SuperdexTM 200Increase 10/300GL色谱柱纯化4种VLP-抗原偶联物,去除其中残留的游离抗原,以用作免疫原。
实施例3:Cap-SpyCatcher003纳米抗原展示载体能显著提高血清抗体水平
1.小鼠免疫
将上述获得的纯化的4种VLP-抗原偶联物(Cap-Cat-GP53、Cap-Cat-COE、Cap-Cat-E2、Cap-Cat-HA中VLP与抗原的摩尔比分别为1:3、1:2、1:3、1:2),分别与MontanideTM ISA201VG佐剂按体积比为1:1进行乳化。通过背部皮下多点注射的方法,在0、21d免疫4组(每组5只)6~8周龄的雌性BALB/c小鼠两次。两次蛋白免疫剂量为10μg VLP-抗原偶联物(Cap-Cat-GP53、Cap-Cat-COE、Cap-Cat-E2、Cap-Cat-HA),总剂量均为200μl/只。并以SpyTag003标签抗原单体(GP53、COE、E2、HA)免疫及PBS注射为对照;分别在首次免疫后第21天和第42天进行小鼠尾部采血。
2.免疫小鼠血清抗体效价测定
用间接ELISA方法检测首次免疫后第21天和第42天采集的4组小鼠血清效价,步骤如下:
(1)包被:用ELISA包被液(CBS)稀释纯化的4种SpyTag003标签装载抗原至1μg/mL,每孔加入50μl包被液,4℃孵育过夜,弃包被液,用PBST洗涤3次,甩干板上残余液体;
(2)封闭:每孔加入200μL的PBST+5%脱脂奶粉,于37℃封闭2h,用同样方法洗板;
(3)一抗:每孔加入50uL用PBST+5%脱脂奶粉以2倍倍比稀释的待检血清各50μL,于37℃孵育1h后,弃上清液,用PBST洗涤6次;
(4)二抗:每孔加入用PBST+5%脱脂奶粉以1:5000稀释的HRP标记的羊抗小鼠IgG各50μL,37℃孵育45min后,弃上清液,用PBST洗涤6次;
(5)显色:向每孔中加入50μL TMB显色液,室温避光作用5-10min后加25μL 2MH2SO4终止反应,并用酶标仪测定450nm处各孔溶液的吸光值。
(6)利用Excel分析数据,并计算出各组小鼠各时间点血清效价的几何平均值。
结果如图7-图10所示,ELISA结果显示,与未连接的相应单体抗原(GP53、COE、E2、HA)相比,由猪圆环病毒2型病毒样颗粒纳米抗原展示载体展示的抗原(Cap-Cat-GP53、Cap-Cat-COE、Cap-Cat-E2、Cap-Cat-HA)诱导产生了5-8倍高的抗体应答水平。
以上仅是本发明的优选实施方式,应当指出的是,上述实施方式不应视为对本发明的限制,对于本领域普通技术人员来说,在不脱离本发明的精神的情况下,还可以进行若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。
Claims (8)
1.基于猪圆环病毒2型病毒样颗粒的通用型模块化纳米抗原展示载体,其特征在于,所述纳米抗原展示载体由与SpyCatcher003融合表达的猪圆环病毒2型病毒样颗粒自组装而成;SpyCatcher003与猪圆环病毒2型病毒样颗粒的融合蛋白的氨基酸序列如SEQ ID NO:1所示。
2.如权利要求1所述的纳米抗原展示载体,其特征在于,SpyCatcher003融合表达在猪圆环病毒2型病毒样颗粒的羧基端。
3.如权利要求1所述的纳米抗原展示载体,其特征在于,所述猪圆环病毒2型病毒样颗粒为PCV2b亚型。
4.如权利要求1所述的纳米抗原展示载体,其特征在于,将SpyCatcher003序列插入到PCV2 Cap蛋白的羧基末端,二者以GSGSGSGSGSGS序列相连,经大肠杆菌偏好性密码子优化,得到氨基酸序列SEQ ID NO:1。
5.利用权利要求1所述的纳米抗原展示载体展示的装载抗原,其特征在于,所述装载抗原为:与SpyTag003标签融合表达的猪源病毒保护性多肽,或与SpyTag003标签融合表达的猪源病毒保护性单体蛋白,或与SpyTag003标签融合表达的猪源病毒保护性二聚体蛋白,或与SpyTag003标签融合表达的猪源病毒保护性三聚体蛋白。
6.如权利要求5所述的装载抗原,其特征在于,与SpyTag003标签融合表达的猪源病毒保护性多肽的氨基酸序列如SEQ ID NO:2所示;
与SpyTag003标签融合表达的猪源病毒保护性单体蛋白的氨基酸序列如SEQ ID NO:3所示;
与SpyTag003标签融合表达的猪源病毒保护性二聚体蛋白的氨基酸序列如SEQ ID NO:4所示;
与SpyTag003标签融合表达的猪源病毒保护性三聚体蛋白的氨基酸序列如SEQ ID NO:5所示。
7.如权利要求5所述的装载抗原,其特征在于,所述装载抗原与纳米抗原展示载体是通过SpyCatcher003与SpyTag003的相互作用而共价连接。
8.如权利要求1所述的纳米抗原展示载体在疫苗制备中的应用。
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