CN112121161A - 一种疫苗免疫佐剂及利用其制备的疫苗和制备方法 - Google Patents
一种疫苗免疫佐剂及利用其制备的疫苗和制备方法 Download PDFInfo
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Abstract
本发明提供一种疫苗免疫佐剂及利用其制备的疫苗和制备方法。所述疫苗免疫佐剂包括人铁蛋白或人铁蛋白突变体,具体序列包括如(I)、(II)或(III)所示的氨基酸序列中的任意一个:(I)如SEQ ID NO.1所示的氨基酸序列;(II)与SEQ ID NO.1所示的氨基酸序列具有至少90%一致性的氨基酸序列;(III)SEQ ID NO.1所示的氨基酸序列经修饰、取代、添加或缺失一个或多个氨基酸获得的氨基酸序列。该疫苗免疫佐剂可以注射使用,且可同时引起体液免疫和细胞免疫,具有免疫迅速、持久、高效的特点,是一种优良的蛋白类通用疫苗免疫增强剂。
Description
技术领域
本发明属于生物医药技术领域,具体涉及一种疫苗免疫佐剂及利用其制备的疫苗和制备方法。
背景技术
疫苗是人类医学领域中的伟大发明,疫苗的使用帮助人类以最小的代价消灭了很多严重的疾病,包括天花、小儿麻痹症等等,使用时一般需要加入佐剂,以提高免疫效果。理想的佐剂能显著减少抗原使用的剂量,同时可以诱导机体产生高效、持久的免疫应答。佐剂有很多种,例如氢氧化铝佐剂、硫柳汞、短小棒状杆菌、脂多糖、细胞因子、明矾等。铝佐剂是当前被国内外使用最广泛的佐剂种类,然而其具有较多的副作用。FDA推荐用于个体的生物学产品中铝含量最多不超过1.25mg。很多含有铝佐剂的婴幼儿疫苗其铝含量大大超出该最大剂量。
市场急需新型的通用免疫佐剂来提高疫苗(特别是新型亚单位疫苗)的安全性,同时保证其有效性。
有别于传统灭活疫苗和减毒疫苗,亚单位疫苗由于其天然的极高安全性,无返毒、致瘤等潜在危险,成为新型疫苗的研究热点。亚单位疫苗有很多优点,但也常见免疫原性较低的例子。提高亚单位疫苗的免疫效果通常需要通过加入佐剂和制备成类病毒颗粒来来实现,而最常用的铝佐剂通常效果较差。
病毒样颗粒疫苗具有与病毒粒子相同的途径激起免疫系统的有效应答,且安全性高,为了提高免疫效果,通常也需要加入佐剂。常见的免疫增强剂机制为:(1)改变抗原物理性状,延长抗原在机体内的存在时间和保持对免疫系统的持续激活作用;(2)使抗原易被巨噬细胞吞噬,刺激单核巨噬细胞系统,增强其对抗原的处理和呈递抗原的能力;(3)促进淋巴细胞的增殖、分化,从而扩大和增强机体的免疫应答的效应。
铁蛋白(Ferritin)为24个亚基组成的纳米颗粒,其空间构型为八面体对称蛋白笼形结构,外表面直径约12纳米,内腔直径约8纳米。铁蛋白广泛存在于各种生物体,主要生物学功能是储存铁,每个铁蛋白分子可以储存多达4500个铁原子,近年来成为生物纳米材料研究领域的热点之一。
铁蛋白独特的纳米尺度和病毒样颗粒结构使得很多的研究聚焦于将各种抗原融合表达于其分子表面,用于更好的展示抗原分子表位。将铁蛋白亚基与目的抗原序列直接融合表达在铁蛋白分子的N端或者C端是最常用的策略,但是也有其他技术采用了体外连接的方式。
CN110201183A和CN108434450A的技术核心在于可以在体外连接抗原至铁蛋白分子表面。其中,CN110201183A利用了内含子intein具有的内切酶特定,用DTT处理两个连有内含子的蛋白以进行酶切连接的步骤,过程繁琐,产品可控性差,其免疫增强作用主要通过连接的鞭毛蛋白等实现;CN108434450A则是利用了两个可以相互特异性识别并产生共价键的多肽分子,分别融合表达于铁蛋白和目的抗原分子上面,体外混合将二者连接,连接效率有待确认,其免疫增强作用通过添加佐剂CpG-1826实现(详见Wang W等.Dual-targetingnanoparticle vaccine elicits a therapeutic antibody response against chronichepatitis B[J].Nature Nanotechnology,2020.)。
目前,铁蛋白在上述载体方面的应用仅利用了铁蛋白分子的纳米颗粒属性,将目的抗原以及可以增强免疫效果的鞭毛蛋白等展示于铁蛋白分子表面。实际上,除了具有纳米属性外,铁蛋白作为体内的功能性分子,还有其独特的有别于其他纳米颗粒特别是人工合成纳米颗粒的生物学属性。
因此,提供一种制备方法简单、免疫增强效果较好且产品可控性较高的疫苗免疫佐剂是本领域亟需解决的问题,而铁蛋白纳米颗粒独有的分子特性则可以满足这一需求。
发明内容
鉴于现有技术中存在的问题,本发明提供一种疫苗免疫佐剂及利用其制备的疫苗和制备方法。
为达此目的,本发明采用以下技术方案:
第一方面,本发明提供一种疫苗免疫佐剂,所述疫苗免疫佐剂包括人铁蛋白(FHC,human Ferritin)或人铁蛋白突变体(FHC-X),具体序列包括如(I)、(II)或(III)所示的氨基酸序列中的任意一个:
(I)如SEQ ID NO.1所示的氨基酸序列;
(II)与SEQ ID NO.1所示的氨基酸序列具有至少90%一致性的氨基酸序列;
(III)SEQ ID NO.1所示的氨基酸序列经修饰、取代、添加或缺失一个或多个氨基酸获得的氨基酸序列。
本发明所述的人铁蛋白(FHC)与常见的作为佐剂使用的细胞因子(如IL1、IL2、IL12、IL15、IFN-α、GM-CSF、TNF或CCL25等)相比,具有更强的免疫增强功能,可同时引起体液免疫和细胞免疫,且具有免疫迅速、持久、高效的特点,其迅速、持久、高效的免疫增强作用来自于所述铁蛋白的体内对免疫细胞主动靶向性和其多聚体形式的纳米粒子特性;因此,可以用做通用疫苗免疫增强剂。同时,其他哺乳动物,特别是灵长类的铁蛋白与人的序列相似度极高,据此推测也有类似的性质,也可以做为通用免疫增强剂。
同时,本发明所述FHC通用疫苗增强剂免疫机制与常见免疫增强剂(如鞭毛蛋白、细胞因子、铝佐剂或弗氏佐剂等)不同,其免疫增强作用为主动靶向淋巴系统,并刺激位于淋巴结处的巨噬细胞、DC细胞等表达特定细胞因子,进一步招募T细胞和B细胞,完成免疫过程。因此,人铁蛋白作为免疫增强剂,能够显著地增强抗原的免疫原性,引起更强的体液、细胞免疫反应,因此人铁蛋白是理想的疫苗免疫佐剂。
作为本发明优选的技术方案,所述疫苗免疫佐剂包括如SEQ ID NO.1所示的氨基酸序列。
人的全H亚基铁蛋白的氨基酸序列(SEQ ID NO.1)如下:
MTTASTSQVRQNYHQDSEAAINRQINLELYASYVYLSMSYYFDRDDVALKNFAKYFLHQSHEEREHAEKLMKLQNQRGGRIFLQDIKKPDCDDWESGLNAMECALHLEKNVNQSLLELHKLATDKNDPHLCDFIETHYLNEQVKAIKELGDHVTNLRKMGAPESGLAEYLFDKHTLGDSD
优选地,所述人铁蛋白的核苷酸序列如SEQ ID NO.2所示。
SEQ ID NO.2如下:
ATGACGACCGCGTCCACCTCGCAGGTGCGCCAGAACTACCACCAGGACTCAGAGGCCGCCATCAACCGCCAGATCAACCTGGAGCTCTACGCCTCCTACGTTTACCTGTCCATGTCTTACTACTTTGACCGCGATGATGTGGCTTTGAAGAACTTTGCCAAATACTTTCTTCACCAATCTCATGAGGAGAGGGAACATGCTGAGAAACTGATGAAGCTGCAGAACCAACGAGGTGGCCGAATCTTCCTTCAGGATATCAAGAAACCAGACTGTGATGACTGGGAGAGCGGGCTGAATGCAATGGAGTGTGCATTACATTTGGAAAAAAATGTGAATCAGTCACTACTGGAACTGCACAAACTGGCCACTGACAAAAATGACCCCCATTTGTGTGACTTCATTGAGACACATTACCTGAATGAGCAGGTGAAAGCCATCAAAGAATTGGGTGACCACGTGACCAACTTGCGCAAGATGGGAGCGCCCGAATCTGGCTTGGCGGAATATCTCTTTGACAAGCACACCCTGGGAGACAGTGATAATGAAAGCTAA
作为本发明优选的技术方案,所述疫苗免疫佐剂包括重链人铁蛋白、轻链人铁蛋白或重链轻链杂合组装而成的多聚体中的任意一种或至少两种的组合。
优选地,所述多聚体包括人铁蛋白的H亚基形成的8聚体、16聚体、24聚体或48聚体中的任意一种或至少两种的组合。
优选地,所述疫苗免疫佐剂单独使用或与其他佐剂配合使用。
第二方面,本发明提供一种疫苗,包括如第一方面所述疫苗免疫佐剂和与所述疫苗免疫佐剂连接的抗原。
优选地,所述连接的方式包括共价键连接和/或非共价键连接。
优选地,所述共价键连接包括二硫键连接和/或酰胺键连接。
由于免疫增强机制的不同,本发明所述FHC做为通用疫苗免疫增强剂时应通过特定方式将抗原与之连接,以将抗原携带至淋巴结等部位的免疫细胞,直接与抗原混合使用不能有效的实现免疫增强效果。
优选地,所述二硫键连接通过利用铁蛋白分子和抗原分子上的半胱氨酸残基的巯基形成二硫键实现;
优选地,所述酰胺键连接通过在体内融合表达在抗原和铁蛋白之间形成肽键实现或通过在体外EDC-NHS连接系统在抗原和铁蛋白分子表面的羧基基团、氨基基团间形成酰胺键实现。
本发明中,连接方法可以为下述任何一种:
1、将抗原与FHC亚基融合表达,FHC自组装成纳米粒子后将抗原展示于表面;
2、将抗原通过连接子与已经组装成纳米粒子的FHC相连接(连接子为具有特异相互作用的两个蛋白/多肽分子,连接后可以形成共价键或者非共价键)。
3、将抗原与FHC通过化学方法连接,这种化学方法可以在两个分子之间通过形成二硫键、酰胺键或者其他化学键达到连接目的。可选择碳二亚胺法、活性酯法、戊二醛法或琥珀酸酐法等任一方法进行连接,从而将传统灭活疫苗、蛋白组分疫苗与FHC连接,以达到增强免疫效果的目的。
优选地,所述疫苗免疫增强剂的给药途径包括静脉注射、皮下注射或肌肉注射中的任意一种或至少两种的组合,优选为静脉注射。
传统的细胞因子类佐剂和铝佐剂等使用时一般与抗原混合,且只能通过皮下或者肌肉注射使用,二者的使用机理均为在注射部位招募免疫细胞达到免疫目的,在注射部位有免疫反应。
本发明所述通用免疫增强剂则可以主动靶向淋巴结等部位的免疫细胞,因此优选静脉注射,但也可以通过其他方式使用,且无注射部位反应。具体的:
1、可以通过静脉注射造成体液免疫,达到快速免疫的效果,初始产生抗体为IgM,随后通过细胞免疫效应产生IgG分子达到长期免疫效果,免疫时间持续200天以上。
2、可以通过皮下注射使用,注射后通过淋巴管快速进入临近注射部位的淋巴结起到免疫效果。
3、可以通过肌肉注射和粘膜给药途径实现免疫,免疫机理同皮下注射。
作为本发明优选的技术方案,所述疫苗包括减毒疫苗、灭活疫苗、核酸疫苗、多肽疫苗或蛋白质疫苗中的任意一种或至少两种的组合。
优选地,所述疫苗免疫增强剂还包括药学上可接受的辅料。
优选地,所述辅料包括载体、稀释剂、赋形剂、填充剂、粘合剂、润湿剂、崩解剂、乳化剂、助溶剂、增溶剂、渗透压调节剂、表面活性剂、包衣材料、着色剂、pH调节剂、抗氧剂、抑菌剂或缓冲剂中的任意一种或至少两种的组合。
第三方面,本发明提供一种如第一方面所述的疫苗的制备方法,所述制备方法包括:
将疫苗免疫佐剂与抗原构建于同一载体上共同表达,得到所述疫苗免疫佐剂与抗原的融合蛋白;
或者,分别合成疫苗免疫佐剂和抗原,再通过共价键和/或非共价键的方式将所述疫苗免疫佐剂与抗原相连,得到所述疫苗。
第四方面,本发明还提供一种新型冠状病毒亚单位疫苗,其特征在于,包括如第一方面所述的疫苗免疫佐剂和与所述疫苗免疫佐剂相连的新型冠状病毒S1蛋白。
优选地,所述新型冠状病毒S1蛋白包括如SEQ ID NO.3所示的氨基酸序列。
COVID-19RBD蛋白片段氨基酸序列(SEQ ID NO.3)如下:
WNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYR
同时,本发明还提供一种HIV病毒亚单位疫苗,包括如第一方面所述的疫苗免疫佐剂和与所述疫苗免疫佐剂相连的HIV S2蛋白。
第五方面,本发明还提供如第一方面所述的疫苗免疫佐剂或如第二方面所述的疫苗在制备抗体或者产生抗体的细胞株中的应用。
本发明中,不仅可以利用所述疫苗免疫佐剂制备疫苗,还可以利用其制备得到的疫苗免疫动物,使其产生抗体从而制备得到相应的抗体;或者利用所述疫苗制备能够产生抗体的细胞株,从而大量生产抗体。
本发明所述的数值范围不仅包括上述列举的点值,还包括没有列举出的上述数值范围之间的任意的点值,限于篇幅及出于简明的考虑,本发明不再穷尽列举所述范围包括的具体点值。
与现有技术相比,本发明至少具有以下有益效果:
(1)本发明提供一种通用型疫苗免疫佐剂,包括人铁蛋白FHC或基于FHC的突变体FHC-X,其显著特点在于该佐剂可以静脉注射使用,可同时引起体液免疫和细胞免疫,且具有免疫迅速、持久、高效的特点;本发明的铁蛋白及其突变体的疫苗佐剂可以用作亚单位疫苗、灭活疫苗、多糖疫苗、多肽疫苗、蛋白质疫苗和小分子疫苗的佐剂;
(2)所述疫苗免疫佐剂在使用时需与抗原连接,通过FHC或FHC-X快速、特异地靶向淋巴结处的巨噬细胞、T细胞等免疫细胞,起到抗原呈递和细胞免疫效果;除了静脉注射使用,还可以皮下注射及肌肉注射使用,具有无注射部位反应,免疫效果好的特点,是一种优良的蛋白类通用疫苗免疫增强剂。
附图说明
图1为H亚基铁蛋白和COVID-19(H-FHC-COVID-19)亚单位疫苗的显微电镜图(标尺100nm)。
图2为H-FHC-COVID-19亚单位疫苗静脉注射免疫小鼠后血清滴度测定曲线图。
图3为H-FHC-COVID-19亚单位疫苗体外连接后的显微电镜图(标尺100nm)。
图4为FHC与HIV S2蛋白亚单位疫苗片段通过二硫键连接后得到的HPLC检测图。
具体实施方式
下面结合附图并通过具体实施方式来进一步说明本发明的技术方案,但下述的实例仅仅是本发明的简易例子,并不代表或限制本发明的权利保护范围,本发明的保护范围以权利要求书为准。
下述实施例中的实验方法,如无特殊说明,均为常规方法。下述实施例中所用的试验材料,如无特殊说明,均为自常规生化试剂商店购买得到的。
以下实施例中,所述室温均为25℃。
实施例1
本实施例提供一种人H亚基铁蛋白(H-FHC)的表达与纯化方法,具体步骤如下:
(1)将人铁蛋白基因序列(来自于PubMed,基因编号:2495;具体序列如SEQ IDNO.1所示)送北京全式金公司合成,片段合成后克隆到pET28a载体上,并经过序列测定证实插入基因片段与设计序列一致;
(2)将上述含有人铁蛋白基因序列的重组质粒转染到感受态大肠杆菌BL21(DE3)内,得到铁蛋白表达菌株;
(3)经过鉴定的菌种与甘油混合冻存于-80℃冰箱中,使用时于37℃水浴中解冻,然后将菌种划线于含有100mg/L氨苄的LB琼脂培养皿中,培养12h;
(4)挑取粗壮单菌落置于摇瓶中培养,37℃、200rpm下培养7h后,在OD值为0.800±0.050时,开始加入终浓度为0.5mM的IPTG诱导FHC蛋白表达,3.5h后收获发酵液,离心获得菌体;
(5)菌体以20mM的磷酸缓冲液重悬,重悬浓度为15%,菌悬液经800Bar高压破碎两次后铁蛋白被释放出来,离心获得上清;上清经70℃水浴加热10分钟,再次离心取上清,该上清中含有粗纯的H-FHC;
(6)H-FHC粗纯液加入硫酸铵至1.5M终浓度,然后经5mL Pheny HP预装柱(GEhealthcare公司)纯化,获得精纯的H-FHC;
(7)精纯的FHC经Superdex 200(GE healthcare公司)再次纯化,获得终产品H-FHC,保存于PBS溶液中。
实施例2
考虑到FHC独有的免疫增强作用,本实施例中设计了基于H-FHC免疫增强剂的COVID-19新型亚单位疫苗。本实施例中提供一种H亚基铁蛋白和COVID-19(H-FHC-COVID-19)亚单位疫苗,具体制备方法如下:
1、COVID-19S1亚单位疫苗与H-FHC融合表达与纯化
(1)COVID-19S1蛋白RBD(receptor binding domain)氨基酸序列如SEQ ID NO.2所示(详见Xiangyang Chi等,A neutralizing human antibody binds to the N-terminal domain of the Spike protein of SARS-CoV-2,Science 07Aug 2020:Vol.369,Issue 6504,pp.650-655,DOI:10.1126/science.abc6952),为病毒与其受体ACE2结合的关键序列。
(2)将其相应的基因序列与FHC经G4S linker连接,序列全长经南京金斯瑞公司合成;合成的DNA序列经确认无误后克隆到pET28a载体上,并进一步转染到BL21(DE3)菌内形成COVID-19-FHC亚单位疫苗表达菌株;
(3)以LB培养基37℃培养过夜,0.5mM IPTG诱导表达5h后,目的蛋白为包涵体形式;
(4)收集的菌体先经20mM Tris缓冲液重悬,重悬浓度为15%,菌悬液经800Bar高压破碎两次,离心收取沉淀;沉淀经含有8M尿素和10mM DTT的20mM Tris缓冲液溶解,然后进行稀释复性,复性缓冲液为:25mM tris,100mM NaCl,1mM GSH,5mM GSSG,11mM EDTA,pH8.0;
(5)复性后的蛋白经TFF浓缩10倍体积后进行4倍体积的换液(20mM PB,pH7.4),换液后的蛋白继续经SP HP阳离子交换层析柱进行精纯,蛋白纯度达到95%以上;
2、融合蛋白的表征(电镜及纳米粒度分析)
(1)将上述纯化后的H-FHC融合COVID-19亚单位疫苗经稀释后做电镜分析;
首先在碳支持膜上孵育1min,然后用质量分数为2%的醋酸双氧铀清洗两遍,最后用2%的醋酸双氧铀处理1min;制备的样品在FEI Tecnai Spirit透射电子显微镜上观察,条件为HT100 kv。
电镜结果如图1所示,所制备的COVID-19亚单位疫苗呈现标准的球形结构,且分散性良好。
实施例3
本实施中使用实施例2中制备的H-FHC-COVID-19亚单位疫苗进行免疫增强试验,具体步骤如下:
免疫途径分为静脉注射和皮下注射,免疫剂量为3μg/只,第一次免疫后间隔一周加强免疫一次;
初次免疫后第6周取小鼠静脉血进行ELISA抗体滴度测定;
测定时以FHC和FHC-COVID-19亚单位疫苗分别包被96孔板,然后将血清稀释后放入包被后的板中孵育,最后在MD酶标仪上进行检测;
检测结果表明,静脉注射和皮下注射效果相当,抗体对FHC-COVID-19亚单位疫苗的滴度超过10万,同时对FHC的滴度几乎为零;
其中,H-FHC-COVID-19亚单位疫苗静脉注射免疫小鼠后,免疫血清吸收值10μg组、免疫血清吸收值30μg组、免疫血清吸收值3μg组与对照组(本底吸收值)的血清滴度测定如图2所示。
实施例4
考虑到H-FHC作为免疫增强剂的广泛适用性,如果仅采用融合表达方法生产亚单位疫苗其使用必然是受限的。为了提高产品的可生产性,本实施例中采用了大规模表达H-FHC后再与候选疫苗体外连接的方法进行基于H-FHC的亚单位疫苗的生产策略。
本实施例中在体外通过酰胺键将H-FHC与COVID-19S1蛋白亚单位疫苗进行连接形成疫苗免疫佐剂。
1、连接试验
连接方法上,优选共价连接,次选非共价连接。
具体的,本实施例中采用了EDC-EHS连接系统,该系统可以有效的在两个分别具有活性羧基和活性氨基的分子间高效的形成酰胺键。具体步骤如下:
(1)将实施例1中制备得到的H-FHC蛋白以缓冲液1(0.1M MES,0.5M NaCl,pH6.0)稀释至10mg/mL,将S1蛋白亚单位疫苗以PBS缓冲液(0.1M磷酸钠,0.15M NaCl,pH7.4)稀释至5mg/mL;
(2)然后将0.5mg EDC加入到1mL FHC蛋白溶液中,将0.8mg NHS加入到1mL S1蛋白亚单位疫苗蛋白溶液中,将二者混合,室温反应15min,然后加入1.5μL巯基乙醇终止反应;
(3)最后将混合液经G-25层析柱脱盐换液到PBS溶液中,得到H-FHC-COVID-19亚单位疫苗。
2、连接后亚单位疫苗的表征(纳米粒度分析)
连接后的疫苗分子经透射电镜分析,分析方法和条件同实施例2所述。
实验结果见图3,由图可知,通过体外酰胺键连接FHC与COVID-19亚单位疫苗,所得疫苗呈现典型且均一的纳米颗粒形状;其与实施例2中采用的方法相比几乎无差异。采用体外连接方式可以实现快速、方便的连接不同的抗原分子从而获得不同免疫效果的疫苗。
实施例5
本实施例中提供一种FHC与HIV S2蛋白亚单位疫苗片段结合形成的亚单位疫苗,且FHC与HIV S2蛋白通过二硫键连接。
通过EDC-EHS连接系统将FHC与候选疫苗分子连接的方法普适性较好,但同时也有其局限性,即连接位点为非特异性,连接后的分子均一性不容易控制。采用二硫键连接的方法进行连接,其连接位点的特异性可与融合表达的方式相当。
具体步骤如下:
1、连接试验
FHC分子结构解析结果表明,其表面半胱氨酸残基(C 102)可以与具有半胱氨酸残基的候选亚单位疫苗形成二硫键连接。
实验前候选亚单位疫苗的半胱氨酸残基以封闭剂(可以选择使用:谷胱甘肽、光胺、半胱胺等。本实验中选择使用半胱胺进行封闭)封闭其活性巯基-SH,以防止在实验过程中产生二聚体。
实验开始前,以5mM TCEP与经保护的HIV S2蛋白亚单位疫苗片段混合反应,并经G-25层析柱脱盐换液至20mM Tris-HCl,pH 8.0溶液中以除去TCEP及保护基团,然后与FHC蛋白溶液等体积混合,在室温慢速搅拌过夜;
2、连接后亚单位疫苗的表征(SEC-HPLC分析)
经TSK G3000swxl分析二硫键连接后的混合样品,如图4所示,可以看到蛋白呈现单一的洗脱峰。
本实施例表明,本发明提供的疫苗免疫佐剂具有普适性,不仅能够与COVID-19亚单位疫苗相连,还能够与HIV S2蛋白亚单位疫苗相连,且连接成功率较高。
综上所述,本发明中提供的疫苗免疫佐剂不仅具有普适性,能够与多种亚单位疫苗通过酰胺键、二硫键或生物合成的方式进行连接;同时,该免疫疫苗佐剂还具有较好的免疫增强效果,将其用于皮下注射和静脉注射时,能够明显提高抗体对亚单位疫苗的滴度。
申请人声明,以上所述仅为本发明的具体实施方式,但本发明的保护范围并不局限于此,所属技术领域的技术人员应该明了,任何属于本技术领域的技术人员在本发明揭露的技术范围内,可轻易想到的变化或替换,均落在本发明的保护范围和公开范围之内。
SEQUENCE LISTING
<110> 北京理工大学
北京晟之美生物科技有限责任公司
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Claims (10)
1.一种疫苗免疫佐剂,其特征在于,所述疫苗免疫佐剂包括人铁蛋白或人铁蛋白突变体,具体序列包括如(I)、(II)或(III)所示的氨基酸序列中的任意一个:
(I)如SEQ ID NO.1所示的氨基酸序列;
(II)与SEQ ID NO.1所示的氨基酸序列具有至少90%一致性的氨基酸序列;
(III)SEQ ID NO.1所示的氨基酸序列经修饰、取代、添加或缺失一个或多个氨基酸获得的氨基酸序列。
2.根据权利要求1所述的疫苗免疫佐剂,其特征在于,所述疫苗免疫佐剂包括如SEQ IDNO.1所示的氨基酸序列。
3.根据权利要求1或2所述的疫苗免疫佐剂,其特征在于,所述疫苗免疫佐剂包括重链人铁蛋白、轻链人铁蛋白或重链、轻链杂合组装而成的多聚体中的任意一种或至少两种的组合;
优选地,所述多聚体包括人铁蛋白的H亚基形成的8聚体、16聚体、24聚体或48聚体中的任意一种或至少两种的组合;
优选地,所述疫苗免疫佐剂单独使用或与其他佐剂配合使用。
4.一种疫苗,其特征在于,包括如权利要求1~3任一项所述疫苗免疫佐剂和与所述疫苗免疫佐剂连接的抗原。
5.根据权利要求4所述的疫苗,其特征在于,所述连接的方式包括共价键连接和/或非共价键连接;
优选地,所述共价键连接包括二硫键连接和/或酰胺键连接。
6.根据权利要求4或5所述的疫苗,其特征在于,所述疫苗的给药途径包括静脉注射、皮下注射或肌肉注射中的任意一种或至少两种的组合,优选为静脉注射。
7.根据权利要求4~6任一项所述的疫苗,其特征在于,所述疫苗包括减毒疫苗、灭活疫苗、核酸疫苗、多肽疫苗或蛋白质疫苗中的任意一种或至少两种的组合;
优选地,所述疫苗还包括药学上可接受的辅料;
优选地,所述辅料包括载体、稀释剂、赋形剂、填充剂、粘合剂、润湿剂、崩解剂、乳化剂、助溶剂、增溶剂、渗透压调节剂、表面活性剂、包衣材料、着色剂、pH调节剂、抗氧剂、抑菌剂或缓冲剂中的任意一种或至少两种的组合。
8.一种如权利要求4~7任一项所述的疫苗的制备方法,其特征在于,所述制备方法包括:
将疫苗免疫佐剂与抗原构建于同一载体上共同表达,得到所述疫苗免疫佐剂与抗原的融合蛋白;
或者,分别合成疫苗免疫佐剂和抗原,再通过共价键和/或非共价键的方式将所述疫苗免疫佐剂与抗原相连,得到所述疫苗。
9.一种新型冠状病毒亚单位疫苗,其特征在于,包括如权利要求1~3任一项所述的疫苗免疫佐剂和与所述疫苗免疫佐剂相连的新型冠状病毒S1蛋白;
优选地,所述新型冠状病毒S1蛋白包括如SEQ ID NO.3所示的氨基酸序列。
10.如权利要求1~3任一项所述的疫苗免疫佐剂或如权利要求4~7任一项所述的疫苗在制备抗体或者产生抗体的细胞株中的应用。
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