CN110354271A - 一种基于内含肽介导纳米载体模块平台及其构建方法与应用 - Google Patents

一种基于内含肽介导纳米载体模块平台及其构建方法与应用 Download PDF

Info

Publication number
CN110354271A
CN110354271A CN201910421408.9A CN201910421408A CN110354271A CN 110354271 A CN110354271 A CN 110354271A CN 201910421408 A CN201910421408 A CN 201910421408A CN 110354271 A CN110354271 A CN 110354271A
Authority
CN
China
Prior art keywords
leu
gly
glu
ser
lys
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201910421408.9A
Other languages
English (en)
Inventor
汤书兵
周伟
袁伟明
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Guangzhou Women and Childrens Medical Center
Original Assignee
Guangzhou Women and Childrens Medical Center
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Guangzhou Women and Childrens Medical Center filed Critical Guangzhou Women and Childrens Medical Center
Priority to CN201910421408.9A priority Critical patent/CN110354271A/zh
Publication of CN110354271A publication Critical patent/CN110354271A/zh
Priority to PCT/CN2020/089924 priority patent/WO2020233460A1/zh
Pending legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/385—Haptens or antigens, bound to carriers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/644—Transferrin, e.g. a lactoferrin or ovotransferrin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6068—Other bacterial proteins, e.g. OMP
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6075—Viral proteins

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Epidemiology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Microbiology (AREA)
  • Mycology (AREA)
  • Molecular Biology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Oncology (AREA)
  • Nanotechnology (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
  • Peptides Or Proteins (AREA)

Abstract

本发明公开了一种基于内含肽介导纳米载体模块平台及其构建方法与应用，该纳米载体模块平台可便捷地在同一个纳米载体上共递送免疫增强剂和抗原，显著提高佐剂助力效果。所述的基于内含肽介导纳米载体模块平台，为蛋白X与接头gbl‑intein的C端融合形成重组蛋白gbl‑inteinc‑X后经分子自组装成的多聚体纳米颗粒，所述蛋白X的N端伸展至纳米颗粒表面。

Description

一种基于内含肽介导纳米载体模块平台及其构建方法与应用
技术领域
本发明涉及药物载体及其构建方法与应用，尤其是涉及一种基于内含肽介导纳米载体模块平台及其构建方法与应用。
背景技术
疫苗是预防和控制传染性疾病最有效、最经济的医疗干预手段。减毒或灭活病原作为传统的经典疫苗取得巨大成功，帮助人类消灭天花和小儿麻痹症。然而，传统的疫苗存在安全隐患且无法有效预防具有高频突变特性的病毒，如HIV、流感病毒等。
亚单位疫苗是利用微生物的某种表面结构成分(抗原)制成不含有核酸、能诱发机体产生抗体的疫苗，因其易于纯化、安全有效、理化性质稳定等优点而备受疫苗学家关注。亚单位疫苗免疫原性差，诱导免疫应答较弱且持续时间短，限制了其临床上的应用。随着技术的发展，该类疫苗通过采用纳米递送系统和免疫佐剂(简称为佐剂)提高机体响应免疫应答的广度和力度。纳米递送载体通常为5-100nm的纳米颗粒，穿透力强，可自由扩散至B细胞和T细胞定居的回流淋巴结，提升抗原被抗原递呈细胞(APCs)捕获概率。纳米颗粒具有纳米尺寸效应，模拟天然病原，偏好性地被APCs吞噬。此外，纳米颗粒促进树突状细胞(DCs)成熟，分泌多种细胞因子，增强B细胞和T细胞免疫应答。另外，纳米颗粒表面高度重复展示的抗原可交联B细胞受体，直接活化B细胞，引起高强度的体液免疫应答。佐剂可助力免疫应答，特别是免疫增强剂，有效激活固有免疫和适应性免疫，调控免疫应答的类型和强度及持久度。这是因为病原体相关分子模式(pathogen associated molecular pattern，PAMP)高度保守，易被天然免疫细胞上的相应受体识别。例如，CpG被Toll样受体9(Toll-likereceptor 9，TLR9)识别，沙门氏菌鞭毛flagellin蛋白被TLR5识别，两者均可依赖DCs内的髓样分化因子88(myeloid differentiation factor 88，MyD88)激活NF-κB信号通路，刺激促炎症因子释放，触发级联免疫应答。但是，免疫增强剂作为刺激因子，非特异性地增强天然免疫和获得性免疫应答，因此可能引起系统炎症反应、发烧甚至肝脾肿大等疾病。有研究表明，免疫增强剂和抗原共定位至同一个APCs可显著提高佐剂的有效浓度进而改善佐剂助力效果，亦可降低佐剂和抗原剂量从而减少非特异性免疫应答引起的副作用。目前，纳米颗粒的修饰方法为基因融合法和化学偶联法。其中，基因融合法负载抗原和佐剂分子效率高，操作方便，大量应用于疫苗的研发。然而负载的抗原可能严重影响纳米颗粒组装，导致重组蛋白形成沉淀，需要一系列的复性-再组装才可重新形成纳米颗粒。此外，基因融合的方法难以将抗原分子和佐剂分子同时整合至同一个纳米颗粒上。化学偶联法可便捷、多态性地修饰纳米颗粒，使其多功能化，实现在纳米颗粒上共递送佐剂与抗原。但是该方法效率较低、特异性差，副反应常伴有副作用。
发明内容
本发明的目的之一是提供一种基于内含肽介导纳米载体模块平台，该纳米载体模块平台具有良好的稳定性，在负载外源蛋白后仍能保持原有结构，可便捷地在同一个纳米载体模块平台上共递送免疫增强剂和抗原，显著提高佐剂助力效果。
为了实现上述技术目的，本发明提供的技术方案是这样的：一种基于内含肽介导的纳米载体模块平台，其为可自组装的蛋白X与接头gbl-intein的C端融合形成的重组蛋白gbl-inteinc-X自组装成的多聚体纳米颗粒，所述蛋白X的N端伸展至纳米颗粒表面暴露于外部环境。
其中，所述的蛋白X为铁蛋白或二氧四氢蝶啶合成酶或细菌噬菌体Qβ核衣壳蛋白或豇豆花叶病毒核衣壳蛋白或戊肝病毒核衣壳蛋白。上述各蛋白在自装配成纳米颗粒后，N端都伸展在纳米颗粒表面暴露于外部环境，故本发明应用所制备的纳米颗粒中蛋白X融合了内含肽(intein)的N端暴露于外部环境，可以经蛋白编辑技术进行修饰。
其中，所述的铁蛋白为人源的重链铁蛋白或强烈火球菌的重链铁蛋白或其他物种来源的铁蛋白。
本发明的目的之二是提供上述纳米载体模块平台的构建方法。
为了实现上述技术目的，本发明提供的技术方案是这样的：一种基于内含肽介导的纳米载体模块平台的构建方法，由可自装配成纳米颗粒的蛋白X的N端与接头gbl-intein的C端融合形成重组蛋白gbl-inteinc-X，所述重组蛋白gbl-inteinc-X自装配成蛋白X的N端伸展至纳米颗粒表面暴露于外部环境的多聚体纳米颗粒。
其中，所述的可自装配成纳米颗粒的蛋白X为铁蛋白或二氧四氢蝶啶合成酶或细菌噬菌体Qβ核衣壳蛋白或豇豆花叶病毒核衣壳蛋白或戊肝病毒核衣壳蛋白。
其中，所述的铁蛋白为人源的重链铁蛋白或强烈火球菌的重链铁蛋白或其他物种来源的铁蛋白。
本发明的目的之三是提供上述纳米载体模块平台的应用。
为了实现上述技术目的，本发明提供的技术方案是这样的：如上所述的基于内含肽介导的纳米载体模块平台作为药物和/或疫苗递送系统的应用。
本发明与现有技术相比，具有以下优点：
(1)本发明提供的纳米载体模块平台为蛋白X的N端引入接头gbl-inteinC形成的重组蛋白gbl-inteinc-X自组装成多聚体纳米颗粒，蛋白X的N端伸展至纳米颗粒表面暴露于外部环境，抗原蛋白、免疫增强剂、多肽表位和抗体等外源蛋白通过内含肽介导的蛋白质编辑技术高效、特异地共价交联于蛋白X的N端，构成药物和/或疫苗递送系统，包括疫苗递送系统、佐剂递送系统以及疫苗-佐剂共递送系统。
(2)本发明提供的纳米载体模块平台具有良好的稳定性，在pH2-8下保持稳定。在负载抗原蛋白、免疫增强剂、多肽表位和抗体等外源蛋白后，纳米颗粒的组装不被破坏，即结构不被破坏，无需再进行复性-再组装。既提高疫苗递送系统等制备效率的同时，又解决了负载蛋白对蛋白自装配纳米颗粒稳定性影响以及纳米颗粒难以共递送佐剂和抗原分子的难题。实现了纳米制剂高效地同时递送抗原和免疫增强剂，同时且具有良好特异性。
(3)本发明提供的纳米载体模块平台负载抗原蛋白、免疫增强剂、多肽表位和抗体等外源蛋白效率高，副反应小。
(4)本发明提供的重组蛋白gbl-inteinc-X在大肠杆菌中有高度表达，易于制备，造价低，大大降低了制造成本。
(5)本发明提供的纳米载体模块平台可用于增强型的预防性亚单位疫苗和个性化多肽表位肿瘤疫苗研发及抗体偶联药物领域。
附图说明
图1为纳米载体模块构建原理的示意图。
图2为引入接头gbl-inteinC不影响纳米颗粒稳定性的分析图；
图2a为三种重组蛋白的电泳图；图2b为gb1-inteinC-HFT和gb1-inteinC-PFT经硫酸铵沉淀后的电泳图；图2c为gb1-inteinC-HFT纯化后的电泳图；图2d为凝胶过滤纯化gb1-inteinC-HFT凝胶的UV图；图2e为TEM检测纯化的gb1-inteinC-HFT的电镜图；图2f为TEM检测纯化的gb1-inteinC-PFT的电镜图。
图3为GFP蛋白和strep2高效共价偶联至HFT后的分析图；
图3a为考马斯亮蓝法(CB assay)检测目的产物GFP-HFT含量的电泳图；图3b为使用Western-blotting检测(WB assay)GFP与HFT偶联效率的电泳图；图3c为考马斯亮兰法检测目的产物strep2-HFT含量的电泳图；图3d为使用Western-blotting分析strep2多肽与HFT偶联效率的电泳图。
图4为通过自剪切的方式负载GFP蛋白和strep2多肽不影响纳米颗粒结构稳定性的分析图；
图4a为凝胶过滤纯化GFP-HFT的考马斯亮蓝染色法电泳分析图；图4b为凝胶过滤纯化GFP-HFT的UV曲线图；图4c为TEM检测纯化的GFP-HFT的电镜图；图4d为凝胶过滤纯化strep2-HFT的电泳分析图；图4e为凝胶过滤纯化strep2-HFT的UV曲线图；图4f为TEM检测纯化的strep2-HFT的电镜图。
图5为本发明提供的纳米颗粒与外源蛋白经内含肽介导蛋白编辑技术剪接制备不同纳米器件示意图。
图6为纳米颗粒可高效负载多肽表位和鞭毛蛋白的分析图；
图6a为纳米疫苗SP70-HFT的凝胶过滤纯化电泳分析图；图6b为凝胶过滤纯化纳米疫苗SP70-HFT的UV曲线图；图6c为纳米疫苗SP70-HFT的透射电镜图；图6d为SP70与CBLB共递送纳米制剂SP70-CBLB-HFT的凝胶过滤纯化电泳分析图；图6e为凝胶过滤纯化共递送纳米制剂SP70-CBLB-HFT的UV曲线图；图6f为共递送纳米制剂SP70-CBLB-HFT的透射电镜图；图6g为纳米佐剂CBLB-HFT的凝胶过滤纯化电泳分析图；图6h为凝胶过滤纯化纳米佐剂CBLB-HFT的UV曲线图；图6i为纳米佐剂CBLB-HFT的透射电镜图。
图7为模块化纳米载体是否可高效负载CpG的分析图；
图7a为rhizavidin-HFT的凝胶过滤纯化电泳分析图；图7b为rhizavidin-HFT凝胶过滤纯化的UV曲线图；图7c为rhizavidin-HFT的透射电镜图；图7d为SP70-rhizavidin-HFT的凝胶过滤纯化电泳分析图；图7e为SP70-rhizavidin-HFT凝胶过滤纯化的UV曲线图；图7f为SP70-rhizavidin-HFT的透射电镜图；图7g为SP70-rhizavidin-HFT的二维类平均图；图7h为目的蛋白rhizavidin-HFT结合生物素化CpG电泳分析图。
图8为以SP70表位为模型分析不同疫苗剂型对免疫效果的影响分析图；
图8a-c为ELISA分析不同方式递送CpG佐剂以及CBLB诱导IgG滴度；图8d体外细胞滴定分析不同疫苗制剂诱导的抗体中和效价；图8e为共递送纳米制剂诱导的免疫应答。
图9为蛋白编辑剪切技术可在HFT/PFT载体上高效负载HA抗原分析图；
图9a为重组基因HA-HFT表达分析电泳图；图9b为凝胶过滤纯化基于蛋白编辑剪切技术制备的HA-HFT的电泳分析图；图9c为凝胶过滤纯化的HA-HFT的UV曲线图；图9d为HA-HFT的透射电镜图；图9e为凝胶过滤纯化基于蛋白编辑剪切技术制备的HA-PFT的电泳分析图；图9f为凝胶过滤纯化的HA-PFT的UV曲线图；图9g为HA-PFT的透射电镜图。
图10为纳米佐剂CpG-HFT增强HA特异性体液免疫应答并调控IgG类型的分析图。
图10a为ELISA分析揭示纳米载体和纳米佐剂均可增强HA特异性体液免疫应答；图10b-d为ELISA分析揭示疫苗诱导抗体可特异性识别H1N1(P)、H3N2和H7H9毒株，且纳米佐剂与纳米疫苗混合制剂诱导的抗体识别能力高于纳米疫苗本身。
具体实施方式
下面结合实验及实施例，对本发明的权利要求做进一步的详细说明，但不构成对本发明的任何限制，任何在本发明权利要求保护范围内所做的有限次修改，仍在本发明的权利要求保护范围内。
本发明的实验及实施例中涉及以下蛋白质、多肽的基因合成在上海吐露港生物科技有限公司，引物合成在南京金斯瑞生物科技有限公司。
其中，HFT为人源的重链铁蛋白(human heavy chain of human ferrtin)，PFT为强烈火球菌的重链铁蛋白(pyrococcus furiosus heavy chain of human ferrtin)。
本发明中GFP-inteinN-gb1、strep2-gp41-1-inteinN-gb1、SP70-gp41-1-inteinN-gb1、rhizavidin-gp41-1-intN-gb1、CBLB-gp41-1-inteinN、SP70-CBLB-gp41-1-inteinN、H1HA10-gp41-1-inteinN-gb1为inteinN相关的“货物”融合蛋白。“货物”融合蛋白的基因通过overlap PCR方法扩增目的基因，使用的5’引物和3’引物分别带有NcoI和XhoI酶切位点。融合基因经酶切后插入相同酶切的线性化pET28a载体，转化至大肠杆菌JM109进行重组克隆构建。gb1-gp41-1-inteinC-HFT、gb1-gp41-1-inteinC-PFT为inteinC相关的“载体”融合蛋白。“载体”融合蛋白的基因亦通过overlap PCR方法扩增目的基因，5’引物和3’引物分别带有EcoRI和XhoI酶切位点。酶切后的片段插入pET28a线性化载体后，转化大肠杆菌JM109。上述重组基因经测序正确后，提取质粒转化至大肠杆菌BL21(DE3)plysS进行蛋白表达。挑取单克隆菌株，接种至20ml的卡纳氯霉素双抗LB培养基中，37℃摇床过夜培养。第二天1:25接种至500ml的卡纳氯霉素双抗的LB培养基，37℃、250rpm培养至OD为0.6-0.8时，加入0.2mM IPTG进行诱导。25℃、250rpm诱导5h后，4000rpm、4℃、30分钟离心收菌。弃上清，菌可用于下一步纯化或-80℃冻存备用。
本发明重组“载体”融合蛋白基因使用的引物及“货物”融合蛋白的基因使用的引物详见表1：
表1：
注：本发明中使用的引物，用于制备不同功能的纳米器件。
本发明涉及的抗原或佐剂蛋白纯化：所有的蛋白均采用Ni2+亲和层析的方法进行纯化。不同的重组蛋白，因其性质不同，溶解度不同，采用的纯化方法略有差异。GFP-inteinN-gb1、strep2-inteinN-gb1、SP70-inteinN-gb1、H1HA10-inteinN-gb1和rhizavidin-inteinN-gb1为可溶性蛋白，细菌裂解液上清液可直接加入Ni2+树脂中开展亲和层析纯化。30ml NT buffer重悬后裂解细菌的上清，与1ml的树脂在4℃摇床上混匀30分钟。而后，非特异性结合蛋白随液体以重力方式流出。接着，加入含10mM、20mM、40mM咪唑的20ml NT buffer洗涤树脂，去掉非特异性结合蛋白。而后，加入500mM咪唑的NT buffer洗脱。最后，洗脱产物在1L的NT buffer中4℃透析过夜。CBLB-inteinN和SP70-CBLB-inteinN大部分形成沉淀，沉淀用含2M尿素的NT buffer重悬后，细菌裂解液上清液用可溶蛋白纯化的方法纯化。CBLB-inteinN和SP70-CBLB-inteinN大部分形成包涵体沉淀，可用含2M尿素的NTbuffer重悬后，重悬液离心后上清用可溶蛋白纯化的方法纯化。
实验例1纳米载体模块平台构建
①内含肽(inteinC)与HFT进行基因融合，获得重组蛋白inteinC-ferrtin(图2a中标记为1)。
②内含肽(intein)引入链球菌G蛋白B1结构域标签(gb1)构建接头gb1-inteinC(图1中示为A)。重链铁蛋白(ferrti，图1中示为C)的N端与接头gb1-inteinC进行基因融合构成重组蛋白gb1-inteinC-ferrtin。分别以HFT和PFT构建重组蛋白gb1-inteinC-HFT(图2a-2b中标记为2)和gb1-inteinC-PFT(图2a-2b中标记为3)。在图2等本申请的各图中，本发明中所有UV profile of gel filtration的横坐标代表洗脱体积，纵坐标代表紫外光A280nm处的吸光度变化。
取上述三种重组蛋白细菌裂解进行Tricine-SDS-PAGE电泳，结果如图2a所示，其中，control：pET28a载体空白对照菌裂解液，1：inteinC-ferrtin，2：gb1-inteinC-HFT，3：gb1-inteinC-PFT，all：细菌裂解液，up：上清液。从图2a可见，35kDa处电泳细菌裂解液有明显条带，但相应位置上清中几乎不可见蛋白条带，表明重组蛋白inteinC-ferrtin形成包涵体。35kDa-40kDa处电泳带非常明显，重组蛋白gb1-inteinC-HFT和gb1-inteinC-PFT表达量均显著增高，且可溶性高。由结果可见，引入gb1能够极大地提高重链铁蛋白在细胞内的表达并改善其可溶性，有利于形成规模性制备，故重组蛋白gb1-inteinC-ferrtin纳米颗粒具有用于制备纳米载体平台的可行性。
用30ml的50mM Tris，pH 7.5，150mM Nacl(NT buffer)重悬500ml的LB培养细菌，而后超声破碎。超声结束后，12000rpm、4℃、30分钟离心，上清转移至50ml的离心管中。加入0.15g/ml的硫酸铵固体，在4℃摇床混匀15分钟后，12000rpm、4℃、30分钟离心。弃上清，得硫酸铵沉淀产物(图2b-2c中标记为pellet)。用10ml的NT buffer重悬硫酸铵沉淀产物，在1L的NT buff中4℃透析过夜。BCA测得gb1-inteinC-HFT和gb1-inteinC-PFT浓度分别为10mg/ml和5mg/ml。硫酸铵沉淀产物用50mM pH8.0Tris及150mM Nacl(NT buffer)重悬后，在上海闪谱生物科技有限公司ClearFirst-3000型蛋白纯化系统上利用superpose6Increase凝胶过滤分离纯化，并进行Tricine-SDS-PAGE进行电泳观察纯度，纯化结果见图2b-2d，其中，图2b为gb1-inteinC-HFT和gb1-inteinC-PFT经硫酸铵沉淀后的电泳图，其中，up：上清液，pellet：硫酸铵沉淀产物；图2c为gb1-inteinC-HFT纯化后的电泳图，其中，pellet：硫酸铵富集沉淀重悬产物，Purification of gb1-inteinC-HFT by gelfiltration：凝胶过滤方法纯化gb1-inteinC-HFT，gel filtration of gb1-inteinC-HFT：凝胶过滤纯化gb1-inteinC-HFT，9-17：分管收集凝胶过滤纯化gb1-inteinC-HFT样品的编号；图2d为凝胶过滤纯化gb1-inteinC-HFT凝胶的UV图，检测波长为280nm，箭头所指的是gb1-inteinC-HFT的分布位置。
通过透射电镜(transmission electron microscope，TEM)观察纯化后的重组蛋白
gb1-inteinC-HFT和gb1-inteinC-PFT，透射电镜放大倍数为110k，标尺长度为20nm。结果详见图2e及2f，其中，图2e为TEM检测纯化的gb1-inteinC-HFT的电镜图(负染制样)；图2f为TEM检测纯化的gb1-inteinC-PFT的电镜图；图2e和图2f显示gb1-inteinC-HFT和gb1-inteinC-PFT均以纳米颗粒形式存在，本发明中透射电镜观察负染样品所用标尺均代表20nm，箭头标示的为纳米颗粒。
两种重组蛋白均以10-20nm的球形纳米颗粒形式存在，可见重组蛋白gb1-inteinC-ferrtin通过分子自组装形成纳米颗粒。经测定，重组蛋白gb1-inteinC-HFT和gb1-inteinC-PFT均能以24单体自组装成直径为18nm纳米颗粒。
实验例2纳米载体模块平台可与外源蛋白高效共价偶联
以实施例1构建的重组蛋白gb1-inteinC-HFT纳米颗粒为例，分别与外源蛋白GFP蛋白和strep2多肽标签(Trp-Ser-His-Pro-Gln-Phe-Glu-Lys)进行内含肽介导蛋白剪接修饰，观察该纳米颗粒的稳定性。
1.GFP蛋白和strep2多肽标签的C端分别通过基因融合引入接头inteinN-gb1，分别构成GFP-inteinN-gb1和strep2-inteinN-gb1。
2.取2μl浓度为10mg/ml的重组蛋白gb1-inteinC-HFT母液加入DTT中，在室温下1小时内，在DTT中逐渐增加浓度为3mg/ml的GFP-inteinN-gb1蛋白母液或5mg/ml的strep2-inteinN-gb1蛋白母液，以3μl、6μl、9μl、12μl、15μl、18μl、21μl、24μl递增添加，并观察目的产物GFP-HFT或strep2-HFT的量。反应条件：反应体系总体积为30μl，2mM DTT，室温1小时。然后分别进行考马斯亮蓝法检测(CB assay)以及western blotting检测(WB assay)
3.检测结果
如图3a及3c所示，随着GFP蛋白和strep2标签的量的逐渐增加，会促进目的产物GFP-HFT(图3a)和strep2-HFT(图3c)的量增加。固定gb1-inteinC-HFT的量，增加GFP-inteinN-gb1或strep2-inteinN-gb1反应浓度，反应底物gb1-inteinC-HFT与GFP-inteinN-gb1或strep2-inteinN-gb1的摩尔比例接近1:1时反应达最大程度，内含肽自剪切效率接近最高，未被剪切gb1-inteinC-HFT几乎无残留。反应完成后，用GFP标签抗体、strep2标签抗体和HFT抗体分别检测反应体系中各反应底物和目的产物的变化。Western-blotting检测数据表明反式剪切效率非常高，反应彻底(图3b和3d)。可见，GFP及strep2均能被高效、特异地共价交联至HFT蛋白上。
反应结束后，所得目的产物在上海闪谱生物科技有限公司ClearFirst-3000型蛋白纯化系统上利用superpose 6increase开展凝胶过滤加以纯化。结果详见图4，其中，图4a为凝胶过滤纯化GFP-HFT的考马斯亮蓝染色法电泳分析图；图4b为凝胶过滤纯化GFP-HFT的UV曲线图；图4c为TEM检测纯化的GFP-HFT的电镜图(负染制样)；图4d为凝胶过滤纯化strep2-HFT的电泳分析图(考马斯亮蓝染色)；图4e为凝胶过滤纯化strep2-HFT的UV曲线图；图4f为TEM检测纯化的strep2-HFT的电镜图(负染制样)；before：内含肽自剪切前，after：内含肽自剪切后，图4a 8-19及4d中的9-20：分管收集凝胶过滤纯化的目的产物样品的编号。
如图4a，4b所示，分子量极为接近的蛋白GFP-HFT和GFP-inteinN-gb1可以很好地分开，且蛋白GFP-HFT洗脱位置比GFP-inteinN-gb1早，图4b中箭头所指为GFP-HFT的峰，显示该蛋白GFP-HFT以大分子聚合物的形式存在。图4d，4e所示，分子量极为接近的蛋白strep2-HFT和strep2-inteinN-gb1也可以很好地分开，且蛋白strep2-HFT洗脱位置比strep2-inteinN-gb1早，图4e中箭头所指为GFP-HFT的峰，显示该蛋白strep2-HFT以大分子聚合物的形式存在。将分离纯化的目的产物GFP-HFT和strep2-HFT通过透射电镜观察，GFP-HFT和strep2-HFT均以纳米颗粒的形式存在(图4c，4f)。综上，蛋白或多肽可高效剪切至HFT纳米颗粒，且不影响纳米颗粒稳定性，纳米载体模块平台可与外源蛋白高效共价偶联。
实验例3纳米载体模块平台的多态性
以人肠道病毒71型(enterovirus 71)的SP70中和性表位为抗原模型，探讨不同的给药系统与佐剂对抗原免疫应答的影响。利用纳米载体模块技术允许多态性地修饰纳米颗粒的特点，制备了不同功能的纳米颗粒，为研究不同疫苗剂型对抗原免疫原性的影响，建立纳米佐剂、疫苗-佐剂共递送纳米制剂和纳米疫苗，详见图5。
以CpG或鞭毛蛋白(flagellin)为佐剂分别制备相应制剂。鞭毛蛋白具体采用CBLB。CBLB是鞭毛蛋白的一个截短体，具备其佐剂功能。
纳米颗粒可高效负载多肽表位和鞭毛蛋白的分析图详见图6：其中，图6a为纳米疫苗SP70-HFT的凝胶过滤纯化电泳分析图；图6b为凝胶过滤纯化纳米疫苗SP70-HFT的UV曲线图；图6c为纳米疫苗SP70-HFT的透射电镜图；图6d为SP70与CBLB共递送纳米制剂SP70-CBLB-HFT的凝胶过滤纯化电泳分析图；图6e为凝胶过滤纯化共递送纳米制剂SP70-CBLB-HFT的UV曲线图；图6f为共递送纳米制剂SP70-CBLB-HFT的透射电镜图(负染制样)；图6g为纳米佐剂CBLB-HFT的凝胶过滤纯化电泳分析图；图6h为凝胶过滤纯化纳米佐剂CBLB-HFT的UV曲线图；图6i为纳米佐剂CBLB-HFT的透射电镜图(负染制样)；before：内含肽自剪切前，after：内含肽剪切后，图6a、6d及6g中的8-19：分管收集凝胶过滤纯化的目的产物样品的编号。
1.纳米疫苗：①内含肽(intein)引入链球菌G蛋白B1结构域标签(gb1)构建接头gb1-inteinC(图1中示为A)。人源重链铁蛋白HFT的N端与接头gb1-inteinC进行基因融合构成重组蛋白gb1-inteinC-HFT。24个重组蛋白gb1-inteinC-HFT分子自组装成24聚体纳米颗粒。②SP70的C端引入inteinN-gb1构成蛋白SP70-inteinN-gb1。在2mM DTT溶液中，按摩尔比为1:1添加纳米颗粒和蛋白SP70-inteinN-gb1，经蛋白剪接得到SP70-HFT纳米颗粒，即为纳米疫苗给药系统。
2.纳米佐剂：
2.1 CBLB的C端引入inteinN构成蛋白CBLB-inteinN，在2mM DTT溶液中，按HFT与CBLB摩尔比1:1添加纳米颗粒和CBLB-inteinN，经蛋白质剪接获得CBLB-HFT，并自组装形成CBLB-HFT纳米颗粒。
2.2 CpG不能直接与HFT共价连接，但生物素化的CpG可通过抗生物素-生物素的高亲结合作用，负载至纳米颗粒表面。生物素化的CpG可通过抗生物素-生物素的高亲结合作用，负载至纳米颗粒表面。抗生物素蛋白rhizavidin的C端引入inteinN-gb1构成蛋白rhizavidin-inteinN-gb1，在2mM DTT溶液中，添加纳米颗粒和rhizavidin-inteinN-gb1，经蛋白质剪接获得rhizavidin-HFT，并自组装形成rhizavidin-HFT纳米颗粒。
模块化纳米载体是否可高效负载CpG的分析图详见图7；其中，图7a为rhizavidin-HFT的凝胶过滤纯化电泳分析图；图7b为rhizavidin-HFT凝胶过滤纯化的UV曲线图；图7c为rhizavidin-HFT的透射电镜图(负染制样)；图7d为SP70-rhizavidin-HFT的凝胶过滤纯化电泳分析图；图7e为SP70-rhizavidin-HFT凝胶过滤纯化的UV曲线图；图7f为SP70-rhizavidin-HFT的透射电镜图(负染制样)；图7g为SP70-rhizavidin-HFT的二维类平均图；图7h为目的蛋白rhizavidin-HFT结合生物素化CpG电泳分析图；before：内含肽自剪切前，after：内含肽剪切后，图7a、7d及7g中的8-19编号：分管收集凝胶过滤纯化的目的产物样品的编号。
如图7a-b所示：rhizavidin可高效偶联至HFT，目的产物可用凝胶过滤加以纯化。凝胶过滤纯化的rhizavidin-HFT经透射电镜观察以球形纳米颗粒的形式存在(图7c)。且rhizavidin与SP70以1:5的摩尔比例偶联，亦不影响纳米颗粒结构，详见图7d-f。如图7g所示，二维类平均(2D-class average)分析，白色圆圈表示目的蛋白自装配成球形纳米尺寸的颗粒，圆圈中离散分布的白色亮斑为rhizavidin蛋白(电子密度比多肽大)，且亮斑平均数目与初始反应的rhizavidin-inteinN-gb1和SP70-inteinN-gb1的浓度一致。如图7h所示，目的产物rhizavidin-HFT与足量的生物素化的CpG结合后，蛋白条带迁移，揭示CpG被成功负载。
因此，生物素化的CpG可通过以下方法与纳米颗粒共价连接。如图5所示，①内含肽(intein)引入链球菌G蛋白B1结构域标签(gb1)构建接头gb1-inteinC(图1中示为A)。人源重链铁蛋白HFT的N端与接头gb1-inteinC进行基因融合构成重组蛋白gb1-inteinC-HFT。24个重组蛋白gb1-inteinC-HFT分子自组装成24聚体纳米颗粒。②rhizavidin的C端引入inteinN-gb1构成蛋白rhizavidin-inteinN-gb1。在2mM DTT溶液中，按摩尔比为1:1:1添加纳米颗粒、生物素化的CpG和蛋白rhizavidin-inteinN-gb1，经蛋白剪接和抗生素蛋白与生物素化CpG高亲和相互作用得到CpG-HFT纳米颗粒，即为纳米佐剂给药系统。
3.疫苗-佐剂共递送纳米制剂
3.1 CBLB与SP70基因融合成蛋白SP70-CBLB，CBLB与SP70的摩尔比为1:1。SP70-CBLB的CBLB的C端引入inteinN构成蛋白SP70-CBLB-inteinN，在2mM DTT溶液中，按HFT与SP70-CBLB摩尔比1:1添加纳米颗粒和SP70-CBLB-inteinN，经蛋白质剪接获得SP70-CBLB-HFT，并自组装形成SP70-CBLB-HFT纳米颗粒。
如图6a-b所示为Tricine-SDS-PAGE和UV曲线分析凝胶过滤分离纯化目的产物SP70-HFT的结果。结果显示：该方法可纯化较纯目的产物；before和after分别表示为：自剪切前后样品，数字8-19代表凝胶过滤纯化目的产物收集样品编号(1ml/管)。图6c：TEM分析目的产物SP70-HFT以纳米颗粒形式存在，箭头指示目的产物。图6d-f和图6g-i分别为目的产物SP70-CBLB-HFT和CBLB-HFT凝胶过滤法纯化及质量检测。图6a、6d和6g显示未被剪切的gb1-inteinC-HFT条带几乎不可见，说明纳米颗粒的自剪切效率高。图6c、6f和6i显示三种目的产物均以纳米颗粒形式存在，表明本发明提供的纳米颗粒可通过自剪切与抗原、佐剂共价交联，从而赋予纳米颗粒不同的功能。
3.2如图5所示，①内含肽(intein)引入链球菌G蛋白B1结构域标签(gb1)构建接头gb1-inteinC(图1中示为A)。人源重链铁蛋白HFT的N端与接头gb1-inteinC进行基因融合构成重组蛋白gb1-inteinC-HFT。24个重组蛋白gb1-inteinC-HFT分子自组装成24聚体纳米颗粒。②SP70的C端引入inteinN-gb1构成蛋白SP70-inteinN-gb1。在2mM DTT溶液中，按摩尔比为1:1添加纳米颗粒和蛋白SP70-inteinN-gb1，经蛋白剪接得到蛋白SP70-HFT。rhizavidin的C端引入inteinN-gb1构成蛋白rhizavidin-inteinN-gb1。在2mM DTT溶液中，添加纳米颗粒、生物素化的CpG、蛋白rhizavidin-inteinN-gb1和蛋白SP70-inteinN-gb1，经蛋白剪接得到SP70-CpG-HFT纳米颗粒，即为纳米共递送给药系统。其中，蛋白rhizavidin-inteinN-gb1和蛋白SP70-inteinN-gb1的摩尔比为1:5，纳米颗粒与蛋白rhizavidin-inteinN-gb1和蛋白SP70-inteinN-gb1的混合物的摩尔比为6:1：5，生物素化的CpG和蛋白rhizavidin-inteinN-gb1的摩尔比为1:1。
实验例4不同的免疫制剂的免疫效果
1.建立小鼠试验组：SP70-CpG-HFT组使用摩尔比为1:1的0.4μg/只生物素化CpG和10μg/只SP70-rhizavidin-HFT冰浴混合30分钟后，免疫小鼠。SP70-HFT+CpG-HFT组使用纳米疫苗SP70-HFT与纳米佐剂CpG-HFT共同免疫小鼠，使用剂量分别为10μg/只和1.6μg/只。纳米疫苗SP70-HFT组：SP70-HFT免疫小鼠，使用剂量分别为10μg/只。SP70-HFT+CpG组：SP70-HFT和0.4μg/只生物素化CpG共同免疫小鼠。各组小鼠5只，3次接种，每次接种间隔2周。第三次免疫2周后眼眶采血开展免疫分析。
表2：
注：SP70-HFT：纳米疫苗；SP70-HFT+CpG：纳米疫苗和游离CpG混合剂；SP70-CpG-HFT：SP70和CpG共递送纳米制剂；SP70-HFT+CpG-HFT：纳米疫苗和纳米佐剂联合制剂。
不同剂型疫苗接种小鼠后，接种3次，每次间隔2周，第三次免疫2周后眼眶取血进行ELISA分析。
如8a所示，ELISA分析不同方式递送CpG佐剂诱导IgG滴度。低剂量游离的0.4μg/只CpG无明显佐剂效果，纳米佐剂CpG-HFT的助力效果明显，SP70-CpG-HFT共递送纳米制剂的佐剂效果最佳。可见，低剂量的0.4μg/只游离CpG佐剂效果不明显。但是，用纳米颗粒递送的纳米佐剂CpG-HFT及抗原-佐剂共递送纳米颗粒SP70-CpG-HFT佐剂效果显著，且共递送纳米制剂诱导B细胞免疫应答最高效。这是因为共递送纳米制剂最大程度保证抗原与佐剂定位至同一个抗原呈递细胞(antigen presenting cell，简称APC)，充分发挥佐剂的助力效应。使用ELISA检测不同亚型的IgG抗体滴度(1:1000)，IgG亚型分析结果显示，共递送纳米制剂SP70-CpG-HFT同时明显增强Th1和Th2型的IgG滴度，见表1，其中Th2型的IgG1占比例最高。
2.根据本实验例的第1点建立小鼠试验组(每组5只小鼠，接种3次，每次间隔2周)，分别为SP70-CBLB-HFT(10μg/只)组以及SP70-HFT(10μg/只)+CBLB-HFT(10μg/只)组，然后取血进行ELISA分析(第3次免疫两周后眼眶取血)。如图8b所示，SP70-CBLB-HFT诱导的抗体滴度低于SP70-HFT+CBLB-HFT。
3.CpG作为佐剂，对B细胞免疫应答的助力效果优于鞭毛蛋白；对比共递送的CpG和鞭毛蛋白，发现CpG诱导抗体滴度更高；详见图8c。
4.分别取本实验例的第1～2点的各试验组小鼠的血清，以2倍为梯度系列稀释血清(50μl)，与100TCID50的EV71 G082(50μl)在37℃的CO2培养箱中放置1h后，加入15000个人胚胎横纹肌肉瘤细胞(human embryo rhabdomyosarcoma cells，RD cells)。感染3天后观察CPE，统计中和抗体效价。如图8d所示，对抗体中和效价分析发现，SP70-CpG-HFT、SP70-HFT+CpG-HFT、SP70-CBLB-HFT以及SP70-HFT+CBLB-HFT的抗体效价均显著高于SP70-HFT，其中以SP70-CpG-HFT诱导的抗体效价最高。
综上，以SP70表位为模型分析不同疫苗剂型对免疫效果的影响分析图详见图8：其中，图8a-c为ELISA分析不同方式递送CpG佐剂以及CBLB诱导IgG滴度；图8a为不同方式递送CpG对SP70IgG抗体滴度的影响，与纳米疫苗SP70-HFT相比，纳米疫苗混合游离的CpG(SP70-HFT+CpG)无明显佐剂效果。但是纳米疫苗与纳米佐剂CpG-HFT混合制剂及SP70与CpG的共递送纳米制剂SP70-CpG-HFT可显著提高特异性抗体滴度。且共递送纳米制剂效果比纳米疫苗与纳米佐剂混合制剂诱导的抗体滴度更高。图8b揭示当CBLB为佐剂，共递送纳米制剂诱导特异抗体滴度高于纳米疫苗SP70-HFT和纳米佐剂CBLB-HFT混合制剂。图8c表明CpG的佐剂效果高于CBLB。图8d体外细胞滴定分析不同疫苗制剂诱导的抗体中和效价。抗体的中和效价结果显示，共递送纳米制剂诱导的抗体中和能力最高，纳米疫苗和纳米佐剂混合制剂刺激产生的抗体中和能力次之，均明显高于纳米疫苗本身。且CpG为佐剂诱导的抗体中和滴度优于CBLB佐剂。图8e：体内致死保护分析揭示共递送纳米制剂可提供最为高效的免疫保护(75％)，纳米疫苗和纳米佐剂提供的保护效果次之(50％)，两者均高于纳米疫苗(25％)。免疫分析结果显示，共递送纳米制剂诱导的免疫应答最有效，纳米佐剂与纳米疫苗混合剂亦有显著佐剂效果。
实验例5纳米载体模块平台的稳定性
流感病毒血凝素茎部区是通用型流感疫苗的重要靶标，其中大肠杆菌表达的H1HA10三聚体诱导的抗体可中和不同亚型的毒株。采用现有的基因融合方法进行H1HA10(简写为HA)与ferrtin融合获得重组蛋白HA-HFT。
结果详见图9：即蛋白编辑剪切技术可在HFT/PFT载体上高效负载HA抗原；其中，图9a为重组基因HA-HFT表达分析电泳图；图中，control：pET28a载体空白对照菌裂解液；all：细菌裂解液；up：细菌裂解液上清；HA-HFT：HA(H1HA10，简写为HA)与HFT直接基因融合形成重组蛋白。图9b为凝胶过滤纯化基于蛋白编辑剪切技术制备的HA-HFT的电泳分析图。图9c为凝胶过滤纯化的HA-HFT的UV曲线图。图9d为HA-HFT的透射电镜图(负染制样)。图9e为凝胶过滤纯化基于蛋白编辑剪切技术制备的HA-PFT的电泳分析图。图9f为凝胶过滤纯化的HA-PFT的UV曲线图。图9g为HA-PFT的透射电镜图(负染制样)。before：内含肽自剪切前，after：内含肽剪切后，图9b及9e中的8-19编号：分管收集凝胶过滤纯化的目的产物样品的编号(1ml/管)。
如图9a所示，菌裂解液的上清液中不含有重组蛋白HA-HFT，可见重组蛋白形成包涵体沉淀。
采用本发明的方法将HA共价偶联至HFT形成HA-HFT，如图9b-d所示基于蛋白编辑剪切制备的HA-HFT以纳米颗粒形式存在。
采用本发明的方法将HA共价偶联至PFT形成HA-PFT，如图9e-g所示基于蛋白编辑剪切制备的HA-PFT同样以纳米颗粒形式存在，也就是说PFT亦可负载HA。
由于H1HA10需要维持天然三聚体构象，因此本发明中仅研究纳米佐剂CpG-HFT对纳米疫苗HA-HFT/PFT免疫原性的影响。
实验例6 CpG-HFT增强针对HA的Th1型IgG应答
建立小鼠试验组：每组小鼠5只，免疫三次，每次接种间隔2周，免疫3周后眼眶采血用于免疫分析。HA组使用H1HA10-inteinN免疫小鼠，使用剂量10μg/只。HA-HFT+CpG-HFT组使用纳米疫苗HA-HFT与纳米佐剂CpG-HFT共同免疫小鼠，使用剂量分别为10μg/只和1.6μg/只。纳米疫苗HA-HFT组：HA-HFT免疫小鼠，使用剂量分别为10μg/只。纳米疫苗HA-PFT组：HA-PFT免疫小鼠，使用剂量分别为10μg/只。HA-PFT+CpG-HFT组使用纳米疫苗HA-PFT与纳米佐剂CpG-HFT共同免疫小鼠，使用剂量分别为10μg/只和1.6μg/只。
不同剂型疫苗接种小鼠后，第3次免疫2周后眼眶取血进行ELISA分析。用纳米颗粒递送HA，比HA诱导抗体滴度提高10倍。CpG-HFT的联合使用，特异性抗体梯度再次提高10倍。以PFT为载体诱导抗体滴度高于HFT。PFT与小鼠的ferrtin相似度低，可能具有一定的佐剂作用。
纳米佐剂CpG-HFT增强HA特异性体液免疫应答并调控IgG类型的分析图详见图10；其中，图10a为ELISA分析揭示纳米载体和纳米佐剂均可增强HA特异性体液免疫应答。与HA抗原相比，纳米疫苗HA-HFT可将HA特异性IgG抗体滴度提高10倍左右，纳米佐剂CpG-HFT与纳米疫苗混合制剂可进一步提高特异性抗体滴度(10倍左右)。另外，PFT为载体效果高于HFT，可能是由PFT与小鼠内铁蛋白ferritin同源性低，有一定的佐剂作用引起。人源HFT与小鼠铁蛋白高度同源(氨基酸序列99％相同)，HFT蛋白自身无佐剂效果。图10b-d ELISA分析揭示，疫苗诱导抗体可特异性识别H1N1(P)、H3N2和H7H9毒株，且纳米佐剂与纳米疫苗混合制剂诱导的抗体识别能力高于纳米疫苗本身。(一抗是接种3次剂量疫苗小鼠的血清，二抗是辣根过氧化物酶HRP标记的鼠二抗)。
图10a显示，纳米颗粒递送HA显著改善机体响应HA的B细胞免疫应答，比HA诱导抗体滴度提高10倍，与纳米佐剂CpG-HFT联合使用，特异性抗体梯度再次提高10倍，进一步提高应答水平。以PFT为载体诱导的抗体滴度高于HFT载体，提示PFT与小鼠的ferrtin相似度低，PFT载体可能具有一定的佐剂功能。图10b-d显示，HA特异性相关抗体可识别不同亚型的流感毒株，识别亚型涵盖：H1NI的(简写P)、H3N2和H7N9(安徽毒株，简写Ahpri)。ELISA分析IgG亚型(1:105稀释接种不同剂型疫苗小鼠的血清)发现，CpG-HFT主要增强Th2型IgG2和IgG3免疫应答，对Th1型的IgG有很强的佐剂效果，见表3。用纳米模块负载CpG制备纳米佐剂，与负载HA的纳米疫苗联合使用，显著增强HA特异性抗体免疫应答，诱导抗体识别不同亚型的流感病毒毒株能力亦增强。
表3：
注：HA：H1HA10-inteinN；HA-HFT：HFT纳米颗粒递送H1HA10；HA-HFT+CpG-HFT：HA-HFT与CpG-HFT联合制剂；HA-PFT：PFT纳米颗粒递送H1HA10；HA-PFT+CpG-HFT：HA-PFT与CpG-HFT联合制剂(备注：由于gb1特异性地结合所有类型的IgG，因此在检测HA特异性抗体滴度时使用H1HA10-inteinN蛋白。在利用蛋白编辑剪切时，基于gb1可显著改善蛋白的可溶性，因此使用H1HA10-inteinN-gb1)。
综上所述，本发明的纳米载体模块平台可在纳米颗粒表面高效展示抗原蛋白、免疫增强剂、多肽表位，用于增强型的预防性亚单位疫苗和个性化多肽表位肿瘤疫苗研发等领域。解决了负载蛋白对蛋白自装配纳米颗粒稳定性影响以及纳米颗粒难以共递送佐剂和抗原分子的难题。
实施例1
取人源重链铁蛋白(HFT)的基因N端融合gbl-inteinC基因，通过overlap PCR方法扩增目的基因，5’引物和3’引物分别带有EcoRI和XhoI酶切位点。EcoRI和XhoI酶切后的片段插入pET28a线性化载体后，转化大肠杆菌JM109。经测序正确后，提取质粒转化至大肠杆菌BL21(DE3)plysS进行蛋白表达。挑取单克隆菌株，接种至20ml的卡纳氯霉素双抗LB培养基中，37℃摇床过夜培养。第二天1:25接种至500ml的卡纳氯霉素双抗的LB培养基，37℃、250rpm培养至OD为0.6-0.8时，加入0.2mM IPTG进行诱导。25℃、250rpm诱导5h后，4000rpm、4℃、30分钟离心收菌。弃上清，菌可用于下一步纯化或-80℃冻存备用。用30ml的50mMTris，pH 7.5，150mM Nacl(NT buffer)重悬500ml的LB培养细菌，而后超声破碎，先经过硫酸铵沉淀法初步纯化后在上海闪谱生物科技有限公司ClearFirst-3000型蛋白纯化系统上利用superpose 6 Increase分子排阻分离纯化，获得重组蛋白gbl-inteinc-HFT，重组蛋白gbl-inteinc-HFT自装配成24聚体纳米颗粒，直径为18nm，HFT的N端及连接的接头gbl-inteinc伸展至纳米颗粒表面。该纳米颗粒为纳米载体模块平台。
多肽表位SP70的C端融合内含肽的N端形成重组蛋白SP70-inteinN-gb1，作为外源蛋白。
重组蛋白SP70-inteinN-gb1与gbl-inteinc-HFT纳米颗粒混合构成反应体系，其中，重组蛋白SP70-inteinN-gb1与人源重链铁蛋白等摩尔比，在反应体系中的浓度分别为30μM。在反应体系中，inteinN特异性识别并切除暴露在纳米颗粒表面的接头gbl-inteinc，多肽表位SP70与人源重链铁蛋白N端共价交联成蛋白SP70-PFT，从而构成单价抗原递送系统。
实施例2
与实施例1不同是：由强烈火球菌的重链铁蛋白PFT基因的N端与接头gbl-intein基因的C端融合，而后通过overlap PCR方法扩增目的基因，5’引物和3’引物分别带有EcoRI和XhoI酶切位点。然后经过构建质粒、转化、经测序正确后，提取质粒转化至大肠杆菌BL21(DE3)plysS进行蛋白表达。经培养、诱导、提取、纯化后获得重组蛋白gbl-inteinC-PFT，重组蛋白gbl-inteinc-PFT自装配成24聚体纳米颗粒，直径为18nm，PFT的N端及连接的接头gbl-inteinC伸展至纳米颗粒表面。该纳米颗粒为纳米载体模块平台。
鞭毛蛋白的C端融合内含肽的N端形成重组蛋白CBLB-inteinN，作为外源蛋白。重组蛋白CBLB-inteinN与gbl-inteinC-PFT的纳米颗粒混合构成反应体系，其中，重组蛋白CBLB-inteinN与PFT等摩尔比，反应体系中，重组蛋白CBLB-inteinN的浓度为20μM，PFT的浓度为10μM。在反应体系中，inteinN特异性识别并切除暴露在纳米颗粒表面的接头gbl-inteinC，CBLB与PFT的N端共价交联成蛋白CBLB-PFT，从而构成单价佐剂递送系统。
实施例3
将同物质的量的负载CpG的rhizavidin蛋白和流感病毒血凝素茎部区H1HA10的C端均融合内含肽的N端形成重组蛋白。重组蛋白与实施例2的纳米颗粒混合构成反应体系，其中，重组蛋白与PFT等摩尔比，反应体系中，重组蛋白的浓度为10μM，PFT的浓度为10μM。在反应体系中，inteinN特异性识别并切除暴露在纳米颗粒表面的接头gbl-inteinC，重组蛋白与PFT的N端共价交联成蛋白重组蛋白-PFT，从而构成单价抗原递送系统。
序列表
<110> 广州市妇女儿童医疗中心
<120> 一种基于内含肽介导纳米载体模块平台及其构建方法与应用
<160> 20
<170> SIPOSequenceListing 1.0
<210> 1
<211> 238
<212> PRT
<213> GFP
<400> 1
Val Ser Lys Gly Glu Glu Leu Phe Thr Gly Val Val Pro Ile Leu Val
1 5 10 15
Glu Leu Asp Gly Asp Val Asn Gly His Lys Phe Ser Val Ser Gly Glu
20 25 30
Gly Glu Gly Asp Ala Thr Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys
35 40 45
Thr Thr Gly Lys Leu Pro Val Pro Trp Pro Thr Leu Val Thr Thr Leu
50 55 60
Thr Tyr Gly Val Gln Cys Phe Ser Arg Tyr Pro Asp His Met Lys Gln
65 70 75 80
His Asp Phe Phe Lys Ser Ala Met Pro Glu Gly Tyr Val Gln Glu Arg
85 90 95
Thr Ile Phe Phe Lys Asp Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val
100 105 110
Lys Phe Glu Gly Asp Thr Leu Val Asn Arg Ile Glu Leu Lys Gly Ile
115 120 125
Asp Phe Lys Glu Asp Gly Asn Ile Leu Gly His Lys Leu Glu Tyr Asn
130 135 140
Tyr Asn Ser His Asn Val Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly
145 150 155 160
Ile Lys Val Asn Phe Lys Ile Arg His Asn Ile Glu Asp Gly Ser Val
165 170 175
Gln Leu Ala Asp His Tyr Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro
180 185 190
Val Leu Leu Pro Asp Asn His Tyr Leu Ser Thr Gln Ser Ala Leu Ser
195 200 205
Lys Asp Pro Asn Glu Lys Arg Asp His Met Val Leu Leu Glu Phe Val
210 215 220
Thr Ala Ala Gly Ile Thr Leu Gly Met Asp Glu Leu Tyr Lys
225 230 235
<210> 2
<211> 8
<212> PRT
<213> strep2
<400> 2
Trp Ser His Pro Gln Phe Glu Lys
1 5
<210> 3
<211> 42
<212> PRT
<213> gp41-1-inteinC
<400> 3
Met Leu Lys Lys Ile Leu Lys Ile Glu Glu Leu Asp Glu Arg Glu Leu
1 5 10 15
Ile Asp Ile Glu Val Ser Gly Asn His Leu Phe Tyr Ala Asn Asp Ile
20 25 30
Leu Thr His Asn Ser Ser Ser Ser Asp Val
35 40
<210> 4
<211> 93
<212> PRT
<213> gp41-1-inteinN
<400> 4
Thr Arg Ser Gly Tyr Cys Leu Asp Leu Lys Thr Gln Val Gln Thr Pro
1 5 10 15
Gln Gly Met Lys Glu Ile Ser Asn Ile Gln Val Gly Asp Leu Val Leu
20 25 30
Ser Asn Thr Gly Tyr Asn Glu Val Leu Asn Val Phe Pro Lys Ser Lys
35 40 45
Lys Lys Ser Tyr Lys Ile Thr Leu Glu Asp Gly Lys Glu Ile Ile Cys
50 55 60
Ser Glu Glu His Leu Phe Pro Thr Gln Thr Gly Glu Met Asn Ile Ser
65 70 75 80
Gly Gly Leu Lys Glu Gly Met Cys Leu Tyr Val Lys Glu
85 90
<210> 5
<211> 16
<212> PRT
<213> SP70
<400> 5
Tyr Pro Thr Phe Gly Glu His Lys Gln Glu Lys Asp Leu Glu Tyr Gly
1 5 10 15
<210> 6
<211> 56
<212> PRT
<213> gb1
<400> 6
Gln Tyr Lys Leu Ile Leu Asn Gly Lys Thr Leu Lys Gly Glu Thr Thr
1 5 10 15
Thr Glu Ala Val Asp Ala Ala Thr Ala Glu Lys Val Phe Lys Gln Tyr
20 25 30
Ala Asn Asp Asn Gly Val Asp Gly Glu Trp Thr Tyr Asp Asp Ala Thr
35 40 45
Lys Thr Phe Thr Val Thr Glu Lys
50 55
<210> 7
<211> 128
<212> PRT
<213> Rhizavidin
<400> 7
Phe Asp Ala Ser Asn Phe Lys Asp Phe Ser Ser Ile Ala Ser Ala Ser
1 5 10 15
Ser Ser Trp Gln Asn Gln His Gly Ser Thr Met Ile Ile Gln Val Asp
20 25 30
Ser Phe Gly Asn Val Ser Gly Gln Tyr Val Asn Arg Ala Glu Gly Thr
35 40 45
Gly Cys Gln Asn Ser Pro Tyr Pro Leu Thr Gly Arg Val Asn Gly Thr
50 55 60
Phe Ile Asp Phe Ser Val Lys Trp Asn Asn Ser Thr Glu Asn Cys Asn
65 70 75 80
Ser Asn Thr Gln Trp Thr Gly Tyr Ala Gln Val Asn Gly Asn Asn Thr
85 90 95
Glu Ile Val Thr Arg Trp Asn Leu Lys Tyr Glu Gly Gly Ser Gly Pro
100 105 110
Ala Ile Trp Gln Gly Gln Asp Thr Phe Gln Tyr Val Pro Thr Thr Glu
115 120 125
<210> 8
<211> 295
<212> PRT
<213> CBLB
<400> 8
Ala Gln Val Ile Asn Thr Asn Ser Leu Ser Leu Leu Thr Gln Asn Asn
1 5 10 15
Leu Asn Lys Ser Gln Ser Ser Leu Ser Ser Ala Ile Glu Arg Leu Ser
20 25 30
Ser Gly Leu Arg Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly Gln Ala
35 40 45
Ile Ala Asn Arg Phe Thr Ser Asn Ile Lys Gly Leu Thr Gln Ala Ser
50 55 60
Arg Asn Ala Asn Asp Gly Ile Ser Ile Ala Gln Thr Thr Glu Gly Ala
65 70 75 80
Leu Asn Glu Ile Asn Asn Asn Leu Gln Arg Val Arg Glu Leu Ser Val
85 90 95
Gln Ala Thr Asn Gly Thr Asn Ser Asp Ser Asp Leu Lys Ser Ile Gln
100 105 110
Asp Glu Ile Gln Gln Arg Leu Glu Glu Ile Asp Arg Val Ser Asn Gln
115 120 125
Thr Gln Phe Asn Gly Val Lys Val Leu Ser Gln Asp Asn Gln Met Lys
130 135 140
Ile Gln Val Gly Ala Asn Asp Gly Glu Thr Ile Thr Ile Asp Leu Gln
145 150 155 160
Lys Ile Asp Val Lys Ser Leu Gly Leu Asp Gly Phe Asn Val Asn Ser
165 170 175
Pro Gly Ile Ser Gly Gly Gly Gly Gly Ile Leu Asp Ser Met Gly Thr
180 185 190
Leu Ile Asn Glu Asp Ala Ala Ala Ala Lys Lys Ser Thr Ala Asn Pro
195 200 205
Leu Ala Ser Ile Asp Ser Ala Leu Ser Lys Val Asp Ala Val Arg Ser
210 215 220
Ser Leu Gly Ala Ile Gln Asn Arg Phe Asp Ser Ala Ile Thr Asn Leu
225 230 235 240
Gly Asn Thr Val Thr Asn Leu Asn Ser Ala Arg Ser Arg Ile Glu Asp
245 250 255
Ala Asp Tyr Ala Thr Glu Val Ser Asn Met Ser Lys Ala Gln Ile Leu
260 265 270
Gln Gln Ala Gly Thr Ser Val Leu Ala Gln Ala Asn Gln Val Pro Gln
275 280 285
Asn Val Leu Ser Leu Leu Arg
290 295
<210> 9
<211> 172
<212> PRT
<213> H1HA10
<400> 9
Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His Ser
1 5 10 15
Val Asn Leu Leu Glu Asp Ser His Gly Ser Ala Asn Ser Ser Leu Pro
20 25 30
Tyr Gln Asn Thr His Pro Thr Thr Asn Gly Glu Ser Pro Lys Tyr Val
35 40 45
Arg Ser Ala Lys Leu Arg Met Val Thr Gly Leu Arg Asn Gly Ser Ala
50 55 60
Gly Ser Ala Thr Gln Asn Ala Ile Asn Gly Ile Thr Asn Lys Val Asn
65 70 75 80
Thr Val Ile Glu Lys Met Asn Ile Gln Asp Thr Ala Thr Gly Lys Glu
85 90 95
Phe Asn Lys Asp Glu Lys Arg Met Glu Asn Leu Asn Lys Lys Val Asp
100 105 110
Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val Leu
115 120 125
Leu Glu Asn Glu Arg Thr Leu Asp Ala His Asp Ser Gln Gly Thr Gly
130 135 140
Gly Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val Arg
145 150 155 160
Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu
165 170
<210> 10
<211> 182
<212> PRT
<213> HFT
<400> 10
Thr Thr Ala Ser Thr Ser Gln Val Arg Gln Asn Tyr His Gln Asp Ser
1 5 10 15
Glu Ala Ala Ile Asn Arg Gln Ile Asn Leu Glu Leu Tyr Ala Ser Tyr
20 25 30
Val Tyr Leu Ser Met Ser Tyr Tyr Phe Asp Arg Asp Asp Val Ala Leu
35 40 45
Lys Asn Phe Ala Lys Tyr Phe Leu His Gln Ser His Glu Glu Arg Glu
50 55 60
His Ala Glu Lys Leu Met Lys Leu Gln Asn Gln Arg Gly Gly Arg Ile
65 70 75 80
Phe Leu Gln Asp Ile Lys Lys Pro Asp Cys Asp Asp Trp Glu Ser Gly
85 90 95
Leu Asn Ala Met Glu Cys Ala Leu His Leu Glu Lys Asn Val Asn Gln
100 105 110
Ser Leu Leu Glu Leu His Lys Leu Ala Thr Asp Lys Asn Asp Pro His
115 120 125
Leu Cys Asp Phe Ile Glu Thr His Tyr Leu Asn Glu Gln Val Lys Ala
130 135 140
Ile Lys Glu Leu Gly Asp His Val Thr Asn Leu Arg Lys Met Gly Ala
145 150 155 160
Pro Glu Ser Gly Leu Ala Glu Tyr Leu Phe Asp Lys His Thr Leu Gly
165 170 175
Asp Ser Asp Asn Glu Ser
180
<210> 11
<211> 173
<212> PRT
<213> PFT
<400> 11
Leu Ser Glu Arg Met Leu Lys Ala Leu Asn Asp Gln Leu Asn Arg Glu
1 5 10 15
Leu Tyr Ser Ala Tyr Leu Tyr Phe Ala Met Ala Ala Tyr Phe Glu Asp
20 25 30
Leu Gly Leu Glu Gly Phe Ala Asn Trp Met Lys Ala Gln Ala Glu Glu
35 40 45
Glu Ile Gly His Ala Pro Arg Phe Tyr Asn Tyr Ile Tyr Asp Arg Asn
50 55 60
Gly Arg Val Glu Leu Asp Glu Ile Pro Lys Pro Pro Lys Glu Trp Glu
65 70 75 80
Ser Pro Leu Lys Ala Phe Glu Ala Ala Tyr Glu His Glu Lys Phe Ile
85 90 95
Ser Lys Ser Ile Tyr Glu Leu Ala Ala Leu Ala Glu Glu Glu Lys Asp
100 105 110
Tyr Ser Thr Arg Ala Phe Leu Glu Trp Phe Ile Asn Glu Gln Val Glu
115 120 125
Glu Glu Ala Ser Val Lys Lys Ile Leu Asp Lys Leu Lys Phe Ala Lys
130 135 140
Asp Ser Pro Gln Ile Leu Phe Met Leu Asp Lys Glu Leu Ser Ala Arg
145 150 155 160
Ala Pro Lys Leu Pro Gly Leu Leu Met Gln Gly Gly Glu
165 170
<210> 12
<211> 405
<212> PRT
<213> strep2-GFP-gp41-1-inteinN-gb1-6xHis
<400> 12
Met Gly Trp Ser His Pro Gln Phe Glu Lys Val Ser Lys Gly Glu Glu
1 5 10 15
Leu Phe Thr Gly Val Val Pro Ile Leu Val Glu Leu Asp Gly Asp Val
20 25 30
Asn Gly His Lys Phe Ser Val Ser Gly Glu Gly Glu Gly Asp Ala Thr
35 40 45
Tyr Gly Lys Leu Thr Leu Lys Phe Ile Cys Thr Thr Gly Lys Leu Pro
50 55 60
Val Pro Trp Pro Thr Leu Val Thr Thr Leu Thr Tyr Gly Val Gln Cys
65 70 75 80
Phe Ser Arg Tyr Pro Asp His Met Lys Gln His Asp Phe Phe Lys Ser
85 90 95
Ala Met Pro Glu Gly Tyr Val Gln Glu Arg Thr Ile Phe Phe Lys Asp
100 105 110
Asp Gly Asn Tyr Lys Thr Arg Ala Glu Val Lys Phe Glu Gly Asp Thr
115 120 125
Leu Val Asn Arg Ile Glu Leu Lys Gly Ile Asp Phe Lys Glu Asp Gly
130 135 140
Asn Ile Leu Gly His Lys Leu Glu Tyr Asn Tyr Asn Ser His Asn Val
145 150 155 160
Tyr Ile Met Ala Asp Lys Gln Lys Asn Gly Ile Lys Val Asn Phe Lys
165 170 175
Ile Arg His Asn Ile Glu Asp Gly Ser Val Gln Leu Ala Asp His Tyr
180 185 190
Gln Gln Asn Thr Pro Ile Gly Asp Gly Pro Val Leu Leu Pro Asp Asn
195 200 205
His Tyr Leu Ser Thr Gln Ser Ala Leu Ser Lys Asp Pro Asn Glu Lys
210 215 220
Arg Asp His Met Val Leu Leu Glu Phe Val Thr Ala Ala Gly Ile Thr
225 230 235 240
Leu Gly Met Asp Glu Leu Tyr Lys Thr Arg Ser Gly Tyr Cys Leu Asp
245 250 255
Leu Lys Thr Gln Val Gln Thr Pro Gln Gly Met Lys Glu Ile Ser Asn
260 265 270
Ile Gln Val Gly Asp Leu Val Leu Ser Asn Thr Gly Tyr Asn Glu Val
275 280 285
Leu Asn Val Phe Pro Lys Ser Lys Lys Lys Ser Tyr Lys Ile Thr Leu
290 295 300
Glu Asp Gly Lys Glu Ile Ile Cys Ser Glu Glu His Leu Phe Pro Thr
305 310 315 320
Gln Thr Gly Glu Met Asn Ile Ser Gly Gly Leu Lys Glu Gly Met Cys
325 330 335
Leu Tyr Val Lys Glu Gln Tyr Lys Leu Ile Leu Asn Gly Lys Thr Leu
340 345 350
Lys Gly Glu Thr Thr Thr Glu Ala Val Asp Ala Ala Thr Ala Glu Lys
355 360 365
Val Phe Lys Gln Tyr Ala Asn Asp Asn Gly Val Asp Gly Glu Trp Thr
370 375 380
Tyr Asp Asp Ala Thr Lys Thr Phe Thr Val Thr Glu Lys Leu Glu His
385 390 395 400
His His His His His
405
<210> 13
<211> 172
<212> PRT
<213> strep2-gp41-1-inteinN-gb1-6xHis
<400> 13
Met Gly Trp Ser His Pro Gln Phe Glu Lys Gly Gly Gly Gly Ser Thr
1 5 10 15
Arg Ser Gly Tyr Cys Leu Asp Leu Lys Thr Gln Val Gln Thr Pro Gln
20 25 30
Gly Met Lys Glu Ile Ser Asn Ile Gln Val Gly Asp Leu Val Leu Ser
35 40 45
Asn Thr Gly Tyr Asn Glu Val Leu Asn Val Phe Pro Lys Ser Lys Lys
50 55 60
Lys Ser Tyr Lys Ile Thr Leu Glu Asp Gly Lys Glu Ile Ile Cys Ser
65 70 75 80
Glu Glu His Leu Phe Pro Thr Gln Thr Gly Glu Met Asn Ile Ser Gly
85 90 95
Gly Leu Lys Glu Gly Met Cys Leu Tyr Val Lys Glu Gln Tyr Lys Leu
100 105 110
Ile Leu Asn Gly Lys Thr Leu Lys Gly Glu Thr Thr Thr Glu Ala Val
115 120 125
Asp Ala Ala Thr Ala Glu Lys Val Phe Lys Gln Tyr Ala Asn Asp Asn
130 135 140
Gly Val Asp Gly Glu Trp Thr Tyr Asp Asp Ala Thr Lys Thr Phe Thr
145 150 155 160
Val Thr Glu Lys Leu Glu His His His His His His
165 170
<210> 14
<211> 185
<212> PRT
<213> SP70-gp41-1-inteinN-gb1-strep2-6xHis
<400> 14
Met Gly Tyr Pro Thr Phe Gly Glu His Lys Gln Glu Lys Asp Leu Glu
1 5 10 15
Tyr Gly Thr Arg Ser Gly Tyr Cys Leu Asp Leu Lys Thr Gln Val Gln
20 25 30
Thr Pro Gln Gly Met Lys Glu Ile Ser Asn Ile Gln Val Gly Asp Leu
35 40 45
Val Leu Ser Asn Thr Gly Tyr Asn Glu Val Leu Asn Val Phe Pro Lys
50 55 60
Ser Lys Lys Lys Ser Tyr Lys Ile Thr Leu Glu Asp Gly Lys Glu Ile
65 70 75 80
Ile Cys Ser Glu Glu His Leu Phe Pro Thr Gln Thr Gly Glu Met Asn
85 90 95
Ile Ser Gly Gly Leu Lys Glu Gly Met Cys Leu Tyr Val Lys Glu Gln
100 105 110
Tyr Lys Leu Ile Leu Asn Gly Lys Thr Leu Lys Gly Glu Thr Thr Thr
115 120 125
Glu Ala Val Asp Ala Ala Thr Ala Glu Lys Val Phe Lys Gln Tyr Ala
130 135 140
Asn Asp Asn Gly Val Asp Gly Glu Trp Thr Tyr Asp Asp Ala Thr Lys
145 150 155 160
Thr Phe Thr Val Thr Glu Lys Trp Ser His Pro Gln Phe Glu Lys Gly
165 170 175
Ser Leu Glu His His His His His His
180 185
<210> 15
<211> 317
<212> PRT
<213> rhizavidin-gp41-1-intN-gb1-6xHis
<400> 15
Met Gly Phe Asp Ala Ser Asn Phe Lys Asp Phe Ser Ser Ile Ala Ser
1 5 10 15
Ala Ser Ser Ser Trp Gln Asn Gln His Gly Ser Thr Met Ile Ile Gln
20 25 30
Val Asp Ser Phe Gly Asn Val Ser Gly Gln Tyr Val Asn Arg Ala Glu
35 40 45
Gly Thr Gly Cys Gln Asn Ser Pro Tyr Pro Leu Thr Gly Arg Val Asn
50 55 60
Gly Thr Phe Ile Asp Phe Ser Val Lys Trp Asn Asn Ser Thr Glu Asn
65 70 75 80
Cys Asn Ser Asn Thr Gln Trp Thr Gly Tyr Ala Gln Val Asn Gly Asn
85 90 95
Asn Thr Glu Ile Val Thr Arg Trp Asn Leu Lys Tyr Glu Gly Gly Ser
100 105 110
Gly Pro Ala Ile Trp Gln Gly Gln Asp Thr Phe Gln Tyr Val Pro Thr
115 120 125
Thr Glu Ala Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala
130 135 140
Ala Lys Ala Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Thr Arg
145 150 155 160
Ser Gly Tyr Cys Leu Asp Leu Lys Thr Gln Val Gln Thr Pro Gln Gly
165 170 175
Met Lys Glu Ile Ser Asn Ile Gln Val Gly Asp Leu Val Leu Ser Asn
180 185 190
Thr Gly Tyr Asn Glu Val Leu Asn Val Phe Pro Lys Ser Lys Lys Lys
195 200 205
Ser Tyr Lys Ile Thr Leu Glu Asp Gly Lys Glu Ile Ile Cys Ser Glu
210 215 220
Glu His Leu Phe Pro Thr Gln Thr Gly Glu Met Asn Ile Ser Gly Gly
225 230 235 240
Leu Lys Glu Gly Met Cys Leu Tyr Val Lys Glu Gln Tyr Lys Leu Ile
245 250 255
Leu Asn Gly Lys Thr Leu Lys Gly Glu Thr Thr Thr Glu Ala Val Asp
260 265 270
Ala Ala Thr Ala Glu Lys Val Phe Lys Gln Tyr Ala Asn Asp Asn Gly
275 280 285
Val Asp Gly Glu Trp Thr Tyr Asp Asp Ala Thr Lys Thr Phe Thr Val
290 295 300
Thr Glu Lys Gly Ser Leu Glu His His His His His His
305 310 315
<210> 16
<211> 408
<212> PRT
<213> CBLB-gp41-1-inteinN-strep2-6xHis
<400> 16
Met Gly Ala Gln Val Ile Asn Thr Asn Ser Leu Ser Leu Leu Thr Gln
1 5 10 15
Asn Asn Leu Asn Lys Ser Gln Ser Ser Leu Ser Ser Ala Ile Glu Arg
20 25 30
Leu Ser Ser Gly Leu Arg Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly
35 40 45
Gln Ala Ile Ala Asn Arg Phe Thr Ser Asn Ile Lys Gly Leu Thr Gln
50 55 60
Ala Ser Arg Asn Ala Asn Asp Gly Ile Ser Ile Ala Gln Thr Thr Glu
65 70 75 80
Gly Ala Leu Asn Glu Ile Asn Asn Asn Leu Gln Arg Val Arg Glu Leu
85 90 95
Ser Val Gln Ala Thr Asn Gly Thr Asn Ser Asp Ser Asp Leu Lys Ser
100 105 110
Ile Gln Asp Glu Ile Gln Gln Arg Leu Glu Glu Ile Asp Arg Val Ser
115 120 125
Asn Gln Thr Gln Phe Asn Gly Val Lys Val Leu Ser Gln Asp Asn Gln
130 135 140
Met Lys Ile Gln Val Gly Ala Asn Asp Gly Glu Thr Ile Thr Ile Asp
145 150 155 160
Leu Gln Lys Ile Asp Val Lys Ser Leu Gly Leu Asp Gly Phe Asn Val
165 170 175
Asn Ser Pro Gly Ile Ser Gly Gly Gly Gly Gly Ile Leu Asp Ser Met
180 185 190
Gly Thr Leu Ile Asn Glu Asp Ala Ala Ala Ala Lys Lys Ser Thr Ala
195 200 205
Asn Pro Leu Ala Ser Ile Asp Ser Ala Leu Ser Lys Val Asp Ala Val
210 215 220
Arg Ser Ser Leu Gly Ala Ile Gln Asn Arg Phe Asp Ser Ala Ile Thr
225 230 235 240
Asn Leu Gly Asn Thr Val Thr Asn Leu Asn Ser Ala Arg Ser Arg Ile
245 250 255
Glu Asp Ala Asp Tyr Ala Thr Glu Val Ser Asn Met Ser Lys Ala Gln
260 265 270
Ile Leu Gln Gln Ala Gly Thr Ser Val Leu Ala Gln Ala Asn Gln Val
275 280 285
Pro Gln Asn Val Leu Ser Leu Leu Arg Thr Arg Ser Gly Tyr Cys Leu
290 295 300
Asp Leu Lys Thr Gln Val Gln Thr Pro Gln Gly Met Lys Glu Ile Ser
305 310 315 320
Asn Ile Gln Val Gly Asp Leu Val Leu Ser Asn Thr Gly Tyr Asn Glu
325 330 335
Val Leu Asn Val Phe Pro Lys Ser Lys Lys Lys Ser Tyr Lys Ile Thr
340 345 350
Leu Glu Asp Gly Lys Glu Ile Ile Cys Ser Glu Glu His Leu Phe Pro
355 360 365
Thr Gln Thr Gly Glu Met Asn Ile Ser Gly Gly Leu Lys Glu Gly Met
370 375 380
Cys Leu Tyr Val Lys Glu Trp Ser His Pro Gln Phe Glu Lys Gly Ser
385 390 395 400
Leu Glu His His His His His His
405
<210> 17
<211> 424
<212> PRT
<213> SP70-CBLB-gp41-1-inteinN-strep2-6xHis
<400> 17
Met Gly Tyr Pro Thr Phe Gly Glu His Lys Gln Glu Lys Asp Leu Glu
1 5 10 15
Tyr Gly Ala Gln Val Ile Asn Thr Asn Ser Leu Ser Leu Leu Thr Gln
20 25 30
Asn Asn Leu Asn Lys Ser Gln Ser Ser Leu Ser Ser Ala Ile Glu Arg
35 40 45
Leu Ser Ser Gly Leu Arg Ile Asn Ser Ala Lys Asp Asp Ala Ala Gly
50 55 60
Gln Ala Ile Ala Asn Arg Phe Thr Ser Asn Ile Lys Gly Leu Thr Gln
65 70 75 80
Ala Ser Arg Asn Ala Asn Asp Gly Ile Ser Ile Ala Gln Thr Thr Glu
85 90 95
Gly Ala Leu Asn Glu Ile Asn Asn Asn Leu Gln Arg Val Arg Glu Leu
100 105 110
Ser Val Gln Ala Thr Asn Gly Thr Asn Ser Asp Ser Asp Leu Lys Ser
115 120 125
Ile Gln Asp Glu Ile Gln Gln Arg Leu Glu Glu Ile Asp Arg Val Ser
130 135 140
Asn Gln Thr Gln Phe Asn Gly Val Lys Val Leu Ser Gln Asp Asn Gln
145 150 155 160
Met Lys Ile Gln Val Gly Ala Asn Asp Gly Glu Thr Ile Thr Ile Asp
165 170 175
Leu Gln Lys Ile Asp Val Lys Ser Leu Gly Leu Asp Gly Phe Asn Val
180 185 190
Asn Ser Pro Gly Ile Ser Gly Gly Gly Gly Gly Ile Leu Asp Ser Met
195 200 205
Gly Thr Leu Ile Asn Glu Asp Ala Ala Ala Ala Lys Lys Ser Thr Ala
210 215 220
Asn Pro Leu Ala Ser Ile Asp Ser Ala Leu Ser Lys Val Asp Ala Val
225 230 235 240
Arg Ser Ser Leu Gly Ala Ile Gln Asn Arg Phe Asp Ser Ala Ile Thr
245 250 255
Asn Leu Gly Asn Thr Val Thr Asn Leu Asn Ser Ala Arg Ser Arg Ile
260 265 270
Glu Asp Ala Asp Tyr Ala Thr Glu Val Ser Asn Met Ser Lys Ala Gln
275 280 285
Ile Leu Gln Gln Ala Gly Thr Ser Val Leu Ala Gln Ala Asn Gln Val
290 295 300
Pro Gln Asn Val Leu Ser Leu Leu Arg Thr Arg Ser Gly Tyr Cys Leu
305 310 315 320
Asp Leu Lys Thr Gln Val Gln Thr Pro Gln Gly Met Lys Glu Ile Ser
325 330 335
Asn Ile Gln Val Gly Asp Leu Val Leu Ser Asn Thr Gly Tyr Asn Glu
340 345 350
Val Leu Asn Val Phe Pro Lys Ser Lys Lys Lys Ser Tyr Lys Ile Thr
355 360 365
Leu Glu Asp Gly Lys Glu Ile Ile Cys Ser Glu Glu His Leu Phe Pro
370 375 380
Thr Gln Thr Gly Glu Met Asn Ile Ser Gly Gly Leu Lys Glu Gly Met
385 390 395 400
Cys Leu Tyr Val Lys Glu Trp Ser His Pro Gln Phe Glu Lys Gly Ser
405 410 415
Leu Glu His His His His His His
420
<210> 18
<211> 340
<212> PRT
<213> H1HA10-gp41-1-inteinN-gb1-6xHis
<400> 18
Met Asp Thr Val Asp Thr Val Leu Glu Lys Asn Val Thr Val Thr His
1 5 10 15
Ser Val Asn Leu Leu Glu Asp Ser His Gly Ser Ala Asn Ser Ser Leu
20 25 30
Pro Tyr Gln Asn Thr His Pro Thr Thr Asn Gly Glu Ser Pro Lys Tyr
35 40 45
Val Arg Ser Ala Lys Leu Arg Met Val Thr Gly Leu Arg Asn Gly Ser
50 55 60
Ala Gly Ser Ala Thr Gln Asn Ala Ile Asn Gly Ile Thr Asn Lys Val
65 70 75 80
Asn Thr Val Ile Glu Lys Met Asn Ile Gln Asp Thr Ala Thr Gly Lys
85 90 95
Glu Phe Asn Lys Asp Glu Lys Arg Met Glu Asn Leu Asn Lys Lys Val
100 105 110
Asp Asp Gly Phe Leu Asp Ile Trp Thr Tyr Asn Ala Glu Leu Leu Val
115 120 125
Leu Leu Glu Asn Glu Arg Thr Leu Asp Ala His Asp Ser Gln Gly Thr
130 135 140
Gly Gly Gly Tyr Ile Pro Glu Ala Pro Arg Asp Gly Gln Ala Tyr Val
145 150 155 160
Arg Lys Asp Gly Glu Trp Val Leu Leu Ser Thr Phe Leu Gly Gly Gly
165 170 175
Ser Gly Gly Gly Ser Thr Arg Ser Gly Tyr Cys Leu Asp Leu Lys Thr
180 185 190
Gln Val Gln Thr Pro Gln Gly Met Lys Glu Ile Ser Asn Ile Gln Val
195 200 205
Gly Asp Leu Val Leu Ser Asn Thr Gly Tyr Asn Glu Val Leu Asn Val
210 215 220
Phe Pro Lys Ser Lys Lys Lys Ser Tyr Lys Ile Thr Leu Glu Asp Gly
225 230 235 240
Lys Glu Ile Ile Cys Ser Glu Glu His Leu Phe Pro Thr Gln Thr Gly
245 250 255
Glu Met Asn Ile Ser Gly Gly Leu Lys Glu Gly Met Cys Leu Tyr Val
260 265 270
Lys Glu Gln Tyr Lys Leu Ile Leu Asn Gly Lys Thr Leu Lys Gly Glu
275 280 285
Thr Thr Thr Glu Ala Val Asp Ala Ala Thr Ala Glu Lys Val Phe Lys
290 295 300
Gln Tyr Ala Asn Asp Asn Gly Val Asp Gly Glu Trp Thr Tyr Asp Asp
305 310 315 320
Ala Thr Lys Thr Phe Thr Val Thr Glu Lys Gly Ser Leu Glu His His
325 330 335
His His His His
340
<210> 19
<211> 316
<212> PRT
<213> gb1-gp41-1-inteinC-HFT
<400> 19
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg
20 25 30
Gly Ser Glu Phe Gln Tyr Lys Leu Ile Leu Asn Gly Lys Thr Leu Lys
35 40 45
Gly Glu Thr Thr Thr Glu Ala Val Asp Ala Ala Thr Ala Glu Lys Val
50 55 60
Phe Lys Gln Tyr Ala Asn Asp Asn Gly Val Asp Gly Glu Trp Thr Tyr
65 70 75 80
Asp Asp Ala Thr Lys Thr Phe Thr Val Thr Glu Lys Met Leu Lys Lys
85 90 95
Ile Leu Lys Ile Glu Glu Leu Asp Glu Arg Glu Leu Ile Asp Ile Glu
100 105 110
Val Ser Gly Asn His Leu Phe Tyr Ala Asn Asp Ile Leu Thr His Asn
115 120 125
Ser Ser Ser Ser Asp Val Thr Thr Ala Ser Thr Ser Gln Val Arg Gln
130 135 140
Asn Tyr His Gln Asp Ser Glu Ala Ala Ile Asn Arg Gln Ile Asn Leu
145 150 155 160
Glu Leu Tyr Ala Ser Tyr Val Tyr Leu Ser Met Ser Tyr Tyr Phe Asp
165 170 175
Arg Asp Asp Val Ala Leu Lys Asn Phe Ala Lys Tyr Phe Leu His Gln
180 185 190
Ser His Glu Glu Arg Glu His Ala Glu Lys Leu Met Lys Leu Gln Asn
195 200 205
Gln Arg Gly Gly Arg Ile Phe Leu Gln Asp Ile Lys Lys Pro Asp Cys
210 215 220
Asp Asp Trp Glu Ser Gly Leu Asn Ala Met Glu Cys Ala Leu His Leu
225 230 235 240
Glu Lys Asn Val Asn Gln Ser Leu Leu Glu Leu His Lys Leu Ala Thr
245 250 255
Asp Lys Asn Asp Pro His Leu Cys Asp Phe Ile Glu Thr His Tyr Leu
260 265 270
Asn Glu Gln Val Lys Ala Ile Lys Glu Leu Gly Asp His Val Thr Asn
275 280 285
Leu Arg Lys Met Gly Ala Pro Glu Ser Gly Leu Ala Glu Tyr Leu Phe
290 295 300
Asp Lys His Thr Leu Gly Asp Ser Asp Asn Glu Ser
305 310 315
<210> 20
<211> 307
<212> PRT
<213> gb1-gp41-1-inteinC-PFT
<400> 20
Met Gly Ser Ser His His His His His His Ser Ser Gly Leu Val Pro
1 5 10 15
Arg Gly Ser His Met Ala Ser Met Thr Gly Gly Gln Gln Met Gly Arg
20 25 30
Gly Ser Glu Phe Gln Tyr Lys Leu Ile Leu Asn Gly Lys Thr Leu Lys
35 40 45
Gly Glu Thr Thr Thr Glu Ala Val Asp Ala Ala Thr Ala Glu Lys Val
50 55 60
Phe Lys Gln Tyr Ala Asn Asp Asn Gly Val Asp Gly Glu Trp Thr Tyr
65 70 75 80
Asp Asp Ala Thr Lys Thr Phe Thr Val Thr Glu Lys Met Leu Lys Lys
85 90 95
Ile Leu Lys Ile Glu Glu Leu Asp Glu Arg Glu Leu Ile Asp Ile Glu
100 105 110
Val Ser Gly Asn His Leu Phe Tyr Ala Asn Asp Ile Leu Thr His Asn
115 120 125
Ser Ser Ser Ser Asp Val Leu Ser Glu Arg Met Leu Lys Ala Leu Asn
130 135 140
Asp Gln Leu Asn Arg Glu Leu Tyr Ser Ala Tyr Leu Tyr Phe Ala Met
145 150 155 160
Ala Ala Tyr Phe Glu Asp Leu Gly Leu Glu Gly Phe Ala Asn Trp Met
165 170 175
Lys Ala Gln Ala Glu Glu Glu Ile Gly His Ala Pro Arg Phe Tyr Asn
180 185 190
Tyr Ile Tyr Asp Arg Asn Gly Arg Val Glu Leu Asp Glu Ile Pro Lys
195 200 205
Pro Pro Lys Glu Trp Glu Ser Pro Leu Lys Ala Phe Glu Ala Ala Tyr
210 215 220
Glu His Glu Lys Phe Ile Ser Lys Ser Ile Tyr Glu Leu Ala Ala Leu
225 230 235 240
Ala Glu Glu Glu Lys Asp Tyr Ser Thr Arg Ala Phe Leu Glu Trp Phe
245 250 255
Ile Asn Glu Gln Val Glu Glu Glu Ala Ser Val Lys Lys Ile Leu Asp
260 265 270
Lys Leu Lys Phe Ala Lys Asp Ser Pro Gln Ile Leu Phe Met Leu Asp
275 280 285
Lys Glu Leu Ser Ala Arg Ala Pro Lys Leu Pro Gly Leu Leu Met Gln
290 295 300
Gly Gly Glu
305

Claims (7)

1.一种基于内含肽介导的纳米载体模块平台，其特征在于，其为蛋白X与接头gbl-intein的C端融合形成重组蛋白gbl-inteinc-X后经分子自组装成的多聚体纳米颗粒，所述蛋白X的N端伸展至纳米颗粒表面。
2.根据权利要求1所述的一种基于内含肽介导的纳米载体模块平台，其特征在于，所述的可由N端伸展至纳米颗粒表面自装配纳米颗粒的蛋白X为铁蛋白或二氧四氢蝶啶合成酶或细菌噬菌体Qβ核衣壳蛋白或豇豆花叶病毒核衣壳蛋白或戊肝病毒核衣壳蛋白。
3.根据权利要求2所述的一种基于内含肽介导的纳米载体模块平台，其特征在于，所述的铁蛋白为人源的重链铁蛋白或强烈火球菌的重链铁蛋白或其他物种来源的铁蛋白。
4.一种基于内含肽介导的纳米载体模块平台的构建方法，其特征在于，可由N端伸展至纳米颗粒表面自装配纳米颗粒的蛋白X与接头gbl-intein的C端融合形成重组蛋白gbl-inteinc-X后自装配成24聚体构成。
5.根据权利要求4所述的一种基于内含肽介导的纳米载体模块平台的构建方法，其特征在于，所述的步骤1中的可由N端伸展至纳米颗粒表面自装配纳米颗粒的蛋白X为铁蛋白或二氧四氢蝶啶合成酶或细菌噬菌体Qβ核衣壳蛋白或豇豆花叶病毒核衣壳蛋白或戊肝病毒核衣壳蛋白。
6.根据权利要求5所述的一种基于内含肽介导的纳米载体模块平台的构建方法，其特征在于，所述的铁蛋白为人源的重链铁蛋白或强烈火球菌的重链铁蛋白或其他物种来源的铁蛋白。
7.如权利要求1-3任一所述的基于内含肽介导的纳米载体模块平台作为药物和/或疫苗递送载体的应用。
CN201910421408.9A 2019-05-21 2019-05-21 一种基于内含肽介导纳米载体模块平台及其构建方法与应用 Pending CN110354271A (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CN201910421408.9A CN110354271A (zh) 2019-05-21 2019-05-21 一种基于内含肽介导纳米载体模块平台及其构建方法与应用
PCT/CN2020/089924 WO2020233460A1 (zh) 2019-05-21 2020-05-13 一种基于内含肽介导纳米载体模块平台及其构建方法与应用

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201910421408.9A CN110354271A (zh) 2019-05-21 2019-05-21 一种基于内含肽介导纳米载体模块平台及其构建方法与应用

Publications (1)

Publication Number Publication Date
CN110354271A true CN110354271A (zh) 2019-10-22

Family

ID=68215211

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201910421408.9A Pending CN110354271A (zh) 2019-05-21 2019-05-21 一种基于内含肽介导纳米载体模块平台及其构建方法与应用

Country Status (2)

Country Link
CN (1) CN110354271A (zh)
WO (1) WO2020233460A1 (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020233460A1 (zh) * 2019-05-21 2020-11-26 广州市妇女儿童医疗中心 一种基于内含肽介导纳米载体模块平台及其构建方法与应用
WO2020233685A1 (zh) * 2019-05-21 2020-11-26 广州市妇女儿童医疗中心 一种基于内含肽介导纳米载体及其应用与一种可同时递送抗原和免疫增强剂的纳米制剂

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1927401A (zh) * 2005-09-07 2007-03-14 中国人民解放军军事医学科学院基础医学研究所 自组装复合纳米颗粒肿瘤疫苗及其制备方法
CN104755502B (zh) * 2012-10-12 2018-05-18 清华大学 多肽的产生和纯化方法
CN108434450A (zh) * 2018-02-06 2018-08-24 中国科学院生物物理研究所 基于铁蛋白纳米颗粒的疫苗及其制备方法

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9441019B2 (en) * 2011-09-23 2016-09-13 The United States Of America, As Represented By The Secretary, Department Of Health And Human Services Influenza hemagglutinin protein-based vaccines
SG11201705264WA (en) * 2014-12-31 2017-07-28 The Usa As Represented By The Secretary Detp Of Health And Human Services Novel multivalent nanoparticle-based vaccines
CN106148374B (zh) * 2016-07-07 2019-12-13 苏州大学 制备无融合标签包涵体蛋白纳米颗粒的方法
CN109512799B (zh) * 2019-01-17 2021-06-15 中国科学院生物物理研究所 一种装载抗肿瘤药物的纳米药物载体、其制备方法及应用
CN110354271A (zh) * 2019-05-21 2019-10-22 广州市妇女儿童医疗中心 一种基于内含肽介导纳米载体模块平台及其构建方法与应用
CN110272498A (zh) * 2019-05-21 2019-09-24 广州市妇女儿童医疗中心 一种基于内含肽介导纳米载体及其应用
CN110201183A (zh) * 2019-05-21 2019-09-06 广州市妇女儿童医疗中心 一种可同时递送抗原和免疫增强剂的纳米制剂

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1927401A (zh) * 2005-09-07 2007-03-14 中国人民解放军军事医学科学院基础医学研究所 自组装复合纳米颗粒肿瘤疫苗及其制备方法
CN104755502B (zh) * 2012-10-12 2018-05-18 清华大学 多肽的产生和纯化方法
CN108434450A (zh) * 2018-02-06 2018-08-24 中国科学院生物物理研究所 基于铁蛋白纳米颗粒的疫苗及其制备方法

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020233460A1 (zh) * 2019-05-21 2020-11-26 广州市妇女儿童医疗中心 一种基于内含肽介导纳米载体模块平台及其构建方法与应用
WO2020233685A1 (zh) * 2019-05-21 2020-11-26 广州市妇女儿童医疗中心 一种基于内含肽介导纳米载体及其应用与一种可同时递送抗原和免疫增强剂的纳米制剂

Also Published As

Publication number Publication date
WO2020233460A1 (zh) 2020-11-26

Similar Documents

Publication Publication Date Title
Stano et al. PPS nanoparticles as versatile delivery system to induce systemic and broad mucosal immunity after intranasal administration
KR100808313B1 (ko) 비형 간염 코어 항원 융합 단백질
CN108434450A (zh) 基于铁蛋白纳米颗粒的疫苗及其制备方法
Morris et al. A modular vaccine platform combining self‐assembled peptide cages and immunogenic peptides
WO2013065009A1 (en) A sortase-click reaction suite for synthesis of multivalent dendrimeric protein assembly
KR20210018205A (ko) 항원성 OspA 폴리펩타이드
CN102892431A (zh) 用于增强疫苗免疫原性的肽、偶联物及方法
CN113354740B (zh) 一种猪瘟病毒自组装蛋白纳米颗粒、制备方法及应用
Gill Nanocarriers, nanovaccines, and nanobacteria as nanobiotechnological concerns in modern vaccines
CN103037898A (zh) 呼吸道合胞病毒抗原组合物和方法
Liu et al. Antigen-conjugated N-trimethylaminoethylmethacrylate chitosan nanoparticles induce strong immune responses after nasal administration
CN110354271A (zh) 一种基于内含肽介导纳米载体模块平台及其构建方法与应用
Brohlin et al. Zeolitic imidazolate framework nanoencapsulation of CpG for stabilization and enhancement of immunoadjuvancy
CN110201183A (zh) 一种可同时递送抗原和免疫增强剂的纳米制剂
Lin et al. Nanoparticular CpG-adjuvanted SARS-CoV-2 S1 protein elicits broadly neutralizing and Th1-biased immunoreactivity in mice
Zykova et al. Nanoparticles based on artificial self-assembling peptide and displaying M2e peptide and stalk HA epitopes of influenza A virus induce potent humoral and T-cell responses and protect against the viral infection
CN109966483A (zh) 一种基于铁蛋白的多抗原通用流感疫苗及其制备方法和应用
CN112641935B (zh) 一种含颗粒载体的亚单位疫苗及其应用
Chung et al. Development of a modular NTA: His tag viral vaccine for co-delivery of antigen and adjuvant
Li et al. Ferritin nanoparticle technology... A new platform for antigen presentation and vaccine development
CN110638787B (zh) 一种防治鼻咽癌的亚单位纳米疫苗及其制备方法
CN115960259B (zh) 一种模块化组装双组分纳米颗粒制备方法及其应用
CN116334139A (zh) 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
CN113633763B (zh) 一种新型冠状病毒s1-e疫苗及其制备方法
Fateh et al. New insight into the application of outer membrane vesicles of Gram-negative bacteria

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication

Application publication date: 20191022

RJ01 Rejection of invention patent application after publication