CN117599209A - 自组装纳米蛋白笼及其制备方法和应用 - Google Patents
自组装纳米蛋白笼及其制备方法和应用 Download PDFInfo
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Abstract
本申请属于纳米疫苗技术领域,尤其涉及自组装纳米蛋白笼及其制备方法和应用。本申请公开自组装纳米蛋白笼,包含:(i)包含单体自组装的纳米蛋白笼,所述单体修饰有阳离子肽;所述阳离子肽包含由以下项组成的氨基酸序列:X1~X6~12,其中X1到X6~12中的任一者是彼此独立的氨基酸,条件是X1到X6~12中彼此独立为精氨酸或赖氨酸;(ii)RNA药物;其中所述RNA药物通过所述阳离子肽被包装到组装的纳米蛋白笼中。本申请的自组装纳米蛋白笼可用于包装和递送mRNA,作为mRNA疫苗递送载体,可显著提高mRNA疫苗的稳定性、安全性和生物相容性,有效克服mRNA疫苗冷链依赖和递送载体专利壁垒。
Description
技术领域
本申请属于纳米疫苗技术领域,尤其涉及自组装纳米蛋白笼及其制备方法和应用。
背景技术
由于在安全性、有效性和工业化生产方面的优势,mRNA疫苗在对抗癌症和病毒性疾病方面具有巨大潜力。然而,它们的应用一直受到mRNA固有的不稳定性限制,其裸体形式很容易被细胞外RNA酶迅速降解。因此,已经开发出多种体外和体内转染试剂,以促进细胞对mRNA的摄取并保护其免受降解,包括基于脂质的递送、基于聚合物的递送、病毒样复制子颗粒递送和阳离子纳米乳液递送。然而,目前主流应用的脂质体递送仍存在靶向性不足、生物相容性低、超低温冷链依赖和专利壁垒等问题,亟需开发mRNA疫苗递送载体新材料。
最近,纳米蛋白笼因其独特和理想的特性在货物包装和递送方面备受关注。纳米蛋白笼(protein nanocage)是一种由生物表达系统快速产生的单一蛋白成分组装而成的高度寡聚结构,可由重复蛋白质亚基的精确自组装产生,笼内部通常具有一定的空间可包装外源性分子。相比非生物纳米材料,蛋白笼具有较高的生物相容性和生物降解性,安全性高。作为用于递送抗原和治疗分子的纳米医疗设备的分子组分,相比非生物纳米材料,纳米蛋白笼显示出许多优点,包括超高的原子级精确组装优势、几何表面修饰性和生物相容性。目前,蛋白笼已被设计成通过互补的静电作用在体外包装各种寡核苷酸,包括siRNA、dsRNA、ssDNA和dsDNA等;在体内自组装过程中捕获各种尺寸的内源性RNA分子。静电相互作用为宿主-客体复合物的形成提供了强大的驱动力,非常适合结合高度阴离子核酸。纳米蛋白笼本身具有超强的耐受性,能够有效保护内部包装的核酸分子免受核酸酶、温度和冻融等极端环境的侵害。
尽管纳米蛋白笼已用于体内外包装各种核酸小分子,但对于mRNA疫苗这类较长的核酸序列目前还没有研究。开发一种包装mRNA疫苗的纳米蛋白笼用于mRNA疫苗递送能够有效克服当前mRNA疫苗递送技术的诸多缺陷,例如冷链依赖、靶向性低和生物相容性差等,具有重要的现实意义。
发明内容
鉴于此,本申请提供自组装纳米蛋白笼及其制备方法和应用,自组装纳米蛋白笼可用于包装和递送mRNA,作为mRNA疫苗递送载体,该递送载体可显著提高mRNA疫苗的稳定性、安全性和生物相容性,有效克服mRNA疫苗冷链依赖和递送载体专利壁垒。
本申请第一方面提供了自组装纳米蛋白笼,包含:
(i)包含单体自组装的纳米蛋白笼,所述单体修饰有阳离子肽;所述阳离子肽包含由以下项组成的氨基酸序列:X1~X6~12,其中X1到X6~12中的任一者是彼此独立的氨基酸,条件是X1到X6~12中彼此独立为精氨酸或赖氨酸;
(ii)RNA药物;
其中所述RNA药物通过所述阳离子肽被包装到自组装的纳米蛋白笼中。
在一些实施例中,所述纳米蛋白笼来自人来源24聚体Ferritin、嗜热菌来源24聚体Ferritin、幽门螺杆菌来源24聚体Ferritin、嗜热菌来源60聚体E2蛋白、嗜热菌来源60聚体AaLS、嗜热菌来源60聚体Tm或嗜热菌来源240聚体Qt。
在一些实施例中,具体的,
所述人来源24聚体Ferritin的PDB编号为4Y08;
所述嗜热菌来源24聚体Ferritin的PDB编号为2JD6;
所述幽门螺杆菌来源24聚体Ferritin的PDB编号为3BVF;
所述嗜热菌来源60聚体E2蛋白的PDB编号为1B5S;
所述嗜热菌来源60聚体AaLS的PDB编号为1HQK;
所述嗜热菌来源60聚体Tm的PDB编号为7KQ5;
所述嗜热菌来源240聚体Qt的PDB编号为6NJ8。
在一些实施例中,所述阳离子肽通过连接肽连接在所述单体的N端、C端或中部,使得所述阳离子肽位于所述纳米蛋白笼的内部;其中所述连接肽为柔性GS linker。
具体的,柔性GS linker具体为(GS)n、(GGS)n、(GGGS)n、(GGGGS)n,n的范围为1~4的整数。
具体的,所述阳离子肽的氨基酸序列如下:
X1X2X3X4X5X6;
X1X2X3X4X5X6X7;
X1X2X3X4X5X6X7X8;
X1X2X3X4X5X6X7X8X9;
X1X2X3X4X5X6X7X8X9X10;
X1X2X3X4X5X6X7X8X9X10X11;
X1X2X3X4X5X6X7X8X9X10X11X12;
X1到X6-12彼此独立地是为精氨酸或赖氨酸。
在一些实施例中,所述阳离子肽包含RKKKRR(SEQ ID NO.1)所示的氨基酸序列。
在一些实施例中,所述单体还修饰有蛋白质纯化标签、穿膜肽、靶向肽和佐剂肽中的一种或多种。
具体的,所述蛋白质纯化标签可以为His-Tag、GST标签、Flag标签、HA标签、MBP标签等。
具体的,穿膜肽(cellpenetrating peptides, CPPs)是一类具有较强穿透细胞膜能力的小分子肽,可携带多肽、蛋白质和核酸等多种大分子物质进入细胞,可以为转录反式激活蛋白(Tat)、VP22、transportan、模型两亲性肽 (MAP)、信号转导肽和富含精氨酸序列肽。
具体的,靶向肽为与目标靶标具有高特异性结合的肽。在癌症治疗中,肿瘤靶向肽可以特异性地识别肿瘤血管或肿瘤相关受体,以实现其靶向性。
具体的,佐剂肽为一类具有疫苗佐剂功效的小分子肽,能增强体液或细胞免疫。在mRNA疫苗制备过程中,通过在封装载体的蛋白笼上增加佐剂肽,可进一步增强mRNA疫苗的免疫效果。
在一些实施例中,所述RNA药物的长度不超过5000 nt;所述RNA药物为进行过化学修饰的mRNA。
具体的,本申请提供的自组装纳米蛋白笼包装的mRNA的含量为未修饰有所述阳离子肽的对应的纳米蛋白笼包装的mRNA含量的1.4-2.7倍。
具体的,mRNA可以为环状mRNA,也可以为线性化mRNA。
具体的,mRNA的化学修饰可以为常规的加帽、5’UTR、3’UTR、假尿嘧啶化学修饰和polyA尾等修饰。
本申请第二方面公开了自组装纳米蛋白笼的制备方法,包括以下步骤:
制备并纯化纳米蛋白笼;
对所述纳米蛋白笼在溶液中进行解聚;
将解聚的纳米蛋白笼与RNA药物混合在溶液中重新自组装,得到内部包装有RNA药物的自组装纳米蛋白笼。
具体的,对所述纳米蛋白笼在溶液中进行解聚具体包括:将所述纳米蛋白笼在缓冲体系的pH(pH=2)在室温放置不小于6 h,或将所述纳米蛋白笼于120℃孵育2 h使所述纳米蛋白笼解聚。
具体的,重新自组装具体包括将缓冲体系pH恢复至中性或将缓冲体系的温度降至4 ℃,立即加入RNA药物并孵育,使得所述单体在自组装过程中包装该RNA药物,获得内部包装有RNA药物的自组装纳米蛋白笼。
在一些实施例中,所述制备并纯化纳米蛋白笼的方法包括:
构建表达单体的表达工程菌;其中所述单体修饰有阳离子肽;所述阳离子肽包含由以下项组成的氨基酸序列:X1~X6~12,其中X1到X6~12中的任一者是彼此独立的氨基酸,条件是X1到X6~12中彼此独立地是为精氨酸或赖氨酸;
将表达单体的表达工程菌在筛选培养基中培养并诱导表达所述单体并使其自组装为纳米蛋白笼多聚体,然后收集、获取并纯化所述表达工程菌内的纳米蛋白笼多聚体,得到纳米蛋白笼。
具体的,所述制备并纯化纳米蛋白笼的方法具体包括:合成编码单体的核苷酸,将该核苷酸克隆到表达质粒中(可以为常规的pET28a质粒),该表达质粒转化到大肠杆菌中进行扩大培养,然后加入诱导剂IPTG,诱导蛋白笼表达。进一步通过亲和层析(Co-NTA/Ni-NTA)和尺寸排阻色谱法(SEC)等常规手段进行蛋白纯化和浓缩,得到纳米蛋白笼。
本申请第三方面公开了所述自组装纳米蛋白笼或所述的制备方法制得的自组装纳米蛋白笼在制备mRNA疫苗中的应用。
本申请通过蛋白库筛选得到多种纳米蛋白笼,纳米蛋白笼可由生物表达系统快速产生的单一蛋白成分组装而成的高度寡聚结构(一种单体自组装成的多聚体蛋白质),笼内部通常具有一定的空间可包装外源性分子。蛋白笼对外部的极端环境条件(包括酶、加热、反复冻融等)具有超高的耐受性,有效保护内部包装的核酸分子免受外部条件的侵害。相比非生物纳米材料,纳米蛋白笼具有较高的生物相容性和生物降解性,安全性高。本申请通过将纳米蛋白笼的内腔改造为正增压(带正电荷的阳离子肽)环境,用于多种RNA药物(mRNA疫苗等)的包装和递送。同时对构建的自组装纳米蛋白笼的稳定性、抗原递呈能力、安全性和免疫效果进行评估,实施例数据可知,本申请提供的自组装纳米蛋白笼能稳定、高效用于包装和递送mRNA。
附图说明
为了更清楚地说明本申请实施例或现有技术中的技术方案,下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍。
图1为本申请实施例提供的嗜热菌来源240聚体Qt纳米蛋白笼的空间结构示意图;
图2为本申请实施例提供的纳米蛋白笼Qt+的序列结构;
图3为本申请实施例提供的重组表达载体的质粒图;
图4为本申请实施例提供的SDS-PAGE/考马斯亮蓝染色纯度鉴定结果;
图5为本申请实施例提供的尺寸排阻色谱法(SEC)纯度鉴定结果;
图6为本申请实施例提供的动态光散射法(DLS)鉴定纳米蛋白笼Qt+结果;
图7为本申请实施例提供的负染色透射电镜(TEM)鉴定纳米蛋白笼Qt+外貌形态结果;
图8为本申请实施例提供的普通RT-PCR鉴定Qt+-OVA蛋白笼内目的mRNA包装情况;
图9为本申请实施例提供的数字PCR定量分析Qt+-OVA蛋白笼内封装的目的mRNA含量;
图10为本申请实施例提供的Qt+-OVA耐酶性测试结果;
图11为本申请实施例提供的Qt+-OVA耐热性测试结果;
图12为本申请实施例提供的Qt+-OVA冻融性测试结果;
图13为本申请实施例提供的CCK8检测细胞相对活性水平(24 h)结果;
图14为本申请实施例提供的CCK8检测细胞相对活性水平(48 h)结果;
图15为本申请实施例提供的CCK8检测细胞相对活性水平(72 h)结果;
图16为本申请实施例提供的流式细胞术检测各组小鼠脾细胞中活化T细胞的百分比结果;
图17为本申请实施例提供的试剂盒检测各组小鼠脾细胞中IFN-γ分泌水平;
图18为本申请实施例提供的3针免疫后小鼠的肿瘤湿重水平。
具体实施方式
本申请提供了自组装纳米蛋白笼及其制备方法和应用,用于解决现有技术中递送RNA药物存在的稳定性、安全性、生物相容性和冷链依赖的技术缺陷。
下面将对本申请实施例中的技术方案进行清楚、完整地描述,显然,所描述的实施例仅仅是本申请一部分实施例,而不是全部的实施例。基于本申请中的实施例,本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例,都属于本申请保护的范围。
其中,以下实施例所用试剂或药物均为市售或自制。
以下实施例以肿瘤模型抗原OVA为例,验证本申请提供的自组装纳米蛋白笼的包装和递送mRNA疫苗的性能。
以下实施例使用的商用化脂质体来源于GenNano™-LNP-mRNA试剂包,其品牌为:迈安纳(上海)仪器科技有限公司(Micro&Nano),其货号为:R-0103,根据该试剂包的说明书包被OVA mRNA,获得脂质体-OVA。
实施例1
本实施例选择嗜热菌来源的240聚体Qt(PDB:6NJ8)用于包装和递送mRNA疫苗,选择肿瘤模型抗原OVA的mRNA作为待包装和递送疫苗抗原。该纳米蛋白笼(以下标记为Qt)的空间结构示意图。
鉴于Qt的亚基N端暴露在蛋白笼内表面,将一段由带正电荷氨基酸组成的阳离子肽(氨基酸序列为RKKKRR(SEQ ID NO.1))于N融合。由此得到的蛋白笼(Qt+)内腔形成2406个带正电荷的正增压环境,能够有效通过静电相互作用吸引带负电的外源性核酸分子进入笼内部,实现核酸包装。同时,由于Qt亚基C端暴露于蛋白笼外表面,将组氨酸蛋白纯化标签(氨基酸序列:HHHHH)于C端融合,便于下游蛋白纯化,改造后的纳米蛋白笼标记为纳米蛋白笼Qt+,其序列结构如图2所示。
实施例2
进一步地,本实施例对上述的纳米蛋白笼Qt+进行表达、纯化与鉴定,具体包括:人工合成编码纳米蛋白笼Qt+的cDNA核苷酸,cDNA序列克隆到pET28a表达质粒中,得到重组表达质粒(重组表达载体的质粒图如图3所示),然后将该重组表达质粒转化到大肠杆菌BL21中进行扩大培养,加入0.5 mM IPTG诱导剂,18℃,200 rpm,16 h,诱导纳米蛋白笼Qt+表达。进一步通过亲和层析(Ni-NTA)和尺寸排阻色谱法(SEC)纯化纳米蛋白笼Qt+,浓缩后采用SDS-PAGE的考马斯亮蓝染色和SEC进行纯度鉴定,并通过动态光散射法(DLS)和负染色透射电镜(TEM)进行颗粒均一度和颗粒外貌形态鉴定。鉴定合格的纳米蛋白笼Qt+保存于-20℃,用于后续分析,鉴定野生型Qt和纳米蛋白笼Qt+的纯度、颗粒和外貌情况。
SDS-PAGE/考马斯亮蓝染色纯度鉴定结果如图4所示,尺寸排阻色谱法(SEC)纯度鉴定结果如图5所示,动态光散射法(DLS)鉴定纳米蛋白笼Qt+结果如图6所示,负染色透射电镜(TEM)鉴定纳米蛋白笼Qt+外貌形态结果如图7所示。图4~图7结果显示,纳米蛋白笼Qt+多肽成功表达,纳米蛋白笼Qt+以高纯度纯化,并在镜下显示均匀一致的球状形态颗粒。
实施例3
本实施例为体外合成OVA mRNA序列和纳米蛋白笼Qt+包装OVA mRNA,具体包括:
OVA mRNA序列于上海捷瑞生物工程有限公司合成。
通过调整纳米蛋白笼Qt+所在缓冲体系的pH(pH=2)在室温放置不小于6 h使蛋白笼解聚,加入上述合成的OVA mRNA序列,将缓冲体系pH恢复至中性,纳米蛋白笼Qt+重新自组装,获得内部包装有OVA mRNA序列的纳米蛋白笼(以下标记为Qt+-OVA)。向包装有OVAmRNA的Qt+中加入终浓度10 μg/mL RNase A和2.5 U/μL苯并酶,放入37℃孵箱消化0.5 h,目的去除未包装或蛋白笼表面粘附的游离mRNA分子。提取RNA,进行RT-PCR,通过GelRed染色的琼脂糖凝胶电泳(2%)分析蛋白笼内目的mRNA包装情况;通过数字PCR鉴定蛋白笼内封装的目的mRNA比例。
图8显示普通RT-PCR鉴定Qt+-OVA蛋白笼内目的mRNA包装情况,图9显示数字PCR定量分析Qt+-OVA蛋白笼内封装的目的mRNA含量,图8和图9显示,相比野生型蛋白笼Qt(未改造),纳米蛋白笼Qt+可有效包装目的mRNA,并表现出更高的包装量。
实施例4
进一步地,本实施例对纳米蛋白笼Qt+进行稳定性评估,具体包括:
1、耐酶性测试:将上述制得的Qt+-OVA分别添加5 U/μL苯丙酶20 μg/mL RNase A,于37℃孵箱处理2 h。提取RNA,利用Nanodrop One检测处理前后RNA含量变化。结果如图10所示。
2、热稳定性测试:将上述制得的Qt+-OVA分别在25℃、37℃、65℃和75℃条件下孵育48 h。提取RNA,利用Nanodrop One检测处理前后RNA含量变化。结果如图11所示。
3、冻融性测试:将上述制得的Qt+-OVA于-80℃反复冻融5次。提取RNA,利用Nanodrop One检测处理前后RNA含量变化。结果如图12所示。
图10~图12的结果表明,Qt+-OVA对核酸酶、加热和反复冻融具有超高的耐受性,经不同条件处理后,纳米蛋白笼Qt+能够有效保护内部包装的mRNA,避免其受到外部极端条件的侵害,表明纳米蛋白笼Qt+赋予了OVA mRNA高的稳定性。
实施例5
进一步地,本实施例对纳米蛋白笼Qt+进行安全性评估,具体包括:
采用不同浓度(2 μg/mL、10 μg/mL、100 μg/mL、200 μg/mL、500 μg/mL)的上述制得的Qt+-OVA和商用化脂质体-OVA处理HK293T细胞不同时间(24 h、48 h和72 h),采用CCK8细胞活性检测试剂盒测定细胞活性水平,结果如图13~图15所示。其中,HK293T细胞分为阴性对照组,不同剂量Qt+-OVA组和不同剂量商用化脂质体-OVA组。阴性对照组不做处理。
图13~图15的结果表明,相比商用化脂质体包装的mRNA疫苗,不同剂量的Qt+-OVA处理细胞不同时间后,对细胞活性几乎不产生影响,表明纳米蛋白笼Qt+构建的mRNA疫苗具有更高的生物相容性和安全性。
实施例6
本实施例对纳米蛋白笼Qt+进行免疫原性评估,具体包括:
1、细胞免疫:C57BL/6小鼠分为3组:阴性对照组、脂质体-OVA和上述制得的Qt+-OVA组,每组5只。脂质体-OVA和Qt+-OVA组采用包装有相同质量OVA mRNA的商用脂质体和Qt+肌肉注射免疫。阴性对照组采用相同体积的PBS肌肉注射。小鼠接种疫苗7 d后,处死收集脾脏,通过流式细胞术评估活化T细胞的百分比(图16),包括CD3+ CD4+ CD69+和CD3+ CD8+CD69+ T细胞;收集脾细胞,体外采用OVA抗原刺激,检测IFN-γ的分泌水平(图17)。
图16~图17的结果表明,与阴性对照组相比,脂质体-OVA和Qt+-OVA均诱导了更高比例的活化T细胞,这些细胞经抗原刺激后分泌更高的IFN-γ,表明产生了更多的特异性T细胞;相比脂质体-OVA,Qt+-OVA没有表现出显著的免疫反应提升,但也表明Qt+-OVA能够和目前商用化mRNA疫苗取得相似的细胞免疫效果。
2、肿瘤生长抑制:C57BL/6小鼠分为3组:阴性对照组、脂质体-OVA和上述制得的Qt+-OVA组,每组5只。免疫前进行肿瘤细胞E.G7-OVA接种,每只小鼠腋下皮下接种1×106个细胞。接种7 d后,脂质体-OVA和Qt+-OVA组采用包装有相同质量OVA mRNA的商用脂质体和Qt+肌肉注射免疫。阴性对照组采用相同体积的PBS肌肉注射。所有小鼠均采用初免-加强-加强方式接种,于第7 d、第14 d和第21 d三剂接种,第23 d对小鼠实施安乐死。完整剥离肿瘤组织,测量肿瘤湿重(图18)。
图18结果显示,与阴性对照组相比,脂质体-OVA和Qt+-OVA均显著抑制了肿瘤细胞生长;相比脂质体-OVA,Qt+-OVA没有表现出显著的肿瘤生长抑制,但也表明Qt+-OVA能够和目前商用化mRNA疫苗取得相似的肿瘤生长抑制效果。
实施例7
本实施例通过筛选还确定了多种纳米蛋白笼修饰阳离子肽(氨基酸序列为RKKKRR(SEQ ID NO.1))后可增加其包装mRNA的含量,这些纳米蛋白笼可以为来自人来源24聚体Ferritin(PDB:4Y08)、嗜热菌来源24聚体Ferritin(PDB:2JD6)、幽门螺杆菌来源24聚体Ferritin(PDB:3BVF)、嗜热菌来源60聚体E2蛋白(PDB:1B5S)、嗜热菌来源60聚体AaLS(PDB:1HQK)、嗜热菌来源60聚体Tm(PDB:7KQ5)。
按照实施例3公开的数字PCR定量分析蛋白笼内封装的目的mRNA含量的方法测定上述这些纳米蛋白笼包装目的mRNA含量,结果如表1。表1可知,纳米蛋白笼经过阳离子肽修饰后可包装更多mRNA。
表1
综上所述,本申请实施例提供的纳米蛋白笼Qt+能够有效封装外源性mRNA疫苗,并保护mRNA免受外部极端环境(核酸酶、加热和反复冻融)的侵害,有效克服当前mRNA疫苗技术的冷链依赖问题;相比商用化脂质体mRNA疫苗,纳米蛋白笼Qt+具有更高的生物相容性和安全性,同时能够表现出与商用化脂质体mRNA疫苗相似的免疫效果,包括细胞免疫和肿瘤生长抑制。以上数据均表明经过阳离子肽修饰的纳米蛋白笼(如人来源24聚体Ferritin、嗜热菌来源24聚体Ferritin、幽门螺杆菌来源24聚体Ferritin、嗜热菌来源60聚体E2蛋白、嗜热菌来源60聚体AaLS、嗜热菌来源60聚体Tm或嗜热菌来源240聚体Qt等)在包装更多mRNA、增强mRNA疫苗稳定性、安全性和诱导有效的免疫反应过程中具有重要的应用价值。
以上所述仅是本申请的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本申请原理的前提下,还可以做出若干改进和润饰,这些改进和润饰也应视为本申请的保护范围。
Claims (10)
1.自组装纳米蛋白笼,其特征在于,包含:
(i)包含单体自组装的纳米蛋白笼,所述单体修饰有阳离子肽;所述阳离子肽包含由以下项组成的氨基酸序列:X1~X6~12,其中X1到X6~12中的任一者是彼此独立的氨基酸,条件是X1到X6~12中彼此独立为精氨酸或赖氨酸;
(ii)RNA药物;
其中所述RNA药物通过所述阳离子肽被包装到自组装的纳米蛋白笼中。
2.根据权利要求1所述的自组装纳米蛋白笼,其特征在于,所述纳米蛋白笼来自人来源24聚体Ferritin、嗜热菌来源24聚体Ferritin、幽门螺杆菌来源24聚体Ferritin、嗜热菌来源60聚体E2蛋白、嗜热菌来源60聚体AaLS、嗜热菌来源60聚体Tm或嗜热菌来源240聚体Qt。
3.根据权利要求2所述的自组装纳米蛋白笼,其特征在于,
所述人来源24聚体Ferritin的PDB编号为4Y08;
所述嗜热菌来源24聚体Ferritin的PDB编号为2JD6;
所述幽门螺杆菌来源24聚体Ferritin的PDB编号为3BVF;
所述嗜热菌来源60聚体E2蛋白的PDB编号为1B5S;
所述嗜热菌来源60聚体AaLS的PDB编号为1HQK;
所述嗜热菌来源60聚体Tm的PDB编号为7KQ5;
所述嗜热菌来源240聚体Qt的PDB编号为6NJ8。
4.根据权利要求1所述的自组装纳米蛋白笼,其特征在于,所述阳离子肽通过连接肽连接在所述单体的N端、C端或中部,使得所述阳离子肽位于所述纳米蛋白笼的内部;其中所述连接肽为柔性GS linker。
5.根据权利要求1所述的自组装纳米蛋白笼,其特征在于,所述阳离子肽包含SEQ IDNO.1所示的氨基酸序列。
6.根据权利要求1所述的自组装纳米蛋白笼,其特征在于,所述单体还修饰有蛋白质纯化标签、穿膜肽、靶向肽和佐剂肽中的一种或多种。
7.根据权利要求1所述的自组装纳米蛋白笼,其特征在于,所述RNA药物的长度不超过5000 nt;所述RNA药物为进行过化学修饰的mRNA。
8.权利要求1至权利要求7任意一项所述的自组装纳米蛋白笼的制备方法,其特征在于,包括以下步骤:
制备并纯化纳米蛋白笼;
对所述纳米蛋白笼在溶液中进行解聚;
将解聚的纳米蛋白笼与RNA药物混合在溶液中重新自组装,得到内部封装有RNA药物的自组装纳米蛋白笼。
9.根据权利要求8所述的制备方法,其特征在于,所述制备并纯化纳米蛋白笼的方法包括:
构建表达单体的表达工程菌;其中所述单体修饰有阳离子肽;所述阳离子肽包含由以下项组成的氨基酸序列:X1~X6~12,其中X1到X6~12中的任一者为彼此独立的氨基酸,条件是X1到X6~12中彼此独立为精氨酸或赖氨酸;
将表达单体的表达工程菌在筛选培养基中培养并诱导表达所述单体并使其自组装为纳米蛋白笼多聚体,然后收集、获取并纯化所述表达工程菌内的纳米蛋白笼多聚体,得到纳米蛋白笼。
10.权利要求1至权利要求7任意一项所述的自组装纳米蛋白笼或权利要求8或9所述的制备方法制得的自组装纳米蛋白笼在制备mRNA疫苗中的应用。
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