CN109666973A - 一种穿过血脑屏障的肽库及其筛选方法 - Google Patents
一种穿过血脑屏障的肽库及其筛选方法 Download PDFInfo
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- CN109666973A CN109666973A CN201811394011.7A CN201811394011A CN109666973A CN 109666973 A CN109666973 A CN 109666973A CN 201811394011 A CN201811394011 A CN 201811394011A CN 109666973 A CN109666973 A CN 109666973A
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Abstract
本发明涉及一种穿过血脑屏障的肽库、利用所述肽库筛选穿血脑屏障肽的方法、获得的穿血脑屏障肽。本发明还提供所述穿血脑屏障肽的编码核酸、核酸构建体、宿主细胞、以及其作为脑部药物递送载体制备脑部药物中的用途。在前期已知穿血脑屏障肽构效关系和生物信息学分析的基础上，充分利用活体动物血脑屏障功能和噬菌体表面展示肽库技术，提供了一种高效穿血脑屏障肽的筛选和制备方法。
Description
技术领域
本发明涉及生物工程及医药领域，特别涉及噬菌体表面展示技术、分子生物学技术、以及药物递送技术领域，具体涉及一种穿过血脑屏障的肽库、利用所述肽库筛选穿血脑屏障肽的方法、获得的穿血脑屏障肽及其用途。
背景技术
大脑是人的高级神经中枢，有由脑毛细血管壁与神经胶质细胞形成的血浆与脑细胞之间的屏障称为血脑屏障，该屏障限制了多种物质进入到脑部。实验和临床证据表明血脑屏障为神经功能维持稳定的化学环境，并保护大脑免受有害物质伤害。血脑屏障因而对很多复杂的问题非常重要，例如药物传递，慢性神经疾病的发病机制，以及生物防御相关的问题。脑部与外界有严格的物质限制进入的血脑屏障的阻挡机制，会导致大部分目前有效药物难以到达作用靶点，而难以对脑部疾病进行有效治疗。
我们基于受体介导途径思路，能通过受体LRP1介导的受体途径，参考牛胰岛抑制剂以及β-淀粉样肽跨血脑屏障蛋白保守的活性中心kunitz区域，选择骨架，利用噬菌体展示技术，体外人工合成构建高通量噬菌体展示文库，从基础获取新型自主知识产权高效穿血脑屏障短肽，利用小鼠体内筛选技术，优化筛选流程，通过三轮筛选，得到高效穿过血脑屏障短肽。
发明内容
为解决上述问题，一方面，本发明提供一种穿过血脑屏障的肽库，其特征在于具有以下结构通式：
TFYGGRX1KRNNFX2X3X4X5X6，
其中所述X1-X6为随机氨基酸，
本发明所述肽库优选为噬菌体展示肽库。
第二方面，本发明还提供所述肽库的编码核酸，其特征在于具有以下序列：
ACCTTTTATGGTGGTCGTNNKAAACGTAATAATTTTNNKNNKNNKNNKNNK，
其中所述的N为A、T、C或G；所述的K为G或T。
第三方面，本发明还提供一种利用所述肽库筛选高效穿血脑屏障肽的方法，包括：
(1)将肽库接种到动物模型；
(2)待肽库在动物模型脑组织中富集程度最高时，取脑组织，回收脑组织中的肽库并进行体外扩增；
(3)回收体外扩增后的肽库；
(4)重复步骤(1)-(3)，直至获得高效穿血脑屏障肽；
(5)确定高效穿血脑屏障肽的序列。
本发明所述筛选高效穿血脑屏障肽的方法，其特征在于：在步骤(1)之前还包括预实验：检测肽库接种后在动物模型脑部富集的最佳时间；
本发明所述步骤(2)根据预实验中测得的肽库在脑部富集的最佳时间来确定取动物模型脑组织的时机。
本发明所述筛选高效穿血脑屏障肽的方法，其特征在于：所述步骤(4)中重复步骤(1)-(3)的次数为2-5次，优选重复3次。
第四方面，本发明提供一种高效穿过血脑屏障的肽，其特征在于具有以下结构通式：
TFYGGRX1KRNNFX2X3X4X5X6，
其中，
X1为P、V、S或R；
X2为L、A、P或T；
X3为R、L、K或A；
X4为G、S、L或V；
X5为I、L、H或S；
X6为R、W、R或A。
本发明所述高效穿过血脑屏障的肽，其特征在于所述肽包括：
TFYGGRPKRNNFLRGIR，TFYGGRVKRNNFALSLW，TFYGGRSKRNNFPKLHR，或TFYGGRRKRNNFTAVSA。
第五方面，本发明还提供一种所述高效穿过血脑屏障肽的编码核酸。
第六方面，本发明还提供包含所述编码核酸的核酸构建体或宿主细胞。
所述核酸构建体包括表达盒、载体，所述宿主细胞包括真核细胞和原核细胞。
本发明还提供所述编码核酸、包含所述编码核酸的构建体或宿主细胞在制备所述高效穿过血脑屏障肽的用途。
本发明还提供生产所述高效穿过血脑屏障肽的方法，其利用了所述高效穿过血脑屏障肽的编码核酸、核酸构建体或重组宿主细胞。
第七方面，本发明还提供所述高效穿过血脑屏障肽、其编码核酸、包含所述编码核酸的构建体或宿主细胞在制备脑部药物中的用途。
所述药物包括脑部成像检测药物及脑部疾病治疗药物。
与现有技术相比，本发明的技术方案具有以下优点：
第一、基于前期大量的穿血脑屏障肽构-效分析，确定了基于kunitz区的中心骨架，在此基础上构建了穿血脑屏障肽库。在筛选血脑屏障肽时，相比于完全随机肽库(如商购肽库)，成功率更高、工作量更小、结果更稳定。
第二、改进了噬菌体文库筛选方法。经典的噬菌体展示技术手册是通过体外结合-洗脱模式逐渐富集与受体亲和力高的噬菌体，然而血脑屏障机制复杂，体外血清学试验中对LRP1亲和力高的多肽未必有高的穿血脑屏障效果。本发明利用动物模型体内的血脑屏障，进行富集筛选，效率更高、更接近临床真实的效果。
第三、本发明所述方法制备的肽能特异性穿过血脑屏障。通常情况下，即使能够穿过血脑屏障的药物在机体其他组织器官也有分布，且分布量高于脑部。本发明筛选制备的穿血脑屏障肽能够高效特异的穿过血脑屏障，并且脑内分布量比非屏障部位肌肉等的分布量高。因此，将本发明所述穿过血脑屏障肽用于制备脑部药物能够极大改善药物分布，提高药物利用率、在确保具备有效浓度的情况下降低全身给药的剂量。
附图说明
通过阅读下文优选实施方式的详细描述，各种其他的优点和益处对于本领域普通技术人员将变得清楚明了。附图仅用于示出优选实施方式的目的，而并不认为是对本发明的限制。
图1：肽库梯度稀释空斑计数
A：稀释度10e2；B：稀释度10e4；C：稀释度10e6。
图2：小鼠脑部噬菌体展示肽给药后最佳富集时间
图3：合成M1的质谱检测图
图4：M1标记cy5.5荧光质谱检测图
图5：M1-cy5.5静脉注射后4h小鼠活体成像检测图
A：M1-cy5.5；B：阴性对照。
图6：器官荧光分布图
图7：器官荧光分布值
具体实施方式
下面将参照附图更详细地描述本公开的示例性实施方式。虽然附图中显示了本公开的示例性实施方式，然而应当理解，可以以各种形式实现本公开而不应被这里阐述的实施方式所限制。相反，提供这些实施方式是为了能够更透彻地理解本公开，并且能够将本公开的范围完整的传达给本领域的技术人员。
根据本发明的实施方式，参考牛胰岛抑制剂以及β-淀粉样肽跨血脑屏障蛋白保守的活性中心kunitz区域，选择骨架，利用噬菌体展示技术，体外人工合成构建高通量噬菌体展示文库，从基础获取新型自主知识产权高效穿血脑屏障短肽，利用小鼠体内筛选技术，优化筛选流程，通过三轮筛选，得到高效穿过血脑屏障短肽。
实施例一、建库
1、基于活性中心kunitz区域的序列分析，找到如下中心骨架序列，设计完成噬菌体展示的多肽库kun-M的构建。
构建方法：
Library 1:TFYGGRXKRNNF XXXXX(SEQ ID NO:1)
X为随机氨基酸。
长引物：
Library1
5’-cccaggtgcagctgcagACCTTTTATGGTGGTCGTNNKAAACGTAATAATTTTNNKNNKNNKNNKNNKtctagaggggacccaggtc-3’(SEQ ID NO:2)
所述的N为A、T、C或G；所述的K为G或T
下划线标出了酶切位点，大写字母为肽库编码核酸序列。
设计第一对引物NAG-F：GCCCAGGTGCAGCTG(Tm 57.24)(SEQ ID NO:3)
第二对引物NAG-R：GACCTGGGTCCCCTCTAG(Tm 57.29)(SEQ ID NO:4)
引物交引物合成公司合成。
2、目的片段的扩增
PCR扩增条件：
PCR建库
加水补到50ul
PCR条件：
30cycle
扩出目的片段在100bp左右，天根PCR纯化回收试剂盒回收PCR产物。测定回收产物的浓度。
3、酶切、回收、纯化
酶切回收PMESY 4-质粒，PCR目的片段。
酶切体系
水补齐到没管50ul
跑1％的琼脂糖胶，切胶回收酶切开的质粒片段。
酶切PCR目的片段ang2nw，采用同样酶切体系，利用生工PCR纯化回收试剂盒回收目的片段。
4、目的片段连接
连接体系,质粒与目的片段按1：3的摩尔量加。
H2O补齐到80ul
16度过夜，将连接产物按产物回收试剂盒回收，用超纯水70度洗脱。
5、转感受态制备：
制备感受态
(1)将过夜培养的ER2738菌按1：100的比例将过夜菌液转接到500ml的LB+TET培养基中，培养到对数期0.6，冰上孵育30min。
(2)大肠杆菌液在4℃，4000rpm下离心10min，弃上清液。
(3)弃上清，离心管中加入少量ddH2O，轻柔的悬浮沉淀后，100ml再将水注满离心管，4℃，4000rpm离心10min。
(4)重复步骤(3)1次。
(5)小心弃上清(沉淀可能会很松散)，再往离心管中加入100ml量10％甘油(灭菌，预冷)，重悬菌体，4℃，4000rpm离心10min。重复步骤5一次
(6)用10％甘油重悬浮细胞至最终体积为2ml，将细胞按100μl等份装入微量离心管，放置于－80℃下保存。
6、电转化：
将电转杯冰上预冷30min，取连接产物进行电转，
电转2.5kv、5ms、20uF.
电转加入0.9ml2YT培养基,37度培养1h，涂amp平板。
取10ul梯度稀释，稀释涂小板，37度孵育过夜，结果如图1所示。
挑噬菌体克隆，部分测序结果如序列SEQ ID NO:9-21所示。
实施例二、肽库体内筛选
筛选之前先通过预实验，确定小鼠脑部最佳富集噬菌体展示肽的时间，结果如图2所示。接种后24h噬菌体脑血比最高，表明在脑的富集程度最高。
筛选方法如下：
1成年balb/c小鼠(18～22g)，取噬菌体库TBS稀释到100ul/1011PFU,尾静脉注射，根据预实验在24h噬菌体在脑的富集脑血比最高。
2取24h时间点，5％的水合氯醛麻醉，小鼠保持无菌，心脏贯流100ml生理盐水，解剖取脑，将脑网状体匀桨超声打碎，离心，过0.45μm滤膜，取上清溶液与培养到对数期的ER2738菌液混匀，37度感染培养4h。
3 12000rpm离心20min，取菌液上清，用PEG/NaCl的方法富集噬菌体，将该富集的库用于下一轮的筛选。
4如上重复至少三轮。在看到输出噬菌体的滴度出现明显富集，可取噬菌体感染克隆，送测序分析展示多肽纳米抗体的序列。挑克隆测序，结果如表1：
表1：克隆测序获得的高频序列
| label | sequence | Frequency |
| M1 | TFYGGRPKRNNFLRGIR | 3 |
| M2 | TFYGGRVKRNNFALSLW | 2 |
| M3 | TFYGGRSKRNNFPKLHR | 2 |
| M4 | TFYGGRRKRNNFTAVSA | 2 |
选取M1进行动物体内验证，将多肽序列TFYGGRPKRNNFLRGIR(MW：2053)送多肽合成公司进行合成多肽合成以及标记荧光纯化。得到超过95％纯度以上的纯度多肽。
无标记多肽质谱检测结果如图3所示。肽分子量为1027.86*2-2＝2053.6，分子量符合。
标记上CY5.5荧光多肽质谱检测结果如图4示。Y5.5标记上荧光分子量873.22*3-3-2053＝564，为cy5.5荧光试剂反应后的分子量。说明M1短体连上一个荧光分子。
2、荧光标记肽的体内检测
连荧光分子短肽用于后续小鼠体内检测。准确称取连短肽，用生理盐水将其溶解，调整稀释连接荧光短肽，到荧光当量为5uM，尾静脉注射裸鼠100ul，在不同时间点，利用IVIS小动物活体成像系统分不同时间点观测成像(图5)。
在尾静脉注射4h，可以明显观测到M1-CY5.5在脑部富集。取小鼠，5ml/min的速度心脏贯流100ml生理盐水，洗掉血液中的荧光干扰，
取小鼠脑，以及其他器官观察，可以看出脑部有明显荧光，且高于肌肉心脏等，脑肌肉荧光比约为3∶1(图6、7)。说明短肽能穿过血脑屏障的阻拦，具有高效的穿血脑屏障的能力，且比没有屏障的部位如肌肉富集量高。
以上所述，仅为本发明较佳的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉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，都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应以所述权利要求的保护范围为准。
SEQUENCE LISTING
<110> 申请人名称 北京大学
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Claims (10)
1.一种穿过血脑屏障的肽库，其特征在于具有以下结构通式：
TFYGGRX1KRNNFX2X3X4X5X6，
其中所述X1-X6为随机氨基酸，
所述肽库优选为噬菌体展示肽库。
2.一种编码权利要求1所述肽库的编码核酸，其特征在于具有以下序列：
ACCTTTTATGGTGGTCGTNNKAAACGTAATAATTTTNNKNNKNNKNNKNNK，
其中所述的N为A、T、C或G；所述的K为G或T。
3.一种利用权利要求1所述肽库筛选高效穿血脑屏障肽的方法，包括：
(1)将肽库接种到动物模型；
(2)待肽库在动物模型脑组织中富集程度最高时，取脑组织，回收脑组织中的肽库并进行体外扩增；
(3)回收体外扩增后的肽库；
(4)重复步骤(1)-(3)，直至获得高效穿血脑屏障肽；
(5)确定高效穿血脑屏障肽的序列。
4.如权利要求3所述筛选高效穿血脑屏障肽的方法，其特征在于：在步骤(1)之前还包括预实验：检测肽库接种后在动物模型脑部富集的最佳时间；所述步骤(2)根据预实验中测得的肽库在脑部富集的最佳时间来确定取动物模型脑组织的时机。
5.如权利要求3筛选高效穿血脑屏障肽的方法，其特征在于：所述步骤(4)中重复步骤(1)-(3)的次数为2-5次，优选重复3次。
6.一种高效穿过血脑屏障的肽，其特征在于具有以下结构通式：
TFYGGRX1KRNNFX2X3X4X5X6，
其中，
X1为P、V、S或R；
X2为L、A、P或T；
X3为R、L、K或A；
X4为G、S、L或V；
X5为I、L、H或S；
X6为R、W、R或A。
7.如权利要求6所述高效穿过血脑屏障的肽，其特征在于所述肽包括：
TFYGGRPKRNNFLRGIR，TFYGGRVKRNNFALSLW，TFYGGRSKRNNFPKLHR，或TFYGGRRKRNNFTAVSA。
8.权利要求6或7所述高效穿过血脑屏障的肽的编码核酸。
9.包含权利要求8所述编码核酸的核酸构建体或宿主细胞，所述核酸构建体包括表达盒、载体，所述宿主细胞包括真核细胞和原核细胞。
10.权利要求6-7所述高效穿过血脑屏障肽、权利要求8所述编码核酸、权利要求9所述核酸构建体或宿主细胞制备脑部药物中的用途，所述药物包括脑部成像检测药物及脑部疾病治疗药物。
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020103960A3 (zh) * | 2018-11-21 | 2020-07-09 | 北京大学 | 一种穿过血脑屏障的肽库及其筛选方法 |
| WO2020103961A3 (zh) * | 2018-11-21 | 2020-07-09 | 江苏集萃分子工程研究院有限公司 | 一种脑部肿瘤靶向肽及其应用 |
| CN113425852A (zh) * | 2021-05-24 | 2021-09-24 | 北京大学 | 一种可穿过血迷路屏障的偶联物及其制备方法 |
| CN114787355A (zh) * | 2019-10-16 | 2022-07-22 | 海阳制药 | 具有血脑屏障穿透能力的肽-核酸复合物及包含该肽-核酸复合物的组合物 |
| CN115536730A (zh) * | 2022-09-20 | 2022-12-30 | 南开大学 | 一种穿越血脑屏障的多肽及其制备方法、纳米结构及其制备方法和应用 |
| CN115819498A (zh) * | 2022-09-07 | 2023-03-21 | 吉林农业大学 | 穿透血脑屏障的抗氧化多肽及其制备方法、应用 |
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| WO2020103961A3 (zh) * | 2018-11-21 | 2020-07-09 | 江苏集萃分子工程研究院有限公司 | 一种脑部肿瘤靶向肽及其应用 |
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| CN113425852A (zh) * | 2021-05-24 | 2021-09-24 | 北京大学 | 一种可穿过血迷路屏障的偶联物及其制备方法 |
| CN115819498A (zh) * | 2022-09-07 | 2023-03-21 | 吉林农业大学 | 穿透血脑屏障的抗氧化多肽及其制备方法、应用 |
| CN115819498B (zh) * | 2022-09-07 | 2023-05-16 | 吉林农业大学 | 穿透血脑屏障的抗氧化多肽及其制备方法、应用 |
| CN115536730A (zh) * | 2022-09-20 | 2022-12-30 | 南开大学 | 一种穿越血脑屏障的多肽及其制备方法、纳米结构及其制备方法和应用 |
| CN115536730B (zh) * | 2022-09-20 | 2023-08-15 | 南开大学 | 一种穿越血脑屏障的多肽及其制备方法、纳米结构及其制备方法和应用 |
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| WO2020103960A3 (zh) | 2020-07-09 |
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| WO2020103960A2 (zh) | 2020-05-28 |
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