SpyTag/SpyCatcher体系蛋白共价连接条件的分析

王蓓; 胡海涛; 楼觉人

doi:10.3760/cma.j.cn311962-20210628-00036

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国际生物制品学杂志

• 工艺研发 •

ENGLISH ABSTRACT

SpyTag/SpyCatcher体系蛋白共价连接条件的分析

作者及单位信息

出版日期： 2021 -10 -10 ·

DOI： 10.3760/cma.j.cn311962-20210628-00036

Analysis of protein covalent bonding conditions in SpyTag/SpyCatcher system

Authors Info & Affiliations

Wang Bei

Research and Development Department, Shanghai Wellvax Co., Ltd., Shanghai 200051, China

Hu Haitao

Research and Development Department, Shanghai Wellvax Co., Ltd., Shanghai 200051, China

Lou Jueren

Research and Development Department, Shanghai Wellvax Co., Ltd., Shanghai 200051, China

No.2 Laboratory, Shanghai Institute of Biological Products Co., Ltd., Shanghai 200051, China

Published： 2021 -10 -10 ·

DOI： 10.3760/cma.j.cn311962-20210628-00036

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摘要

目的比较不同实验条件下SpyTag/SpyCatcher体系共价连接的效率，分析得出优化后的连接条件，为今后相关纳米颗粒疫苗的开发提供实验依据。

方法用大肠埃希菌表达分别带有SpyTag和SpyCatcher结构的两种蛋白，摸索共价连接的反应时间、温度、缓冲体系和混合比例，采用非还原型凝胶电泳检测共价连接的效率。

结果反应时间对共价连接的影响有统计学意义( F＝22.028， P<0.05)，带有SpyTag和SpyCatcher结构的两种蛋白质可形成稳定的共价键，6 h后连接效率约为50%，偶联蛋白在25 ℃稳定存在至少72 h。反应温度和缓冲体系对共价连接的影响没有统计学意义。SpyTag和SpyCatcher结构蛋白比例为物质的量比1.0∶2.0混合。

结论SpyTag/SpyCatcher共价连接体系的关键工艺参数为反应时间，且该体系能适用于多种温度和缓冲条件。

关键词： SpyTag/SpyCatcher;共价键;共价连接效率

ABSTRACT

ObjectiveTo investigate the covalent bonding efficiency of SpyTag/SpyCatcher system under different experimental conditions.

MethodsTwo proteins with the structure of SpyTag and SpyCatcher respectively were expressed in E. coli. To detect the efficiency of covalent bonding by non-reducing gel electrophoresis when the reaction time, temperature, buffer system, and mixing ratio were changed.

ResultsThe reaction time had a significant effect on covalent bonding ( F=22.028, P<0.05). When SpyTag and SpyCatcher were mixed, covalent bonding occurred immediately, the bonding efficiency reached about 50% after 6 h, and the new protein was stable at 25 ℃ for at least 72 h. The reaction temperature and buffer system had no significant effect on covalent bonding. In this study, the appropriate mixing molar ratio of the two proteins was 1.0∶2.0.

ConclusionThe critical process parameter of SpyTag/SpyCatcher covalent bonding system is reaction time, and this system can be applied to a variety of temperature and buffer conditions.

KEYWORDS： SpyTag/SpyCatcher;Covalent bond;Covalent bonding efficiency

Corresponding author: Lou Jueren, Email: mocdef.mabrahponis1nereujuol

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All articles published represent the opinions of the authors, and do not reflect the official policy of the Chinese Medical Association or the Editorial Board, unless this is clearly specified.

引用本文

王蓓,胡海涛,楼觉人. SpyTag/SpyCatcher体系蛋白共价连接条件的分析[J]. 国际生物制品学杂志,2021,44(05)：271-275.

DOI:10.3760/cma.j.cn311962-20210628-00036

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版权归中华医学会所有。

未经授权，不得转载、摘编本刊文章，不得使用本刊的版式设计。

除非特别声明，本刊刊出的所有文章不代表中华医学会和本刊编委会的观点。

SpyTag/SpyCatcher体系是近些年发展起来的一种快速稳定的不可逆肽-蛋白质连接方法，源于CnaB2结构域，基于化脓性链球菌纤黏连蛋白FbaB分离得到 ^{[
1
]}。CnaB2含一个具有特殊稳定性的肽键，将其裂解成肽和蛋白片段后进行合理的修饰，可以得到SpyTag(含13个氨基酸残基)和SpyCatcher(含138个氨基酸残基)两个部分。SpyTag中的天冬氨酸能和SpyCatcher上的赖氨酸在与赖氨酸相邻的谷氨酸催化下，自发反应生成异肽共价键，即使在pH2的缓冲体系或100 ℃时仍保持折叠 ^{[
2
,

3
]}。SpyTag/SpyCatcher体系广泛应用于多项基础研究和应用科学中，如通过该体系结合能显著增强免疫应答的蛋白质，诱导针对纳米颗粒疫苗中弱免疫原性表位的高效抗体应答 ^{[
4
,

5
,

6
]}；提高多酶复合体系中酶级联的效率，同时保持甚至提高生物催化剂的稳定性和可重用性 ^{[
7
,

8
]}；表征蛋白质-RNA相互作用，作为紫外交联免疫沉淀的替代方法 ^{[
9
]}；将化学合成的电压敏感染料靶向于特定细胞，进行活体细胞电压的动态光学测量 ^{[
10
]}；修饰蛋白水凝胶 ^{[
11
]}等。

SpyTag/SpyCatche体系与化学偶联、酶联等传统方法相比，具有反应条件简便、特异性高、效率高、反应产物稳定等优点。利用该体系研发的基于铁蛋白纳米颗粒的HBVpreS1纳米疫苗，可以显著增强针对弱抗原表位的特异性抗体水平，在小鼠模型中诱导高水平、高亲和力、持久的抗体应答和免疫记忆 ^{[
12
]}。本研究的目的是表达含SpyTag结构的SpyTag-Fe蛋白及含SpyCatcher结构的SpyCatcher-Btch1小分子蛋白，通过改变两个蛋白质的共价连接反应发生的时间、温度、缓冲体系及混合比例，检测并比较产物蛋白的形成效率得出关键工艺参数，优化该体系稳定合成蛋白连接产物的合适条件，为应用该体系开发相关纳米颗粒疫苗提供实验依据。

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通信作者：楼觉人，Email: mocdef.mabrahponis1nereujuol

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