CN111875701A - 一种SARS-CoV-2病毒的单链抗体及用途 - Google Patents
一种SARS-CoV-2病毒的单链抗体及用途 Download PDFInfo
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- CN111875701A CN111875701A CN202010820233.1A CN202010820233A CN111875701A CN 111875701 A CN111875701 A CN 111875701A CN 202010820233 A CN202010820233 A CN 202010820233A CN 111875701 A CN111875701 A CN 111875701A
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Abstract
本发明公开了一种SARS‑CoV‑2病毒的单链抗体的序列结构，所述单链抗体与抗原S1‑RBD特异性结合。还公开了制备所述单链抗体的方法，在药学上、试剂盒、组合物中的应用。所述单链抗体的功能是与S1‑RBD结合，阻止S1‑RBD与细胞上受体ACE2结合可应用于中和抗体的开发。
Description
技术领域
本发明总体涉及疫苗领域，更具体地，涉及一种SARS-CoV-2病毒的单链抗体及用途。
背景技术
冠状病毒属的病毒是具有外套膜包裹的RNA病毒，其直径约100-160nm，遗传物质在所有RNA病毒中最大。包裹病毒粒子的脂肪膜表面有三个糖蛋白：棘突糖蛋白(S，是受体结合位点、溶细胞作用和主要抗原位点)；小包膜糖蛋白(E，较小，与胞膜结合的蛋白)；膜糖蛋白(M，负责营养物质的跨膜运输、新生病毒出芽释放与病毒外包膜的形成)。
S蛋白被酶解为S1和S2，S1内有一部分区域为受体结合域(RBD)与 ACE2紧密结合，是病毒和受体相互作用的关键因素。
SARS-CoV-2属于一种新型冠状病毒，感染病毒的人会出现程度不同的症状，或是发烧或轻微咳嗽，或发展为肺炎，早期的百分比显示，该病毒致死率约为2％到4％。在SARS-CoV-2感染中，与SARS-CoV(主要感染纤毛支气管上皮细胞和II型肺细胞)感染相同，血管紧张素转化酶II(ACE2)被证明是细胞受体。SARS-CoV-2可以进入表达ACE2的细胞，但不能进入没有ACE2的细胞。因此，ACE2在SARS-CoV-2感染中起着至关重要的作用。
针对SARS-CoV-2病毒，需要提供特异性良好的抗体或者小分子抗体，便于检测和预防。
发明内容
为了解决上述问题，本发明提供了一种SARS-CoV-2病毒的单链抗体，所述单链抗体与抗原S1-RBD的SEQ NO：1序列特异性结合。
根据本发明的一个实施方式，所述的单链抗体，包括：重链的可变区的序列为SEQNO：2所示的氨基酸序列；轻链的可变区的序列为SEQ NO：3所示的氨基酸序列。
根据本发明的一个实施方式，重链的可变区的序列与SEQ NO：2所示的氨基酸序列具有95％相同。
根据本发明的一个实施方式，轻链的可变区的序列与SEQ NO：3所示的氨基酸序列95％相同。
根据本发明的一个实施方式，连接肽的序列为SEQ NO：4所示的序列。
根据本发明的一个实施方式，连接肽的序列与SEQ NO：4所示的序列 95％相同。
根据本发明的另一个方面，提供了一种制备SARS-CoV-2病毒的单链抗体的方法，包括，选取SARS-CoV-2抗原S-RBD，从全人源抗体文库中筛选得到单链抗体，所述单链抗体。
根据本发明的另一个方面，提供了一种试剂盒，所述试剂盒包括权利要求1-7中任一项所述的单链抗体。
根据本发明的另一个方面，提供了一种在药学上可接受的载体，其中包含上述任一方案所述的单链抗体。
根据本发明的另一个方面，提供了一种包含两种或多种上述任一方案所述的单链抗体的组合物，所述组合物与抗原S1-RBD的SEQ NO：1序列相结合。
本发明中的ScFv，靶向S1-RBD，阻止S1-RBD与ACE2的结合，可用于中和抗体的开发；而且，本发明的ScFv为全人源单抗，没有免疫原性反应。
附图说明
图1是阳性克隆显色在深孔板的示意图；
图2是表1中的阳性克隆的OD450数值折线图。
具体实施方式
下面结合附图对本发明的较佳实施例进行详细阐述，参考标号是指本发明中的组件、技术，以便本发明的优点和特征在适合的环境下实现能更易于被理解。下面的描述是对本发明权利要求的具体化，并且与权利要求相关的其它没有明确说明的具体实现也属于权利要求的范围。
本发明中，抗原S1-RBD来自恺佧生物科技(上海)有限公司。
抗体文库使用编号:Hu001—Hu010，来自成都仁域生物技术有限公司。
Hu001—Hu010是全人源抗体文库编号。
本发明以从噬菌体展示抗体文库中通过亲和筛选的方法为例进行说明其获取方法。
包被：将抗原S1-RBD用包被液稀释至浓度5ug/ml，(例如PBS包被液)，加入到ELISA板的孔位中，在4℃条件下持续12小时以上，使抗原S1-RBD 连接到ELISA板上。
洗涤：倒去ELISA板中的包被液，用无菌吸水纸拍干ELISA板，并用 PBS洗涤3次，洗涤去除未结合的抗原S1-RBD。
封闭：然后加入350ul封闭液，对ELISA板进行封闭，37℃放置1小时，避免抗体库中非目标蛋白与ELISA板结合。封闭完成后，倒掉封闭液，利用无菌吸水纸将ELISA板拍干，用PBS洗涤3次，以除去残留的封闭液。封闭液可以采用现有的或将来发明的封闭液，本发明不予限定。
扣除抗体库背景：取100ul抗体库+400ul 2％脱脂奶粉(溶于PBS)混匀，37℃放置1小时，用于与一部分非特异性单链抗体结合，避免这些单链抗体与抗原S1-RBD结合，影响特异性单链抗体富集。
结合：将扣除背景的抗体库加入带有抗原S1-RBD的ELISA板孔中，按照100ul/孔，在37℃条件下，以100rpm转速缓摇1小时，使抗体库中的目标单链抗体与抗原S1-RBD特异性结合。
洗涤：洗涤带有抗原S1-RBD的ELISA板，将未与抗原S1-RBD结合的抗体及杂质除去。例如：PBS洗1次，0.1％PBST洗板9次，PBS洗1 次，每次1min。从而，在ELISA板上，与抗原S1-RBD特异性结合的单链抗体被富集。
加入洗脱液：将与抗原S1-RBD特异性结合的单链抗体从ELISA板孔内洗脱。例如：加入洗脱液，100ul/孔，室温缓摇10min，用枪头吸出洗脱液，加入0.5倍体积的中和液中，混匀，静置1h，4℃保存。
第一轮筛选结束，洗脱物中含有较为富集的目标单链抗体。
针对第一轮筛选的产物进行滴度测定：测量洗脱液中单链抗体的浓度。例如：在2YT(培养基)中梯度稀释洗脱的抗体库(第一轮10E2～3，第二轮10E4～5)。
第一步，挑取TG1单克隆，加入10ml 2YT中，37℃、200rpm培养至对数期OD600＝0.6-0.8，放置在37℃温箱备用；
第二步，向200ul处于上述对数期的TG1中加入10E1稀释的抗体库， 37℃放置30min，然后涂布在2YTAG(培养基加入Amp和Kana两种抗生素)平板上，37℃过夜培养，第二天计算菌落数。
根据第一轮筛选洗脱物的滴度测定结果，将洗脱物混合后用于第一轮产物扩增。
第一轮洗脱产物扩增，将得到的单链抗体进行了扩增。
例如：
(1)挑取平板上的TG1单克隆，加入20ml 2YT中，37℃、200rpm 培养至对数期OD600＝0.6-0.8。
(2)加入洗脱的抗体库混匀，37℃放置60min。
(3)补加入4ul Amp，37℃180rpm培养60min。
(4)向培养物中加入M13KO7(辅助噬菌体：TG1>＝10：1)，37 ℃放置30min。
(5)补加30ml 2YT和6ul Amp，37℃180rpm培养60min。
(6)室温5000rpm离心10min，去掉上清液，收集细菌沉淀，加入新的50ml 2YTAK(Amp:100ug/ml，Kana:50ug/ml)重悬沉淀，在200ml锥形瓶中30℃220rpm过夜培养。
以扩增的洗脱产物为原料，重复上述筛选过程，直至目标产物达到理想滴度。
实施例：
为了检测单链抗体与抗原结合强度，本发明进行了单克隆ELISA，步骤如下：
(1)准备单克隆phage上清
为了有足够的数量的空白对照组，本发明从第二次筛选测定滴度的平板上随机挑取96个单克隆于96孔深孔板中，加入300ul的2YT培养基180rpm， 37℃培养至对数期，加入M13K07辅助噬菌体侵染，换液，30℃过夜培养。
(2)准备靶蛋白，将靶蛋白包被在ELISA板上。
抗原包被用包被液(PBS9.0)将靶蛋白稀释为2ug/m的浓度，然后加入96孔ELISA板，50ul/孔，4℃静置过夜。洗涤、封闭、再洗涤ELISA 板。
(3)结合：phage上清结合：取50ul上清+50ul PBS-1％脱脂奶粉混匀，加入ELISA板中，室温下缓摇1h；弃去液体，拍干，用PBST(0.1％)清洗5次，PBS清洗1次；
(4)二抗结合：将Anti-M13-HRP抗体以1∶5000稀释于封闭液1％脱脂奶粉-PBS中，60ul/孔，加入ELISA板中，37℃静置1h，弃去液体，拍干，用PBST(0.1％)清洗5次，PBS清洗1次；
(5)加入100ul TMB，避光反应10分钟，加入100ul 2M盐酸终止反应，酶标仪中读取OD450数值。
图1示出了阳性克隆显色在深孔板的示意图。
表1示出了OD450数值。
表1.
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 | |
| A | 0.075 | 0.087 | 0.079 | 0.076 | 0.077 | 0.076 | 0.073 | 0.073 | 0.935 | 0.073 | 2.759 | 0.092 |
| B | 0.076 | 0.083 | 0.081 | 2.468 | 0.092 | 0.08 | 0.073 | 0.094 | 0.082 | 0.093 | 0.089 | 0.166 |
| C | 2.701 | 0.074 | 2.646 | 0.066 | 0.08 | 0.077 | 0.066 | 0.252 | 0.069 | 0.079 | 0.074 | 0.077 |
| D | 0.076 | 0.076 | 0.072 | 0.069 | 0.074 | 0.067 | 0.075 | 0.068 | 0.074 | 0.081 | 0.087 | 0.092 |
| E | 0.072 | 0.066 | 0.065 | 0.057 | 1.961 | 0.067 | 0.069 | 0.068 | 0.065 | 0.071 | 0.077 | 0.095 |
| F | 0.097 | 0.082 | 0.081 | 0.091 | 0.078 | 0.094 | 0.073 | 0.091 | 0.462 | 0.149 | 0.081 | 2.324 |
| G | 0.078 | 1.431 | 2.734 | 0.079 | 0.09 | 1.599 | 0.077 | 0.08 | 0.106 | 0.078 | 0.081 | 0.1 |
| H | 0.098 | 0.118 | 2.105 | 0.089 | 0.108 | 0.116 | 2.538 | 0.114 | 0.119 | 0.117 | 0.104 | 0.176 |
图2示出了表1中的阳性克隆的OD450数值折线图。
如图2所示，其中，第10、11、12为到第二次筛选后浓度较低的单克隆抗体抗原结合检测中，OD450的值通常在0.5-3之间。图1中，OD450值高于1.961的占阳性克隆的64％，高于2的占57％。
应该注意的是，上述实施例对本发明进行说明而不是对本发明进行限制，并且本领域技术人员在不脱离所附权利要求的范围的情况下可设计出替换实施例。在权利要求中，不应将位于括号之间的任何参考符号构造成对权利要求的限制。
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Claims (10)
1.一种SARS-CoV-2病毒的单链抗体，所述单链抗体与抗原S1-RBD的SEQ NO：1序列特异性结合。
2.根据权利要求1所述的单链抗体，包括：重链的可变区的序列为SEQ NO：2所示的氨基酸序列；
轻链的可变区的序列为SEQ NO：3所示的氨基酸序列。
3.根据权利要求2所述的单链抗体，重链的可变区的序列与SEQ NO：2所示的氨基酸序列具有95％相同。
4.根据权利要求2所述的单链抗体，轻链的可变区的序列与SEQ NO：3所示的氨基酸序列95％相同。
5.根据权利要求2所述的单链抗体，其中，连接肽的序列为SEQ NO：4所示的序列。
6.根据权利要求5所述的单链抗体，其中，连接肽的序列与SEQ NO：4所示的序列95％相同。
7.一种制备SARS-CoV-2病毒的单链抗体的方法，包括，
选取SARS-CoV-2抗原S-RBD，从全人源抗体文库中筛选得到单链抗体，所述单链抗体。
8.一种试剂盒，所述试剂盒包括权利要求1-7中任一项所述的单链抗体。
9.一种在药学上可接受的载体，其中包含权利要求1-7中任一项权利要求所述的单链抗体。
10.一种包含两种或多种权利要求1-7中任一项所述的单链抗体的组合物，所述组合物与抗原S1-RBD的SEQ NO：1序列相结合。
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| WO2022154267A3 (ko) * | 2021-01-13 | 2022-09-09 | 재단법인대구경북과학기술원 | ACE2에 결합하는 SARS-CoV2에 대한 항 바이러스 항체 및 이의 용도 |
| CN114395033A (zh) * | 2021-12-17 | 2022-04-26 | 江苏中慧元通生物科技有限公司 | 一种SARS-CoV-2病毒的单链抗体及筛选方法和用途 |
| CN114805559A (zh) * | 2022-04-02 | 2022-07-29 | 浙江大学 | 全人源抗新冠病毒受体结合域单链抗体No4及其应用 |
| CN114805559B (zh) * | 2022-04-02 | 2023-06-02 | 浙江大学 | 全人源抗新冠病毒受体结合域单链抗体No4及其应用 |
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