CN116751285A - 一种特异性识别重组猴痘病毒a35r蛋白的纳米抗体 - Google Patents
一种特异性识别重组猴痘病毒a35r蛋白的纳米抗体 Download PDFInfo
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Abstract
本发明属于分子生物学技术领域,具体公开一种特异性识别重组猴痘病毒A35R蛋白的纳米抗体。本发明提供的纳米抗体包含至少三组互补决定区;所述互补决定区包括SEQ ID NO:1所示的CDR1,SEQ ID NO:2所示的CDR2和SEQ ID NO:3所示的CDR3。本发明的纳米抗体具有独特的CDR1、CDR2和CDR3区序列,使所述纳米抗体对重组猴痘病毒A35R蛋白具有特异的识别和结合能力,而与其他非特异性交叉反应性蛋白没有反应;本发明提供的纳米抗体还具有高耐性、高稳定性和高亲和性的特点,对猴痘病毒具有高度特异的识别和结合能力,具有制备猴痘病毒的预防、治疗或检测试剂的应用前景。
Description
技术领域
本发明属于分子生物学技术领域,特别涉及一种特异性识别重组猴痘病毒A35R蛋白的纳米抗体。
背景技术
近期,猴痘成为全球关注的焦点,猴痘是由猴痘病毒引起的急性出疹性人畜共患病,以往主要发生在非洲地区。此次猴痘病例不同寻常地在多个非流行国家或地区暴发,而且有人际传播倾向,引起高度重视。目前对于猴痘病毒感染尚无特效疗法,主要是对症支持和并发症的治疗,早期诊断对于感染病人的及早发现和猴痘疫情的防控都至关重要。
猴痘病毒(MPXV)是一种包膜双链DNA病毒,病毒颗粒有两种感染形式:胞外包膜病毒(Enveloped Virion,EV)和胞内成熟毒粒(Mature Virion,MV),MV形式最为丰富,在环境中也最稳定,是病毒在宿主间传播的主要形式。目前已有20多种结构蛋白被鉴定,其中A29L是猴痘病毒MV的膜蛋白,与细胞表面糖胺聚糖结合,介导病毒和细胞的融合;M1R也是MV的膜蛋白,在已知痘病毒中高度保守;EV表面的包膜糖蛋白A35R能够诱导产生中和抗体,阻断病毒在细胞间的传播,这些蛋白被认为是猴痘病毒研究的重要靶点。
目前,重组猴痘病毒蛋白A35R的检测技术主要有酶联免疫吸附试验(EL ISA)、血清中和试验、SPA协同凝集试验等,检测中所使用的抗体大多是单克隆抗体和多克隆抗体,但单克隆抗体的研发和生产过程及其繁琐和复杂;多克隆抗体来源有限,这使得检测成本较高,不适合于基层医院进行大规模初筛和床旁快检。因此,提供一种的特异性好、耐性好且稳定性强的抗体对实现猴痘病毒早期检测和诊断具有重要意义。
发明内容
鉴于此,本发明提供一种特异性识别重组猴痘病毒A35R蛋白的纳米抗体,该纳米抗体的特异性好、耐性好且稳定性强,为猴痘病毒的检测和治疗药物的开发提供了基础。
为解决上述技术问题,本发明提供的技术方案是:
本发明在第一个方面提供一种特异性识别重组猴痘病毒A35R蛋白的纳米抗体,其特征在于,所述纳米抗体包含至少三组互补决定区;
所述互补决定区包括SEQ ID NO:1所示的CDR1,SEQ ID NO:2所示的CDR2和SEQ IDNO:3所示的CDR3。
相对于现有技术,本发明提供的纳米抗体,具有独特的CDR1、CDR2和CDR3区序列,使所述纳米抗体对重组猴痘病毒A35R蛋白具有特异的识别和结合能力,而与其他非特异性交叉反应性蛋白没有反应;本发明提供的纳米抗体还具有高耐性、高稳定性和高亲和性的特点。
所述SEQ ID NO:1所示的CDR1的氨基酸序列如下所示:
GRAFRDYA;
所述SEQ ID NO:2所示的CDR2的氨基酸序列如下所示:
VSKTGYAI;
所述SEQ ID NO:3所示的CDR3的氨基酸序列如下所示:AADRTHRLGYSLRVDPLGYDD。
优选的,所述纳米抗体包含与SEQ ID NO:4中任一序列具有至少90%的序列相同性的氨基酸序列。
优选的,所述纳米抗体包含SEQ ID NO:4所示的氨基酸序列。
所述SEQ ID NO:4的氨基酸序列如下所示:AVQLVESGGGLAQTGGSLRLSCAASGRAFRDYAMAWFRQAPGKEREFVAAV SKTGYAIRYEDSVKGRFTISRDNAKNTVSLQMASLKVEDTAVYYCAADRTHRLGYSLRVDPLGYDDWGQGTQVTVSS。
本发明在第二个方面提供编码上述纳米抗体的核酸分子。
优选的,所述核酸分子包含SEQ ID NO:5所示的核苷酸序列。
所述SEQ ID NO.5的核苷酸序列如下所示:
本发明第三个方面提供包含上述核酸分子及其表达调控原件的表达载体。
本发明第四个方面提供包含上述核酸分子并进行表达的宿主细胞。
本发明第五个方面提供一种药物组合物,包括上述纳米抗体或其抗原结合片段和药学上可接受的载体和/或赋形剂。
本发明第六个方面提供上述纳米抗体在制备预防、治疗或诊断猴痘病毒感染的试剂盒或药物中的用途。
本发明第七个方面提供一种用于检测猴痘病毒的试剂盒,包含上述纳米抗体。
本发明提供的纳米抗体,对猴痘病毒具有高度特异的识别和结合能力,具有制备猴痘病毒的预防、治疗或检测试剂的应用前景。
附图说明
图1为本发明实施例提供的VHH-A35R纳米抗体鉴定结果示意图;
图2为本发明实施例提供的VHH-A35R纳米抗体对猴痘病毒A35蛋白的ELISA结果图;
图3为本发明实施例提供的VHH-A35R纳米抗体的特异性试验结果图;
图4为本发明实施例提供的VHH-A35R纳米抗体在KD值为54±6nM时的亲和力测定图;
图5为本发明实施例提供的VHH-A35R纳米抗体在不同温度下处理2h并于4℃下放置2天后的活力测定图;
图6为本发明实施例提供的VHH-A35R纳米抗体在不同pH下处理2h并于4℃放置2天的活力测定图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白,以下结合实施例和附图,对本发明进行进一步详细说明。应当理解,此处所描述的具体实施例仅仅用以解释本发明,并不用于限定本发明。
实施例1
VHH-A35R纳米抗体的淘选与鉴定
1.采用固相亲和淘选的方法从天然噬菌体文库中获得抗针对MPXV A35R的纳米抗体。向每个酶标孔中加入100μL用PBS稀释的MPXV A35R蛋白,将抗原包被免疫管(50μg/管,包被液为CBS,pH9.6,2mL/管)于4℃缓慢旋转过夜;将过夜包被的免疫管中的液体弃掉,加入2mL PBS缓冲液室温清洗免疫管3次,每次旋转5min;加入2mL封闭液(3%牛奶)溶液,室温旋转封闭2h;将免疫管中液体丢弃,并加入2mL PBS缓冲液室温清洗免疫管3次,每次5min;弃掉免疫管中的清洗液,加入2mL PBS缓冲液,加入制备的噬菌体文库100μL作为筛选输入噬菌体文库,室温旋转孵育1h;将免疫管内液体弃掉,加入2mL PBST(1×PBS加0.1%Tween-20)缓冲液室温清洗免疫管20次,每次5min;将免疫管内液体弃掉,加入1mL浓度为0.25mg/mL的Trypsin溶液,室温旋转洗脱30min;加入10mL 10% AEBSF终止洗脱,将免疫管中的溶液转移至新的1.5mL离心管中,取10μL用于滴度测定,500μL洗脱物扩增后用于下一轮淘选,剩余4℃保存;
2.经三轮淘选后,采用单克隆ELISA检测对随机挑取的单克隆进行检测,得到可表达展示抗体可变区噬菌体颗粒的阳性菌上清,再用间接phage-ELISA测定展示抗体可变区噬菌体颗粒的阳性菌上清的结合活性和特异性,实验设定对照,具体加样步骤见表1:
间接phage-ELISA加样表
将ELISA阳性克隆送生物技术服务公司进行序列测定,得到插入片段的D NA序列,其编码针对MPXV A35R的纳米抗体。
实施例2
VHH-A35R纳米抗体的规模制备
1.编码VHH-A35R纳米抗体的DNA片段的获取:采用诺唯赞公司 MaxSuper-Fidelity DNA Polymeras,经PCR获得重链抗体的可变区编码基因(采用的引物见表2),琼脂糖凝胶电泳回收VHH-A35R纳米抗体基因;
表2扩增重链抗体可变区引物
注:下划线标记的序列为限制性酶切位点,斜体部分为6×His标签。
2.将得到的VHH-A35R纳米抗体基因片段克隆至表达载体pNFCG1-Fc Ig G1-FC,经PCR和酶切鉴定,构建完成VHH-A35R纳米抗体的大肠杆菌表达质粒;
3.将表达质粒转化至感受态大肠杆菌DH5α中,然后提取质粒DNA;将无菌质粒DNA转染HEK293悬浮细胞;取一支离心管加入1mL KPM(无血清细胞转染缓冲溶液)和20μg无菌质粒DNA,轻轻吹打混匀;取另一支离心管,加入1mL KPM和100mL的TA-293转染试剂,轻轻吹打混匀;将含有转染试剂的离心管中所有液体转移至含质粒的离心管中,轻轻吹打混匀;室温下静置10分钟制备出质粒-载体复合物;从恒温摇床中取出HEK293细胞(2×106个/mL,20mL),边摇边加入制备好的质粒-载体复合物,放回CO2恒温摇床中震荡培养;3小时后可加入20μL青链霉素混合液;转染24小时后加入0.6%的293细胞蛋白表达增强剂(KE-293)(0.6mL/100mL)120μL,瞬时转染营养添加剂(KT-Feed 50×)400μL;转染后37℃、120r/min振荡培养5-6天,1000rpm,10min收集细胞上清测定产物表达量,以及进行亲和层析纯化和SDS-PAGE电泳分析。
实施例3
VHH-A35R纳米抗体的反应性试验
1.通过ELISA试验对VHH-A35R纳米抗体的反应性进行测定,实验方法如下:
向每个酶标孔中加入100μL用CBS稀释的MPXV A35R蛋白,4℃,包被过夜,吸出包被液,PBST(含0.1%Tween-20)洗板5次,每孔加入100μL PBS M(含2%脱脂奶粉的PBS),37℃,封闭1h;PBST(含0.1%Tween-20)洗板5次,加入100μL VHH-A35R纳米抗体(10μg/mL,1μg/mL,10-1μg/mL,10-2μg/mL,10-3μg/mL,10-4μg/mL,10-5μg/mL,10-6μg/mL),4℃,孵育过夜;吸出未结合的VHH-A35R纳米抗体,用PBST(含0.1%Tween-20)洗板3-5次,加入100μL抗6×His标签鼠单克隆抗体,37℃,孵育1h;用PBST(含0.1%Tween-20)洗板3-5次,加入100μL Goatanti-mouse IgG(HRP),37℃,孵育1h;用PBS T(含0.1%Tween-20)洗板3-5次,每孔加入100μL TMB显色液,37℃显色30m in,每孔加入100μL 1M H2SO4终止反应,测定OD450。
结果如图2所示,VHH-A35R纳米抗体的最大稀释浓度可达到10-2μg/mL,具有较好的反应性。
实施例4
VHH-A35R纳米抗体的特异性试验
通过ELISA试验对VHH-A35R纳米抗体的特异性进行测定,实验方法如下:
本实验除包被MPXV A35R蛋白,选取3%BSA作为对照,TIM3作为非相关蛋白进行特异性实验。
向每个酶标孔中加入100μL用CBS稀释的MPXV A35R蛋白和3%BSA蛋白,4℃,包被过夜,吸出包被液,PBST(含0.1%Tween-20)洗板5次,每孔加入100μL PBSM(含2%脱脂奶粉的PBS),37℃,封闭1h;PBST(含0.1%Tween-20)洗板5次,加入100μL VHH-A35R纳米抗体,4℃,孵育过夜;吸出未结合的VHH-A35R纳米抗体,用PBST(含0.1%Tween-20)洗板3-5次,加入100μL抗6×His标签鼠单克隆抗体,37℃,孵育1h;用PBST(含0.1%Tween-20)洗板3-5次,加入100μL Goat anti-mouse IgG(HRP),37℃,孵育1h;用PBST(含0.1%Tween-20)洗板3-5次,每孔加入100μL TMB显色液,37℃显色30min,每孔加入100μL 1M H2SO4终止反应,测定OD450。
结果如图3所示,VHH-A35R纳米抗体专一有效识别MPXV A35R蛋白,而与其他蛋白不发生反应。
实施例5
VHH-A35R纳米抗体的亲和力试验测定
通过亲和力试验对VHH-A35R纳米抗体与MPXV A35R蛋白的亲和力进行测定,实验方法如下:
1.准备VHH-A35R纳米抗体(1ng/μL),MPXV A35R蛋白(10ng/μL,5ng/μL,2.5ng/μL,1.25ng/μL,0.625ng/μL),稀释液均为PBST(含0.02%Tween-20,0.01%BSA)。实验所需ProA探针需预湿10min以上;
2.实验设置Baseline,60s;Loading,300s;Baseline2,120s;Association,300s;Dissciation,300s;Regeneration,30s;
3.实验结果经Octet Analysis Studio 12.2分析获得。
实施例6
温度对VHH-A35R纳米抗体稳定性影响
准备VHH-A35R纳米抗体(0.01μg/mL),-80℃、-20℃、4℃、37℃、50℃、80℃处理两个小时后,ELISA试验对VHH-A35R纳米抗体的稳定性进行测定:向每个酶标孔中加入100μL用CBS稀释的MPXV A35R蛋白,3%BSA蛋白,4℃,包被过夜,吸出包被液,PBST(含0.1%Tween-20)洗板5次,每孔加入100μLPBSM(含2%脱脂奶粉的PBS),37℃,封闭1h;PBST(含0.1%Tween-20)洗板5次,加入处理过的100μL VHH-A35R纳米抗体,4℃,孵育过夜;吸出未结合的VHH-A35R纳米抗体,用PBST(含0.1%Tween-20)洗板3-5次,加入100μL抗6×His标签鼠单克隆抗体,37℃,孵育1h;用PBST(含0.1%Tween-20)洗板3-5次,加入100μL Goatanti-mouse IgG(HRP),37℃,孵育1h;用PBST(含0.1%Tween-20)洗板3-5次,每孔加入100μL TMB显色液,37℃显色30min,每孔加入100μL 1M H2SO4终止反应,测定OD450。
实验结果如图5所示,本发明提供的VHH-A35R纳米抗体在-80℃-50℃之间稳定性好。
实施例7
pH对VHH-A35R纳米抗体稳定性影响
不同pH处理VHH-A35R纳米抗体2小时,通过ELISA试验对VHH-A35R纳米抗体的稳定性进行测定:
准备VHH-A35R纳米抗体(0.01μg/mL),将PBS的pH分别调至pH=2、pH=4、pH=6、pH=10、pH=12处理两个小时后,置于4℃放置2天;ELISA试验对VHH-A35R纳米抗体的稳定性进行测定:向每个酶标孔中加入100μL用CBS稀释的MPXV A35R蛋白,3%BSA蛋白,4℃,包被过夜,吸出包被液,PBST(含0.1%Tween-20)洗板5次,每孔加入100μL PBSM(含2%脱脂奶粉的PBS),37℃,封闭1h;PBST(含0.1%Tween-20)洗板5次,加入处理过的100μL VHH-A35R纳米抗体,4℃,孵育过夜;吸出未结合的VHH-A35R纳米抗体,用PBST(含0.1%Tween-20)洗板3-5次,加入100μL抗6×His标签鼠单克隆抗体,37℃,孵育1h;用PBST(含0.1%Tween-20)洗板3-5次,加入100μL Goat anti-mouse IgG(HRP),37℃,孵育1h;用PBST(含0.1%Tween-20)洗板3-5次,每孔加入100μL TMB显色液,37℃显色30min,每孔加入100μL 1M H2SO4终止反应,测定OD450。
实验结果如图6所示,在pH=4、pH=6、pH=10、pH=12对VHH-A35R纳米抗体活性影响较小,这也表明本发明提供的VHH-A35R具有优异的耐酸碱性。
以上所述仅为本发明的较佳实施例而已,并不用以限制本发明,凡在本发明的精神和原则之内所作的任何修改、等同替换或改进等,均应包含在本发明的保护范围之内。
Claims (9)
1.一种特异性识别重组猴痘病毒A35R蛋白的纳米抗体,其特征在于,所述纳米抗体包含至少三组互补决定区;
所述互补决定区包括SEQ ID NO:1所示的CDR1,SEQ ID NO:2所示的CDR2和SEQ ID NO:3所示的CDR3。
2.如权利要求1所述的特异性识别重组猴痘病毒A35R蛋白的纳米抗体,其特征在于,所述纳米抗体包含与SEQ ID NO:4中任一序列具有至少90%的序列相同性的氨基酸序列。
3.如权利要求1所述的特异性识别重组猴痘病毒A35R蛋白的纳米抗体,其特征在于,所述纳米抗体包含SEQ ID NO:4所示的氨基酸序列。
4.编码权利要求1-3任一项所述的纳米抗体的核酸分子。
5.包含权利要求4所述的核酸分子及其表达调控原件的表达载体。
6.包含权利要求4所述的核酸分子并进行表达的宿主细胞。
7.一种药物组合物,其特征在于,包括权利要求1或2所述的纳米抗体或其抗原结合片段和药学上可接受的载体和/或赋形剂。
8.一种权利要求1或2所述的特异性识别重组猴痘病毒A35R蛋白的纳米抗体在制备预防、治疗或诊断猴痘病毒感染的试剂盒或药物中的用途。
9.一种用于检测猴痘病毒的试剂盒,其特征在于,包含权利要求1-3任一项所述的纳米抗体。
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CN117659177B (zh) * | 2024-01-31 | 2024-05-14 | 深圳湾实验室 | 抗猴痘病毒的抗体或其抗原结合片段及其应用 |
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