CN114656554B - 基孔肯雅热病毒e2蛋白的纳米抗体及其应用 - Google Patents
基孔肯雅热病毒e2蛋白的纳米抗体及其应用 Download PDFInfo
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Abstract
本发明公开了基孔肯雅热病毒E2蛋白的纳米抗体及其应用,属于生物技术领域。本发明的基孔肯雅热病毒E2蛋白的纳米抗体,所述纳米抗体为3C5纳米抗体或2E8纳米抗体中的一种;所述2E8纳米抗体的核苷酸序列如SEQ ID NO.1所示;所述3C5纳米抗体的核苷酸序列如SEQ ID NO.2所示。本发明的纳米抗体特异性针对CHIKV‑E2抗原,可利用原核表达系统进行大量表达,具备可溶性好、成本低及良好的生物活性,为首个报道的抗CHIKV‑E2蛋白的纳米抗体,可应用于制备快速、便捷的CHIKV检测试剂。
Description
技术领域
本发明属于生物技术领域,具体涉及基孔肯雅热病毒E2蛋白的纳米抗体及其应用。
背景技术
基孔肯雅热(Chikungunya Fever,CHIKF)是一种由基孔肯雅病毒(ChikungunyaVirus,CHIKV)感染所引起的以发热、皮疹、肌肉痛和关节痛等为主要症状的虫媒传染病。CHIKV通常在非人类灵长类动物或哺乳动物宿主和伊蚊之间以曲线式循环传播。在城市流行期间,CHIKV可以通过感染的伊蚊叮咬传播给人类。该病毒于1953年在坦桑尼亚南部地区被首次发现,一直未受到重视,直至2004年,在非洲和亚洲局部地区暴发了疫情才引起研究者们的关注。医学文献里第一次报告的大规模暴发是于2006年发生在留尼汪岛的疫情,据估计,岛上感染CHIKV的人数超过25万。到目前为止,CHIKV仍在非洲、亚洲和美洲等热带地区小规模流行,并由热带返回的旅行者多次传播到温带地区,偶尔还会在美国南部、意大利和法国发生由白纹伊蚊介导的本土病例传播。2010年10月,广东省东莞市暴发了我国首起CHIKF社区聚集性疫情,打破了长期以来以散在输入性病例为特征的流行现状。2019年德宏州瑞丽市发生1起输入和本地病例并存的CHIKF流行,此为云南省首次报告CHIKF本地暴发疫情。考虑到全球流行和非流行地区的蚊子频繁流动,可能已经具备传播病毒的能力,因此需要提高我国对该种疾病的认知水平和监测能力。
CHIKV是一种含有包膜的单股正链RNA,在系统分类上属披膜病毒科(Alphavirusgenus)甲病毒属(Togaviridae family),其长度约为11.8kb,编码两种多聚蛋白:一种为非结构多蛋白,即nsP1、nsP2、nsP3和nsP4,参与基因组复制、封帽和多聚蛋白的加工;另一种为结构蛋白,包括衣壳蛋白(Capsid)和包膜糖蛋白(Envelope)E1、E2和E3。其中E2是受体识别的关键蛋白,E1是pH依赖的融合蛋白,它们分别是病毒附着和进入宿主细胞的主要决定因素,属于信号识别颗粒和信号肽酶,分别识别E3中的信号序列,以内质网膜(ER)为靶点并定位于ER上的I型膜蛋白。在内质网中,E2和E1形成异源二聚体。E3、E2和E1在高尔基体内切割成N-糖基化E2-E1复合物。E2由位于N-末端结构域A,侧端结构域B和C-末端结构域C三个免疫球蛋白(Ig)样结构域组成。结构域A和结构域B被认为是靶细胞上Mxra8受体的结合位点,鉴于此种结构优势,CHIKV-E2蛋白表现出良好的免疫原性,同时具备多个可以鉴定的抗原表位,其诱导产生的抗体具有更高的诊断潜力。
重链抗体,是由骆驼、羊驼及其近亲物种天然重链抗体的可变区组成的单域抗体,该抗体通常只包含重链可变区(VHH),因其分子量小,约为~15kDa,也被称为纳米抗体。与传统抗体比较,具有结构稳定、亲和力高、不易聚集、可识别潜在抗原表位、易于人源化等优点,已被用作结构生物学、细胞生物学和发育生物学强有力的研究工具。尽管纳米抗体在生物医学研究中开辟了重要的可能性,但迄今为止,大部分的纳米抗体都是通过免疫驼类动物而获得,其研发过程漫长且昂贵,对大多数实验室来说是一个巨大的门槛。天然纳米抗体库无需免疫动物,节约时间及成本,在筛选纳米抗体时表现出如下优势,(1)可以分离出多样性抗体单克隆;(2)可以分离出针对那些不引起机体免疫反应的弱抗原、自身抗原和具有毒性抗原的抗体;(3)可以用于多种抗原筛选而获得多种抗体产生;(4)在库容量足够大的时候可以获得高亲和力抗体。
发明内容
本发明的目的在于克服现有技术的不足,提供基孔肯雅热病毒E2蛋白的纳米抗体,该纳米抗体具有分子量小、免疫原性小及稳定等特点,可以克服传统单克隆抗体无法在原核体系表达,研发周期长,生产成本高等劣势,该株纳米抗体在靶向CHIKV诊断中具有良好的发展前景。
为实现上述目的,本发明采取的技术方案为:基孔肯雅热病毒E2蛋白的纳米抗体,所述纳米抗体为3C5纳米抗体或2E8纳米抗体中的一种;所述2E8纳米抗体的核苷酸序列如SEQ ID NO.1所示;所述3C5纳米抗体的核苷酸序列如SEQ ID NO.2所示。本发明采用天然纳米抗体文库,以CHIKV-E2蛋白作为抗原筛选纳米抗体,最终淘选出特异性纳米抗体,分别将其命名为3C5纳米抗体和2E8纳米抗体。
作为本发明所述的基孔肯雅热病毒E2蛋白的纳米抗体的优选实施方式,所述2E8纳米抗体的氨基酸序列如SEQ ID NO.3所示;所述3C5纳米抗体的氨基酸序列如SEQ IDNO.4所示。
作为本发明所述的基孔肯雅热病毒E2蛋白的纳米抗体的优选实施方式,所述2E8纳米抗体具有3个互补决定区CDR1、CDR2、CDR3,其中CDR1序列由SEQ ID NO.5所示氨基酸序列组成,CDR2序列由SEQ ID NO.6所示氨基酸序列组成,CDR3序列由SEQ ID NO.7所示氨基酸序列组成。本申请发明人研究发现,将该株纳米抗体基因序列插入到pET-SUMO表达载体的BamHI和XhoI酶切位点之间,使其与SUMO标签融合表达,可使纳米抗体基因以分泌的方式表达,SUMO的N端添加了6×His tag,便于纯化,获得可溶性纳米抗体片段,纯度>90%。
作为本发明所述的基孔肯雅热病毒E2蛋白的纳米抗体的优选实施方式,所述3C5纳米抗体具有3个互补决定区CDR1、CDR2、CDR3,其中CDR1序列由SEQ ID NO.8所示氨基酸序列组成,CDR2序列由SEQ ID NO.9所示氨基酸序列组成,CDR3序列由SEQ ID NO.10所示氨基酸序列组成。本申请发明人研究发现,将该株纳米抗体基因序列插入到pET-SUMO表达载体的BamHI和XhoI酶切位点之间,使其与SUMO标签融合表达,可使纳米抗体基因以分泌的方式表达,SUMO的N端添加了6×His tag,便于纯化,获得可溶性纳米抗体片段,纯度>90%。
作为本发明所述的基孔肯雅热病毒E2蛋白的纳米抗体的优选实施方式,所述2E8纳米抗体还包括框架区FR;所述框架区FR包括FR1、FR2、FR3和FR4,其中FR1的氨基酸序列如SEQ ID NO.11所示,FR2的氨基酸序列如SEQ ID NO.12所示,FR3的氨基酸序列如SEQ IDNO.13所示,FR4的氨基酸序列如SEQ ID NO.14所示。
作为本发明所述的基孔肯雅热病毒E2蛋白的纳米抗体的优选实施方式,所述3C5纳米抗体还包括框架区FR;所述框架区FR包括FR1、FR2、FR3和FR4,其中FR1的氨基酸序列如SEQ ID NO.15所示,FR2的氨基酸序列如SEQ ID NO.16所示,FR3的氨基酸序列如SEQ IDNO.17所示,FR4的氨基酸序列如SEQ ID NO.18所示。
作为本发明所述的基孔肯雅热病毒E2蛋白的纳米抗体的优选实施方式,所述互补决定区CDR识别氨基酸序列如SEQ ID NO.19所示的抗原表位。本发明提供的纳米抗体2E8对CHIKV-E2抗原具有特异的识别和结合能力,采用LSPR技术分析了纳米抗体2E8与E2蛋白的结合亲和力和动力学参数,结果显示本纳米抗体结合的亲和力为1.01×10-7M,结合常数为2.59×104 1/Ms,解离常数为2.62×10-3 1/s,显示本纳米抗体可以与抗原结合。
作为本发明所述的基孔肯雅热病毒E2蛋白的纳米抗体的优选实施方式,所述互补决定区CDR1识别氨基酸序列如SEQ ID NO.20所示的抗原表位。本发明提供的纳米抗体3C5对CHIKV-E2抗原具有特异的识别和结合能力,采用LSPR技术分析了纳米抗体3C5与E2蛋白的结合亲和力和动力学参数,结果显示本纳米抗体结合的亲和力为3.68×10-7M,结合常数为2.7×104 1/Ms,解离常数为9.91×10-3 1/s,显示本纳米抗体可以与抗原结合。
本发明还提供一种核苷酸分子,所述核苷酸分子编码所述的纳米抗体。
本发明还提供所述的纳米抗体在制备检测基孔肯雅热病毒的试剂或试剂盒中的应用。
本发明的有益效果:提供基孔肯雅热病毒E2蛋白的纳米抗体,本发明的纳米抗体为3C5纳米抗体或2E8纳米抗体中的一种,本发明的纳米抗体特异性针对CHIKV-E2抗原,可利用原核表达系统进行大量表达,具备可溶性好、成本低及良好的生物活性,为首个报道的抗CHIKV-E2蛋白的纳米抗体,可应用于制备快速、便捷的CHIKV检测试剂。
附图说明
图1为pET-SUMO-2E8表达质粒载体图谱。
图2为pET-SUMO-3C5表达质粒载体图谱。
图3为2E8纳米抗体的纯化SDS-PAGE图。
图4为3C5纳米抗体的纯化SDS-PAGE图。
图5为2E8纳米抗体的ELISA活性鉴定图。
图6为3C5纳米抗体的ELISA活性鉴定图。
图7为2E8纳米抗体线性表位识别结果图。
图8为3C5纳米抗体线性表位识别结果图。
图9为2E8纳米抗体与CHIKV感染细胞结合荧光显微镜镜检图。
图10为3C5纳米抗体与CHIKV感染细胞结合荧光显微镜镜检图。
图11为LSPR检测2E8纳米抗体与CHIKV E2结合动力学活性。
图12为LSPR检测3C5纳米抗体与CHIKV E2结合动力学活性。
图13为2E8纳米抗体与CHIKV E2结合的潜在抗原表位图。
图14为3C5纳米抗体与CHIKV E2结合的潜在抗原表位图。
具体实施方式
为更好地说明本发明的目的、技术方案和优点,下面将结合具体实施例对本发明作进一步说明。
实施例1靶向E2蛋白纳米抗体的筛选及制备
1、纳米抗体天然噬菌体展示文库构建原理
本发明使用的天然纳米抗体文库由深圳康体生命科技有限公司提供,即采集103只成年健康羊驼外周血细胞(PBMC),提取总RNA,逆转录成cDNA,利用特异性引物扩增重链抗体VHH基因片段,将其连接至噬菌体质粒pADL-10b中,构建含有扩增纳米片段的噬菌体质粒库,通过多次电转化至SS320感受态细胞,在辅助噬菌体M13K07扩增、救援下,将VHH区片段展示在噬菌体表面,获得羊驼天然噬菌体文库,抗体文库容量为2×109,效价为1.28×1013pfu/mL。根据构建的天然文库来筛选能特异性结合CHIKV-E2抗原的纳米序列。
2、利用噬菌体展示技术淘选和富集CHIKV-E2纳米抗体
用CBS包被液(pH7.6)包被50μg CHIKV-E2(Ser326-Gln666)抗原,抗原菌株来源为SL-CK1,NCBI登陆号为ADG95913.1(北京义翘神州科技股份有限公司提供),4℃包被过夜。次日用3%BSA封闭液于室温封闭2h,弃封闭液。每孔加入100μl噬菌体抗体库,室温孵育1h,弃去未结合的噬菌体,用含0.1%Tween-20的PBS清洗免疫管20次。每孔加入1ml 0.25mg/mLTrypsin溶液,室温洗脱30min。加入10μl 10%AEBSF终止洗脱液,收集免疫管中的溶液,即为第一轮筛选噬菌体洗脱液。将洗脱的噬菌体立即加入5mlOD600为0.5-0.6的SS320菌液中,37℃培养30min,涂布于2YT-GA(终浓度100μg/ml AMP,2%葡萄糖)平板,37℃培养过夜。次日用涂布棒刮取过夜培养的全部菌落,取刮下的300μl细菌加入转接中至100ml 2×YT液体培养基中(含10μg/ml Tet和100μg/ml AMP),37℃,250rpm培养至菌液生长至OD600为0.5-0.6,加入辅助噬菌体M13K07(滴度为1013/ml)20μl,220rpm培养30min,分别加入终浓度为50μg/ml KAN和0.2mMIPTG,30℃,250rpm培养过夜。次日离心后,取上清加入20%PEG/2.5M NaCl沉淀噬菌体。收集得到第一轮噬菌体子文库,用于下一轮的筛选。共重复淘筛3次,使表面表达的纳米抗体噬菌体单克隆得到富集。三轮筛选结果如表1所示。
表1 CHIKV-E2三轮淘选噬菌体富集比
3、CHIKV-E2 Phage-ELISA检测阳性克隆
ELISA板包被1ng/μl CHIKV-E2抗原,同时平行包被BSA作对照,4℃包被过夜。挑取396个单个克隆至无菌96孔细胞板中,每孔加入200μl 2×YT液体培养基(含有100μg/mlAMP和10μg/ml Tet),37℃培养过夜,于次日取过夜菌液2μl转接至新的200μl 2×YT液体培养基(含有100μg/ml AMP和10μg/ml Tet)96孔细胞板中,37℃静置培养3-5h,加入辅助噬菌体M13K07,使噬菌体数:细菌个数达20:1,37℃静置培养30min,分别加入终浓度为50μg/mlKAN和0.2mM IPTG,30℃,250rpm培养过夜。于次日培养液离心后收集上清用来检测。取包被的ELISA板,每孔加入3%BSA室温封闭1h,室温清洗酶标板3次后,实验组和对照组每孔分别加入等量单克隆上清噬菌体,室温孵育2h。每孔加入200μl PBST共洗3次,加入M13Bacteriophage Antibody(HRP),室温1h。PBST洗5次,每孔加入TMB显色液,避光显色2-3min,加入1M HCl终止液,用酶标仪读取OD450值,当实验孔与BSA对照比值大于10,且实验孔吸光值≥1时,判定为阳性克隆。
4、阳性克隆基因序列分析
通过phage-ELISA确定针对CHIKV-E2的纳米抗体,经DNA测序分析,确定重链抗体的框架区(FR)和互补决定区(CDR),分别将其命名为3C5纳米抗体和2E8纳米抗体。所述2E8纳米抗体的核苷酸序列如SEQ ID NO.1所示;所述3C5纳米抗体的核苷酸序列如SEQ IDNO.2所示。所述2E8纳米抗体的氨基酸序列如SEQ ID NO.3所示;所述3C5纳米抗体的氨基酸序列如SEQ ID NO.4所示。所述2E8纳米抗体具有3个互补决定区CDR1、CDR2、CDR3,其中CDR1序列由SEQ ID NO.5所述氨基酸序列组成,CDR2序列由SEQ ID NO.6所述氨基酸序列组成,CDR3序列由SEQ ID NO.7所述氨基酸序列组成。所述3C5纳米抗体具有3个互补决定区CDR1、CDR2、CDR3,其中CDR1序列由SEQ ID NO.8所述氨基酸序列组成,CDR2序列由SEQ ID NO.9所述氨基酸序列组成,CDR3序列由SEQ ID NO.10所述氨基酸序列组成。所述2E8纳米抗体还包括框架区FR;所述框架区FR包括FR1、FR2、FR3和FR4,其中FR1的氨基酸序列如SEQ ID NO.11所示,FR2的氨基酸序列如SEQ ID NO.12所示,FR3的氨基酸序列如SEQ ID NO.13所示,FR4的氨基酸序列如SEQ ID NO.14所示。所述3C5纳米抗体还包括框架区FR;所述框架区FR包括FR1、FR2、FR3和FR4,其中FR1的氨基酸序列如SEQ ID NO.15所示,FR2的氨基酸序列如SEQID NO.16所示,FR3的氨基酸序列如SEQ ID NO.17所示,FR4的氨基酸序列如SEQ ID NO.18所示。
实施例2 CHIKV-E2纳米抗体可溶性原核表达及纯化
1、pE-SUMO-2E8原核表达载体质粒、pET-SUMO-3C5原核表达载体质粒的构建
SUMO标签蛋白是一种小分子泛素相关修饰蛋白,是存在于真核生物中高度保守的参与蛋白质小泛素化相关修饰的一类蛋白。在大肠杆菌表达系统中可用作融合标签,通过融合标签可以促进靶蛋白的表达和提高靶蛋白的可溶,可以生产获得具有天然结构的靶蛋白。为了实现2E8纳米抗体和3C5纳米抗体可溶性原核表达,分别将2E8纳米抗体和3C5纳米抗体基因序列插入到pET-SUMO表达载体的BamHI和XhoI酶切位点之间,使其与SUMO标签融合表达,SUMO的N端添加了6×His tag,可用于融合蛋白的纯化。pE-SUMO-2E8的载体图谱如图1所示,pE-SUMO-3C5的载体图谱如图2所示,委托上海捷瑞生物工程有限公司合成该质粒。
2、2E8纳米抗体、3C5纳米抗体可溶性原核表达
取上述构建成功的重组表达质粒穿刺菌均匀涂布于含LB固体平板(KAN终浓度为50μg/mL),37℃培养12-14h。挑单菌落扩大培养于含10mL的LB液体培养基的试管中,220rpm、37℃条件下过夜摇菌获取种子液;取种子菌液转接扩大培养,37℃220rpm振荡培养。待菌体OD600生长至0.6-0.8时,向菌液中加入IPTG(终浓度为0.5mmol/L)诱导,18℃培养12-14h。10,000rpm离心20min弃上清,用预冷PBS重悬菌体,采用超声波细胞粉碎机进行裂解,13,000rpm 4℃离心10min,分别收集上清和菌体沉淀。
3、纳米抗体3C5 CO2+亲和层析柱纯化
融合蛋白亲和标签为His,利用TALON Metal affinity Resin(含CO2+)纯化纳米抗体3C5,取CO2+柱沉淀树脂,灭菌ddH2O清洗2-3次,含10mM咪唑缓冲液平衡CO2+柱,将制备的超声裂解物加入CO2+柱中,震荡混匀,4℃孵育1h,2倍柱体积的洗脱液液(含20mM咪唑)洗去杂蛋白,最后用等体积的洗脱液(含250mM咪唑)洗脱目的蛋白,并收集洗脱液;采用PALL浓缩管,进行样品浓缩。12%SDS-PAGE检测蛋白纯化情况。
实验结果如图3和图4所示。
实施例3间接ELISA检测纳米抗体的结合活性
ELISA板包被终浓度分别为5μg/ml CHIKV-E2和10μg/ml灭活CHIKV病毒颗粒,以pET-SUMO破碎液作对照,4℃孵育过夜。于次日弃包被液,PBST(含0.05%Tween-20)洗板3次,每次5min;3%BSA-PBST,37℃封闭1h;弃封闭液,PBST洗板3次;2倍倍比稀释抗体(抗体稀释起始浓度为100μg/ml),37℃孵育1h;弃掉抗体稀释液,PBST洗板3次,每次5min;每孔加入1:2000稀释的SMT3-HRP单克隆抗体,37℃孵育1h;弃掉孔内液体,PBST洗板5次,每次5min;TMB避光显色10min;2M H2SO4终止反应。结果判定:样品OD450值大于阴性对照2倍以上判断为阳性,稀释度作三个重复孔,取平均值。
实验结果如图5和图6所示。由图5可知,所构建纳米抗体2E8能与CHIKV-E2抗原特异性结合,与对照相比有显著差异;由图6可知,所构建纳米抗体3C5能与CHIKV-E2抗原特异性结合,与对照相比有显著差异。
实施例4 Western Blot鉴定纳米抗体识别抗原线性表位
以CHIKV-E2抗原分别对纯化后的2E8纳米抗体、3C5纳米抗体进行Western Blot验证。5ug CHIKV-E2抗原和灭活CHIKV病毒颗粒上样,进行SDS-PAGE并转膜。5%BSA封闭液室温封闭1h,以纯化后纳米抗体作为一抗,用封闭液稀释至1:1000,4℃孵育过夜,以1:1000稀释的HRP标记的SMT3-HRP,室温孵育1h;洗膜,ECL显影,曝光。
实验结果如图7和图8所示。由图7可知,所构建纳米抗体2E8能识别CHIKV-E2抗原,条带位置正确,发光强烈,说明纯化获得的样品浓度和纯度都比较高。由图8可知,所构建纳米抗体3C5能识别CHIKV-E2抗原,条带位置正确,发光强烈,说明纯化获得的样品浓度和纯度都比较高。
实施例5间接免疫荧光试验鉴定纳米抗体的功能活性
CHIKV感染Vero细胞(MOI=1)72h后,4%多聚甲醛中固定细胞20min;1%TritonX-100透化细胞,用含1%BSA的PBS在室温下静置1h;每孔加入纳米抗体(50μg/ml)和CHIKV病毒粒子免疫后的兔血清(1:1000),37℃下孵育30min,使用Alexa Fluor 488抗6×His标记抗体或Cy3标记的山羊抗兔IgG(H+L)在37℃下孵育30min。PBS洗涤后,晾干,荧光显微镜下观察细胞,用商品化CHIKV-E1鼠抗(R&D)作阳性对照。
实验结果如图9和图10所示。由图9可知,纳米抗体2E8与CHIKV感染靶细胞内的免疫荧光为阳性,结合率与预期相符,光点密集清晰可见。由图10可知,纳米抗体3C5与CHIKV感染靶细胞内的免疫荧光为阳性,结合率与预期相符,光点密集清晰可见。
实施例6 LSPR检测纳米抗体结合E2蛋白的动力学特性
按照OpenSPRTM标准程序,将COOH芯片安装在OpenSPRTM仪器上。以最大流速(150μL/min)运行,检测缓冲液为PBS(PH7.4),达到信号基线后,200μL异丙醇上样,运行10s排出气泡,达到基线后,PBS冲洗样本环,并用空气排空。在信号达到基线后,调整缓冲液流速到20μL/min。加载200μL EDC/NHS(1:1)溶液以激活COOH传感器芯片,稀释的200μL的配体E2运行4min,PBS冲洗样本环,并用空气排空。200μL Blocking溶液上样,PBS冲洗样本环,并用空气排空。观察基线5分钟,以确保稳定性。纳米抗体用缓冲液稀释,分别将2E8纳米抗体、3C5纳米抗体稀释成一系列不同浓度的溶液,并以20μL/min上样,蛋白与配体结合时间均为240s,自然解离360s。使用Trace Drawer软件计算和分析结合反应的动力学参数,One ToOne分析模型。
实验结果如图11和图12所示。由图11可知,2E8纳米抗体结合的亲和力为1.01×10-7M,结合常数为2.59×104 1/Ms,解离常数为2.62×10-3 1/s,显示本纳米抗体可以与抗原结合。由图12可知,3C5纳米抗体结合的亲和力为3.68×10-7M,结合常数为2.7×104 1/Ms,解离常数为9.91×10-3 1/s,显示本纳米抗体可以与抗原结合。
实施例7多肽ELISA鉴定纳米抗体与CHIKV-E2结合的潜在抗原表位
人工合成覆盖CHIKV-E2(Ser326-Gln666)蛋白,以5个氨基酸残基重叠且长度为10-mer的短肽,纯度达90%以上。将以上68条多肽分别稀释到10μg/ml作为捕获抗原包被ELISA板,以SARS-CoV-2多肽作为阴性对照,4℃孵育过夜。于次日用含5%BSA的PBST封闭液,37℃温育1h。通过分别与1μg/ml 2E8纳米抗体、3C5纳米抗体温育来表征包被肽的结合。37℃温育2h后,PBST洗板3次,每孔加入1:2000稀释的HRP-SMT2单克隆抗体,37℃孵育1小时,洗板5次;TMB室温避光反应15min,1M HCl终止反应,酶标仪读取OD450值,以检测结合的潜在表位。
实验结果如图13和图14所示。由图13和图14可知,2E8纳米抗体、3C5纳米抗体可以识别CHIKV-E2抗原结合表位。
最后应当说明的是,以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制,尽管参照较佳实施例对本发明作了详细说明,本领域的普通技术人员应当理解,可以对本发明的技术方案进行修改或者等同替换,而不脱离本发明技术方案的实质和范围。
SEQUENCE LISTING
<110> 中山大学
<120> 基孔肯雅热病毒E2蛋白的纳米抗体及其应用
<130> 2022.03.03
<160> 20
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ccagggaagg agcgtgagtg gatggcgtct attgtgcgga gtggtgggcc cacatactat 180
gcagactccg tgaaggaccg attcaccatc tccagagaaa acgccaggaa cacggtgtat 240
ctgcaaatga acagcctgaa acctgaggac acggccgttt attactgtgc agcagacctg 300
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ccagggaagg agcgtgagtg gatggcgtct attgtgcgga gtggtgggcc cacatactat 180
gcagactccg tgaaggaccg attcaccatc tccagagaaa acgccaggaa cacggcgtat 240
ctgcaaatga acagcctgaa acctgaggac acggccgttt attactgtgc agcagacctg 300
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Claims (8)
1. 基孔肯雅热病毒E2蛋白的纳米抗体,其特征在于,所述纳米抗体为3C5纳米抗体或2E8纳米抗体中的一种;所述2E8纳米抗体的编码核苷酸序列如SEQ ID NO.1所示;所述3C5纳米抗体的编码核苷酸序列如SEQ ID NO.2所示。
2. 根据权利要求1所述的基孔肯雅热病毒E2蛋白的纳米抗体,其特征在于,所述2E8纳米抗体的氨基酸序列如SEQ ID NO.3所示;所述3C5纳米抗体的氨基酸序列如SEQ ID NO.4所示。
3. 根据权利要求1所述的基孔肯雅热病毒E2蛋白的纳米抗体,其特征在于,所述2E8纳米抗体具有3个互补决定区CDR1、CDR2、CDR3,其中CDR1序列由SEQ ID NO.5所示氨基酸序列组成,CDR2序列由SEQ ID NO.6所示氨基酸序列组成,CDR3序列由SEQ ID NO.7所示氨基酸序列组成。
4. 根据权利要求1所述的基孔肯雅热病毒E2蛋白的纳米抗体,其特征在于,所述3C5纳米抗体具有3个互补决定区CDR1、CDR2、CDR3,其中CDR1序列由SEQ ID NO.8所示氨基酸序列组成,CDR2序列由SEQ ID NO.9所示氨基酸序列组成,CDR3序列由SEQ ID NO.10所示氨基酸序列组成。
5. 根据权利要求1所述的基孔肯雅热病毒E2蛋白的纳米抗体,其特征在于,所述2E8纳米抗体还包括框架区FR;所述框架区FR包括FR1、FR2、FR3和FR4,其中FR1的氨基酸序列如SEQ ID NO.11所示,FR2的氨基酸序列如SEQ ID NO.12所示,FR3的氨基酸序列如SEQ IDNO.13所示,FR4的氨基酸序列如SEQ ID NO.14所示。
6. 根据权利要求1所述的基孔肯雅热病毒E2蛋白的纳米抗体,其特征在于,所述3C5纳米抗体还包括框架区FR;所述框架区FR包括FR1、FR2、FR3和FR4,其中FR1的氨基酸序列如SEQ ID NO.15所示,FR2的氨基酸序列如SEQ ID NO.16所示,FR3的氨基酸序列如SEQ IDNO.17所示,FR4的氨基酸序列如SEQ ID NO.18所示。
7.一种核苷酸分子,其特征在于,所述核苷酸分子编码权利要求1所述的纳米抗体。
8.根据权利要求1~6任一项所述的纳米抗体在制备检测基孔肯雅热病毒的试剂或试剂盒中的应用。
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