CN114702577A - 一种广谱识别西尼罗病毒以及寨卡病毒e蛋白diii的抗体 - Google Patents
一种广谱识别西尼罗病毒以及寨卡病毒e蛋白diii的抗体 Download PDFInfo
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- CN114702577A CN114702577A CN202210627096.9A CN202210627096A CN114702577A CN 114702577 A CN114702577 A CN 114702577A CN 202210627096 A CN202210627096 A CN 202210627096A CN 114702577 A CN114702577 A CN 114702577A
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Abstract
发明属于生物医药领域，涉及抗病毒抗体，具体涉及一种广谱识别西尼罗病毒以及寨卡病毒E蛋白DIII的抗体。本发明所述抗体的重链CDR、轻链CDR依次如SEQ ID NO:1‑3、SEQ ID NO:4‑6所示。
Description
技术领域
本发明属于生物医药领域，涉及抗病毒抗体，具体涉及一种广谱识别西尼罗病毒以及寨卡病毒E蛋白DIII的抗体。
背景技术
西尼罗病毒属于黄病毒科黄病毒属，与乙型脑炎、圣路易脑炎、黄热病、登革热、丙型肝炎等病毒同属。有囊膜，单链线形核糖核酸，RNA为正链，约有10000～11000个碱基对，具有感染性，可引起人和动物发生西尼罗河热和西尼罗病毒脑膜脑炎，库蚊属的蚊子是其主要的传播媒介，鸟类是该病毒的中间宿主，而人和马则是终端宿主。
寨卡病毒属黄病毒科，黄病毒属，单股正链RNA病毒，直径20nm，是一种通过蚊虫进行传播的虫媒病毒，宿主不明确，主要在野生灵长类动物和栖息在树上的蚊子，如非洲伊蚊中循环。寨卡病毒感染者中，约20%会表现轻微症状，典型的症状包括急性起病的低热、斑丘疹、关节疼痛（主要累及手、足小关节）、结膜炎，其他症状包括肌痛、头痛、眼眶痛及无力。另外少见的症状包括腹痛、恶心、呕吐、黏膜溃疡和皮肤瘙痒。症状通常较温和，持续不到一周，需要住院治疗的严重病情并不常见。2013年和2015年分别在法属波利尼西亚和巴西塞卡疫情期间，有报道称寨卡病毒病可能会造成神经和自身免疫系统并发症。
抗原的检测可以通过细胞培养分离技术和噬斑滴定、传统的RT-PCR、实时荧光RT-PCR和免疫组织化学等方法进行。其中RT-PCR和实时荧光RT-PCR可以快速检测病毒抗原，但该方法成本昂贵，容易出现由于样品污染而呈假阳性结果；通过细胞培养技术分离病毒和检测样品中的病毒噬斑也可以检测病毒抗原，但该方法检测周期长，且需要专业的技术人员以及BSL-3生物安全试验室，因而在大范围检测中难以普及；而利用抗原捕获ELISA、间接免疫荧光、免疫组化等方法可以快速、灵敏地检测抗原，适于在大规模的监测系统中应用。抗原捕获ELISA、间接免疫荧光、免疫组化等检测方法的应用依赖于针对抗原特异性的单克隆抗体的研制。
发明内容
本发明的主要目的在于提供一种抗体，所述抗体是以寨卡病毒E蛋白DIII以及西尼罗河病毒E蛋白DIII作为抗原制备而成，也即本发明提供了一种广谱识别西尼罗病毒以及寨卡病毒E蛋白DIII的抗体。
为了实现上述目的，本发明采用了如下技术方案：
本发明第一方面，提供了一种识别西尼罗病毒和/或寨卡病毒的抗体，所述抗体包括轻链CDR1、轻链CDR2、轻链CDR3、重链CDR1、重链CDR2和重链CDR3；
所述重链CDR1的氨基酸序列如SEQ ID NO:1所示；
所述重链CDR2的氨基酸序列如SEQ ID NO:2所示；
所述重链CDR3的氨基酸序列如SEQ ID NO:3所示；
所述轻链CDR1的氨基酸序列如SEQ ID NO:5所示；
所述轻链CDR2的氨基酸序列如SEQ ID NO:6所示；
所述轻链CDR3的氨基酸序列如SEQ ID NO:7所示。
更优选地，所述抗体的重链可变区的氨基酸序列如SEQ ID NO:4所示；所述抗体的轻链可变区的氨基酸序列如SEQ IDNO:8所示。
更具体地，所述寨卡病毒是指寨卡病毒E蛋白DIII结构域（Envelope DomainⅢ，EDⅢ）；所述西尼罗病毒是指西尼罗河病毒E蛋白DIII结构域（Envelope DomainⅢ，EDⅢ）。
优选地，所述抗体可以包含一个或多个合成的氨基酸，所述合成的氨基酸在本领域是已知的，并且包括但不限于氨基环己羧酸、正亮氨酸、α-氨基n-癸酸、高丝氨酸、S-乙酰氨甲基-半胱氨酸、反式-3-和反式-4-羟脯氨酸、4-氨基苯丙氨酸、4-硝基苯丙氨酸、4-氯苯丙氨酸、4-羧基苯丙氨酸、β-苯基丝氨酸β-羟基苯丙氨酸、苯基甘氨酸、α-萘基丙氨酸、环己基丙氨酸、环己基甘氨酸、吲哚啉-2-羧酸、1,2,3,4-四氢异喹啉-3-羧酸、氨基丙二酸、氨基丙二酸单酰胺、N’-苯甲基-N’-甲基-赖氨酸、N’,N’-二苄基-赖氨酸、6-羟赖氨酸、鸟氨酸、α-氨基环戊烷羧酸、α-氨基环己羧酸、α-氨基环庚烷羧酸、α-（2-氨基-2-降莰烷）-羧酸、α,γ-二氨基丁酸、α,β-二氨基丙酸、高苯丙氨酸以及α-叔丁基甘氨酸。
本领域技术人员应理解如本文所用，术语 “抗体”、“肽”、“多肽”、和“蛋白质”可互换使用，并且皆是指具有通过肽键方式共价连接的氨基酸残基的化合物。
如本文所述术语“抗体”包括完整抗体及其任何抗原结合片段（即“抗原结合部分”）或单链。“抗体”指包括通过二硫键互连的至少2条重（H）链和2条轻（L）链的糖蛋白，或其抗原结合部分。每条重链包括重链可变区（本文缩写为VH）和重链恒定区。重链恒定区包括3个结构域——CH1、CH2和CH3。每条轻链包括轻链可变区（本文缩写为VL）和轻链恒定区。轻链恒定区包括一个结构域——CL。VH和VL区可以进一步再分成称为互补性决定区（CDR）的高变区域，散布于称为构架区（FR）的更保守区域之中。每个VH和VL由3个CDRs和4个FRs组成，从氨基末端到羧基末端以下述顺序排列：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。重链和轻链的可变区包括与抗原相互作用的结合结构域。抗体的恒定区可以通过Fc受体（例如，效应细胞）和经典补体系统的第一种组分（C1q）介导免疫球蛋白与宿主组织或因子的结合，包括免疫系统的各种细胞。
本发明第二方面，提供了一种编码前述抗体的多核苷酸。
优选地，编码前述重链可变区的多核苷酸序列如SEQ ID NO:9所示。
优选地，编码前述轻链可变区的多核苷酸序列如SEQ ID NO:10所示。
本发明所述的“多核苷酸”是可以被化学修饰的。具体地，“化学修饰”是指与自然的DNA相比，本发明提供的多核苷酸可被化学修饰（chemical modification）。多核苷酸的化学修饰是通过引入或除去任何化学基因而使其共价结构发生改变的现象或方法。多核苷酸的化学修饰部位可发生在磷酸二酯键、核糖和碱基。多核苷酸的化学修饰可发生在5’端或3’端，可在合成时或合成后进行。
本发明第三方面，提供了一种含有前述多核苷酸的重组载体。
优选地，所述载体包括质粒（表达质粒、克隆载体、小环、微载体、双微小染色体）、慢病毒载体、腺病毒载体或逆转录病毒载体。
优选地，所述载体还包括一种或多种调控元件。
优选地，所述调控元件包含启动子、增强子、翻译起始的核糖体结合位点、终止子、多聚腺苷酸序列、筛选标记基因。
优选地，所述启动子是诱导型启动子、组成型启动子、组织特异性启动子、自杀型启动子或其任意组合。
如本文所用，术语“载体”是指包含完整复制子的非染色体核酸，使得当置于允许的细胞内时，载体可以被复制，例如通过转化过程。
本发明第四方面，提供了一种宿主细胞，所述宿主细胞含有前述重组载体或细胞的基因组中整合了外源性的前述多核苷酸。
在一种实施方式中，宿主细胞包括大肠杆菌，链霉菌、农杆菌、酵母细胞、植物细胞、动物细胞或病毒中的一种或多种。示例性的，可应用于本发明的宿主细胞还包括但不限于：CHO细胞、293细胞、CHO-K1细胞、HEK293细胞、Caco2细胞、U2-OS细胞、NIH 3T3细胞、NSO细胞、SP2细胞、CHO-S细胞、DG44细胞、K-562细胞、U-937细胞、MRC5细胞、IMR90细胞、Jurkat细胞、HepG2细胞、HeLa细胞、HT-1080细胞、HCT-116细胞、Hu-h7细胞、Huvec细胞、Molt 4细胞等细胞系。
在另一实施方式中，所述动物是哺乳动物；更具体地，所述动物包括人。
本发明第五方面，提供了一种生产本发明所述抗体的方法，所述方法包括培养前述宿主细胞、收集抗体的步骤。
可以使用本领域常用的方法从宿主细胞中收集抗体。例如，离心分离培养基和宿主细胞，高压匀浆破碎细胞、离心过滤去除细胞碎片，亲和层析纯化抗体。对于分离纯化所得的产物，可以使用本领域常用的方法进行纯度鉴定。例如，考马斯亮蓝法、凯氏定氮法、双缩脲法、lowry法、紫外吸收法、亲和层析、抗原抗体法、电泳分析（例如十二烷基磺酸钠聚丙烯酰胺凝胶电泳）、沉降分析、扩散分析、恒溶度法、蛋白质谱等。
本发明第六方面，提供了一种药物组合物，其含有本发明的抗体。
优选地，所述药物组合物还包括药学上可接受的载体、稀释剂或赋形剂。
优选地，所述药学上可接受的载体、稀释剂或赋形剂包括但不限于已经由美国食品和药品管理局或中国食品药品监督管理局批准可用于人或家畜的任何佐剂、载体、赋形剂、助流剂、甜味剂、稀释剂、防腐剂、染料/着色剂、增味剂、表面活性剂、润湿剂、分散剂、悬浮剂、稳定剂、等渗剂、溶剂、表面活性剂或乳化剂。
优选地，所述药学上可接受的载体包括但不限于糖，例如乳糖、葡萄糖和蔗糖；淀粉，例如玉米淀粉和马铃薯淀粉；纤维素及其衍生物，例如羧甲基纤维素钠、乙基纤维素和醋酸纤维素；黄芪胶；麦芽；明胶；滑石；可可脂，蜡，动植物油脂，石蜡，有机硅，膨润土，硅酸，氧化锌；油，例如花生油、棉籽油、红花油、芝麻油、橄榄油、玉米油和豆油；二醇，例如丙二醇；多元醇，例如甘油、山梨糖醇、甘露醇和聚乙二醇；酯类，例如油酸乙酯和月桂酸乙酯；琼脂；缓冲剂，例如氢氧化镁和氢氧化铝；海藻酸；无热原水；等渗盐水；林格氏溶液；乙醇；磷酸盐缓冲液；以及药物制剂中使用的任何其他相容性物质。
优选地，所述药物组合物可以为片剂，丸剂，粉剂，颗粒剂，胶囊剂，锭剂，糖浆剂，液体，乳剂，混悬剂，控制释放制剂，气雾剂，膜剂，注射剂，静脉滴注剂，透皮吸收制剂，软膏剂，洗剂，粘附制剂，栓剂，小药丸，鼻制剂，肺制剂，眼睛滴剂等等，口服或胃肠外制剂。
本发明第七方面，提供了一种缀合物，所述缀合物可以是以化学方法或者通过基因工程将本发明的抗体与其他因子、可检测标记物缀合而成。
优选地，所述可检测标记物包括荧光染料、荧光分子、化学发光标记物、染料分子、磷光分子、生物素、放射性同位素、在UV光谱中进行吸收的分子、在近红外辐射中能够进行吸收的分子或远红外辐射中能够进行吸收的分子中的一种或多种。
这些因子或标记可以提供将抗体靶向所需功能位点的作用或者为抗体提高或提供其他性能。
根据以上提示，本发明还提供了一种检测试剂盒，所述检测试剂盒包括本发明所述抗体。
本发明第八方面，提供了一种非诊断目的的检测西尼罗病毒和/或寨卡病毒的方法，所述方法包括如下步骤：
（1）提取待检测样品；
（2）将步骤（1）获取的样品与本发明的抗体接触；
（3）检测样品与本发明的抗体的免疫反应。
更具体地，所述寨卡病毒是指寨卡病毒E蛋白DIII结构域（Envelope DomainⅢ，EDⅢ）；所述西尼罗病毒是指西尼罗河病毒E蛋白DIII。
优选地，所述免疫反应的检测方法包括但不限于：蛋白质印迹、放射免疫测定、ELISA（酶联免疫吸附测定）、“夹心”免疫测定、免疫沉淀测定、沉淀素反应、凝胶扩散沉淀素反应、免疫扩散测定、凝集测定、补体结合测定、免疫放射分析、荧光免疫测定等。
优选地，所述待检测样品来源于疑似西尼罗病毒和/或寨卡病毒感染的受试者，优选地受试者为人。
本发明第十方面，提供了一种抗西尼罗病毒和/或寨卡病毒感染的方法，包括步骤：向需要的对象施用有效量的本发明的抗体或其抗原结合部分。
本文中使用的术语“对象”包括任何动物（例如，哺乳动物），所述哺乳动物包括但不限于人、非人灵长类动物、啮齿类动物等，其将成为特定治疗的接受者。
优选地，所述受试者是人。
本发明第十一方面，提供了一种本发明的抗体的用途，所述用途包括以下任一项所述的用途：
（1）制备用于检测西尼罗病毒和/或寨卡病毒的产品中的用途；
（2）制备抗西尼罗病毒和/或寨卡病毒感染的药物中的用途；
（3）制备预防或治疗西尼罗病毒和/或寨卡病毒感染导致的疾病的药物中的用途。
同时本发明还提供了前述药物组合物、缀合物、检测试剂盒的以下任一项所述的用途：
（1）制备用于检测西尼罗病毒和/或寨卡病毒的产品中的用途；
（2）制备抗西尼罗病毒和/或寨卡病毒感染的药物中的用途；
（3）制备预防或治疗西尼罗病毒和/或寨卡病毒感染导致的疾病的药物中的用途。
更具体地，所述寨卡病毒是指寨卡病毒E蛋白DIII结构域（Envelope DomainⅢ，EDⅢ）；所述西尼罗病毒是指西尼罗河病毒E蛋白DIII。
附图说明
图1显示本发明的抗体的SDS-PAGE电泳图，其中A：非还原，B：还原。
图2显示利用ELISA检测本发明的抗体的抗原结合特异性的结果图。
具体实施方式
下面结合具体实施例对本发明作更进一步的说明，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，根据本发明的技术方案及其发明构思加以等同替换或改变，都应涵盖在本发明的保护范围之内。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
实施例1、抗体的制备
1、实验步骤
（1）本发明所提供抗体的重链可变区、轻链可变区序列的结构如表1、表2所示，利用基因合成的方法合成抗体重链可变区序列（氨基酸序列如SEQ ID NO：4所示，核酸序列如SEQ ID NO：9所示）、轻链可变区序列（氨基酸序列如SEQ ID NO:8所示，核酸序列如SEQ IDNO：10所示），并将其利用分子克隆的方法将片段克隆入PRT/KIgGl载体中。
表1、本发明所述抗体重链可变区的结构
表2、本发明所述抗体轻链可变区的结构
（2）将步骤1构建的抗体表达重组载体转染对数生长期的293T细胞，转染后6-8小时换新鲜培养基，并在37℃ 8% CO2培养箱中培养96小时。收集转染上清，4000rpm离心1小时，利用蛋白（Protein）A亲和层析法进行纯化。利用SDS-PAGE和Western Blot实验检验抗体的表达及纯化情况。
2、结果
结果见图1A-B，证实得到较纯蛋白，并可清察到解链后的抗体轻、重链，说明抗体制备成功。
实施例2、抗体结合活性测定
包被液稀释寨卡病毒E蛋白DIII或西尼罗河病毒E蛋白DIII至1μg/mL，100μL每孔加入酶联板中，置于湿盒中4℃过夜。洗板机清洗酶联板3次，1.5%酪蛋白封闭，每孔200μL，湿盒37℃封闭1h。用1xPBS稀释抗体到不同浓度，并以100μL每孔加入酶联板中，湿盒中37℃反应1h，清洗酶联板3次，加入羊抗人（Fab'）2-HRP二抗室温反应45min，清洗酶联板5次并加入100μL TMB底物显色，反应3 min并用100 μL 2N H2SO4终止反应，酶联免疫检测仪450nm读数。并以抗体浓度为横坐标，OD值为纵坐标绘制抗体-抗原结合曲线。
结果如图2所示，本发明制备的抗体够特异性识别固相载体上的抗原寨卡病毒E蛋白DIII或西尼罗河病毒E蛋白DIII抗原分子，且随着抗体浓度的增加抗原和抗体之间结合具有良好的剂量依赖性。
以上内容是结合具体的优选实施方式对本发明所作的进一步详细说明，不能认定本发明的具体实施只局限于这些说明。对于本发明所属技术领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干简单推演或替换，都应当视为属于本发明的保护范围。
序列表
<110> 中国人民解放军军事科学院军事医学研究院
<120> 一种广谱识别西尼罗病毒以及寨卡病毒E蛋白DIII的抗体
<141> 2022-05-31
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cgctttaccg gcagcggcag cggcaccgat tttaccctga ccattagcag cctggaaccg 240
gaagattttg cggtgtatta ttgccagcag cgcagccgct ggccgctgac ctttggcggc 300
ggcaccaaag tggaaattaa a 321
Claims (10)
1.一种识别西尼罗病毒和/或寨卡病毒的抗体，所述抗体包括轻链CDR1、轻链CDR2、轻链CDR3、重链CDR1、重链CDR2和重链CDR3；
所述重链CDR1的氨基酸序列如SEQ ID NO:1所示；
所述重链CDR2的氨基酸序列如SEQ ID NO:2所示；
所述重链CDR3的氨基酸序列如SEQ ID NO:3所示；
所述轻链CDR1的氨基酸序列如SEQ ID NO:5所示；
所述轻链CDR2的氨基酸序列如SEQ ID NO:6所示；
所述轻链CDR3的氨基酸序列如SEQ ID NO:7所示。
2.如权利要求1所述的抗体，所述抗体的重链可变区的氨基酸序列如SEQ ID NO:4所示；所述抗体的轻链可变区的氨基酸序列如SEQ IDNO:8所示。
3.一种编码权利要求1或2所述的抗体的多核苷酸。
4.如权利要求3所述的多核苷酸，编码权利要求1所述的抗体的重链可变区的多核苷酸序列如SEQ ID NO:9所示，编码权利要求1所述的抗体的轻链可变区的多核苷酸序列如SEQID NO:10所示。
5.一种含有权利要求3所述的多核苷酸的重组载体。
6.一种宿主细胞，所述宿主细胞含有权利要求5所述的重组载体或细胞的基因组中整合了权利要求3或4所述的多核苷酸多核苷酸。
7.一种生产权利要求1或2所述的抗体的方法，所述方法包括培养权利要求6所述的宿主细胞的步骤。
8.一种含有权利要求1或2所述的抗体的药物组合物、缀合物或检测试剂盒。
9.一种非诊断目的的检测西尼罗病毒和/或寨卡病毒的方法，所述方法包括如下步骤：
（1）提取待检测样品；
（2）将步骤（1）获取的样品与权利要求1或2所述的抗体接触；
（3）检测样品与本发明的抗体的免疫反应。
10.一种权利要求1或2所述的抗体的用途，所述用途包括以下任一项所述的用途：
（1）制备用于检测西尼罗病毒和/或寨卡病毒的产品中的用途；
（2）制备抗西尼罗病毒和/或寨卡病毒感染的药物中的用途；
（3）制备预防或治疗西尼罗病毒和/或寨卡病毒感染导致的疾病的药物中的用途。
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