CN110467672A - 一种针对sftsv的全人源单克隆中和抗体及其应用 - Google Patents
一种针对sftsv的全人源单克隆中和抗体及其应用 Download PDFInfo
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- CN110467672A CN110467672A CN201910770022.9A CN201910770022A CN110467672A CN 110467672 A CN110467672 A CN 110467672A CN 201910770022 A CN201910770022 A CN 201910770022A CN 110467672 A CN110467672 A CN 110467672A
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Abstract
本发明公开了一种针对SFTSV的全人源单克隆中和抗体及其应用,本发明的单克隆抗体能特异性与SFTSV的Gn蛋白结合,且具有较强的中和活性。本发明的抗体可以用于治疗SFTSV感染引起的疾病,在临床上具有广泛的应用前景。
Description
技术领域
本发明属于细胞免疫学、分子生物学领域,涉及一种针对SFTSV的全人源单克隆中和抗体及其应用。
背景技术
发热伴血小板减少综合征(severe fever with thrombocytopenia syndrome,SFTS)属于新型蜱传虫媒出血热,是由新发现和命名的布尼亚病毒目白蛉纤细病毒科白蛉病毒属病毒-发热伴血小板减少综合征病毒(SFTSV)感染而引起的一种新发、自然疫源性、急性传染病,俗称“蜱虫病”,自2009年在我国中东部地区被发现以来,随着监测力度的加强,许多地方相继报道了确诊病例,日本、韩国、美国和阿拉伯联合酋长国等也时有病案报道,SFTS已对全球人类健康构成了严重威胁。
布尼亚病毒目成员大多由媒介生物,如蜱、螨、蚊、鼠等传播,引起地区或全球性流行。在我国主要有汉坦病毒(汉坦病毒科)和新疆出血热病毒(内罗病毒科)引起地方性流行。2011年中国疾病预防控制中心从SFTS急性期病患血液中分离出SFTSV,经过基因序列比对和同源性分析,认定该病毒属于白蛉病毒属,目前该病毒基因组已被解析:由小(S)、中(M)、大(L)三个单股负链RNA片段组成,与布尼亚病毒目其他病毒相似,病毒基因组3′末端和5′末端序列互补。S片段属双义RNA,主要编码核蛋白NP和非结构蛋白NSS,L片段编码由2084个氨基酸组成的RNA依赖的RNA聚合酶;M片段编码具有1073个氨基酸的膜蛋白前体,翻译后经宿主细胞内蛋白酶修饰形成的Gn和Gc两个糖蛋白介导了病毒感染宿主的全过程,是刺激宿主产生中和抗体的关键抗原分子体,Gn和Gc已成为目前SFTS疫苗研究的重要靶点。
SFTS患者通常具有蜱虫叮咬史,感染后典型表现是起病急,高热伴全身乏力、头痛、肌肉关节酸痛,其典型临床特征为白细胞和血小板明显降低,转氨酶升高,血清乳酸脱氢酶明显升高,凝血酶原时间延长,钠、钾、氯等电解质偏低。病例大都生活于丘陵地区,首发病例多为有野外工作经历的中老年人,平均病死率约为10%,死亡原因主要是多脏器功能衰竭。该病例多为散发,亦可引起家庭聚集性暴发,SFTS存在严重人传人现象,接触患者的血液或分泌物可以感染SFTSV。
由于该病是一新发自然疫源性传染病,疫情不可能短期内消失;目前尚无有效的疫苗用于预防,人感染了SFTSV,亦无特效药用于临床,主要以对症治疗为主;该病临床始发症状与普通流感无异,且病人多集中在农村地区,交通不便,卫生医疗水平薄弱,等到确诊,病情大多发展到病毒血症、多脏器衰竭,此时临床只能采取对症治疗手段。而作为化学治疗的补充,由抗体介导的预防和治疗病毒感染的措施已显现良好的效果,其应用前景得到专家的认同。抗体作为人体内一种最重要的抗病毒免疫介质,抗体分子可以通过阻断病毒颗粒与其受体的结合、激活巨噬细胞、NK细胞等杀伤细胞、激活补体等多种机制来杀伤、清除病毒颗粒及受感染细胞。抗体制剂不仅可以中和病人体内大量的病毒,降低荷载,转归病情,对病人的密切接触者,如陪护、医护人员进行紧急被动免疫,防止二代、三代感染者的出现。
研究表明,临床上使用病毒特异性的康复人血浆,可有效中和病毒,防止病毒在体内各器官扩散,避免发生致死性的多脏器衰竭,对病人病程的转归也起了重要作用。但多抗血浆不仅来源有限,同时其临床应用也受到诸如难以质控、供受体血型不匹配、潜在的传染性因子等条件的限制。鼠源单抗制备简单,治疗机理明确,但是其异源性阻碍了在人体内的应用。而人源单克隆抗体可有效克服上述问题。
目前国内外尚没有上市的抗SFTSV抗体,因此建立和发展具有自主知识产权的以抗体为基础的检测和诊治用品,对于多种相关性疾病的干预具有重要的现实意义。
发明内容
本发明的目的之一在于提供一种特异性结合SFTSV蛋白的单克隆抗体,具有高效和安全的特点。
本发明的目的之二在于提供上述抗体的重链、轻链或其片段。
本发明的目的之三在于提供编码上述单克隆抗体或其抗原结合片段的核酸分子或其片段,以及插入这些核酸分子以用于重组表达上述抗体的重组载体和重组细胞。
本发明的目的之四在于提供上述单克隆抗体的制备方法和用途。
为了实现上述目的,本发明采用了如下技术方案:
本发明提供了一种特异性结合SFTSV蛋白的单克隆抗体或其抗原结合片段,所述单克隆抗体或其抗原结合片段包括轻链CDR1、轻链CDR2、轻链CDR3、重链CDR1、重链CDR2和重链CDR3;
重链CDR1具有SEQ ID NO:1所示的氨基酸序列或与其具有至少80%同源性的氨基酸序列;
重链CDR2具有SEQ ID NO:2所示的氨基酸序列或与其具有至少80%同源性的氨基酸序列;
重链CDR3具有SEQ ID NO:3所示的氨基酸序列或与其具有至少80%同源性的氨基酸序列;
轻链CDR1具有SEQ ID NO:4所示的氨基酸序列或与其具有至少80%同源性的氨基酸序列;
轻链CDR2具有SEQ ID NO:5所示的氨基酸序列或与其具有至少80%同源性的氨基酸序列;
轻链CDR3具有SEQ ID NO:6所示的氨基酸序列或与其具有至少80%同源性的氨基酸序列。
优选地,重链CDR1具有SEQ ID NO:1所示的氨基酸序列;重链CDR2具有SEQ ID NO:2所示的氨基酸序列;重链CDR3具有SEQ ID NO:3所示的氨基酸序列;轻链CDR1具有SEQ IDNO:4所示的氨基酸序列;轻链CDR2具有SEQ ID NO:5所示的氨基酸序列;轻链CDR3具有SEQID NO:6所示的氨基酸序列。
进一步,所述单克隆抗体的重链可变区具有SEQ IDNO:7所示的氨基酸序列或与其具有至少80%同源性的氨基酸序列,所述单克隆抗体的轻链可变区具有SEQ ID NO:8所示的氨基酸序列或与其具有至少80%同源性的氨基酸序列。
优选地,所述单克隆抗体的重链可变区具有SEQ IDNO:7所示的氨基酸序列;所述单克隆抗体的轻链可变区具有SEQ ID NO:8所示的氨基酸序列。
包括优选抗体氨基酸序列的保守序列变体的抗体也包括在本发明的范围之内。保守氨基酸序列变体包括不显著改变本发明的单克隆抗体结合性质的氨基酸序列的修饰,如源于本领域熟知的相似氨基酸替代的变体,氨基酸的缺失、增加导致的变体。
本发明的单克隆抗体还包括人源与非人源抗体,以及具有与上述单克隆抗体相同功能或改造及优化的一切抗体。
进一步,所述单克隆抗体的抗原结合片段包括Fab、Fab′、F(ab′)2、Fv或单链抗体。
Fab是指含有一条轻链的可变区和恒定区和一条重链的可变区和恒定区经二硫键结合起来的抗体分子的一部分。
Fab’是指包含了部分铰链区的Fab片段。
F(ab’)2指的是Fab’的二聚体。
Fv指的是含有抗体重链可变区、轻链可变区并具有全部抗原结合位点的最小抗体片段。
单链抗体指的是由轻链可变区与重链可变区直接相连或通过一个肽链连接而成的工程抗体。
本发明的公开的单克隆抗体可以在重链和轻链可变区包含一个或多个糖基化位点,如本领域内熟知的,在可变区中存在的一个或多个糖基化位点可以导致增强的抗体免疫原性,或者由于改变了抗原结合而改变抗体的药物动力学。
本发明的单克隆抗体可以被设计为在Fc区域内包含修改,通常是改变抗体的1个或多个功能特性,如血清半衰期、补体结合、Fc受体结合、和/或抗原依赖的细胞毒性。此外,本发明的抗体可以被化学修饰(如,可以将一个或多个化学基团连接于抗体),或被修饰以改变其糖基化,从而再改变抗体的一个或多个功能特性。
本发明的单克隆抗体可以被设计的另一个修饰是聚乙二醇化。抗体可被聚乙二醇化,从而,例如,增加抗体的生物(如血清)半衰期。为了使抗体聚乙二醇化,该抗体或其片段通常在适合于一个或多个聚乙二醇(PEG)基团连接到该抗体或抗体片段的条件下,与PEG反应,如聚乙二醇的活性酯或醛衍生物。优选地,该聚乙二醇化是通过与活性的PEG分子(或类似的活性水溶性聚合物)进行酰化反应或烷基化反应而实现。
本发明还提供了编码前面所述的单克隆抗体或其抗原结合片段的核酸分子,所述核酸分子包括编码单克隆抗体轻链可变区的核苷酸序列,编码单克隆抗体重链可变区的核苷酸序列,编码单克隆抗体重链的核苷酸序列,或编码单克隆抗体轻链的核苷酸序列。
本发明的编码前面所述的单克隆抗体或其抗原结合片段的核酸分子包括具有优选的核苷酸序列的保守核苷酸序列变体的核酸分子。所谓的保守核苷酸序列变体源于遗传密码简并和沉默的变体,核苷酸的替代、缺失和增加也包含在内。
具体地,编码重链CDR1的核酸分子序列如SEQ ID NO:9所示,编码重链CDR2的核酸分子序列如SEQ ID NO:10所示,编码重链CDR3的核酸分子序列如SEQ ID NO:11所示,编码重链可变区的核酸分子序列如SEQ ID NO:12所示;编码轻链CDR1的核酸分子序列如SEQ IDNO:13所示,编码轻链CDR2的核酸分子序列如SEQ ID NO:14所示,编码轻链CDR3的核酸分子序列如SEQ ID NO:15所示,编码轻链可变区的核酸分子序列如SEQ ID NO:16所示。
本发明还提供了一种重组载体,所述重组载体包含前面所述的核酸分子,此外,还包括与所述核酸分子序列操作性相连的表达调控序列。
本发明中“载体”一词指的是,可将编码某蛋白的多聚核苷酸插入其中并使蛋白获得表达的一种核酸运载工具。载体可通过转化、转导或转染宿主细胞,使其携带的遗传物质元件在宿主细胞内得以表达。举例来说,载体包括:质粒;噬菌粒;柯斯质粒;人工染色体如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC);噬菌体如λ噬菌体或M13噬菌体及动物病毒等。用作载体的动物病毒种类有逆转录病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。一种载体可能含有多种控制表达的元件,包括启动子序列、转录起始序列、增强子序列、选择元件及报告基因。另外,载体还可含有复制起始位点。载体还有可能包括协助其进入细胞的成分,如病毒颗粒、脂质体或蛋白外壳,但不仅仅只有这些物质。
本发明还提供了一种重组细胞,所述重组细胞中导入了前面所述的核酸分子或转染了前面所述的重组载体。
本发明中“重组细胞”一词指的是导入核酸分子或载体的细胞,包括如下许多细胞类型,如大肠杆菌或枯草菌等原核细胞,如酵母细胞或曲霉菌等真菌细胞,如S2果蝇细胞或Sf9等昆虫细胞,或者如纤维原细胞,CHO细胞,COS细胞,NSO细胞,HeLa细胞,BHK细胞,HEK293细胞或人细胞的动物细胞。
在本发明的具体实施方案中,所述重组细胞是293细胞。
本发明还提供了一种利用前面所述的重组细胞产生抗体或抗原结合片段的方法,所述方法包括在合适的条件下培养前面所述的重组细胞并回收所述抗体。
本发明还提供了一种通过上述方法产生的抗体或抗原结合片段。
本发明还提供了一种检测产品,所述检测产品包括本发明前面所述的单克隆抗体或其抗原结合片段。
所述检测产品包括但不限于检测试剂、试剂盒、芯片或试纸。凡是能够检测出SFTSV的检测产品均包括在本发明的范围之内。
本发明还提供了一种药物组合物,所述药物组合物包括治疗有效量的本发明的前面所述的单克隆抗体或其抗原结合片段。
进一步,所述药物组合物还包括可药用载体或稀释剂。可以用任何适当的药用载体施用根据本发明的药物组合物用于治疗。
用于肠胃外给药和局部给药时,本发明的药物组合物包括无菌水或非水溶剂、悬液和乳剂。非水溶剂的例子是丙二醇、聚乙二醇、植物油、鱼油、和可注射的有机酯。水性载体包括水、水-乙醇溶液,其中包括盐和缓冲的一般肠胃外载体,其包括氯化钠溶液、林格氏葡萄糖溶液、葡萄糖加氯化钠溶液、含有乳糖的林格氏液、或非挥发性油。静脉载体包括流体和营养补充剂、电解质补充剂,如那些基于林格氏葡萄糖等的电解质补充剂。组合物可能包括其它赋形剂,如稳定化试剂或防腐剂。实用的稳定赋形剂包括表面活性剂(聚山梨酯20&80、泊咯沙姆407)、聚合物(聚乙二醇,聚乙烯吡咯酮)、糖类(蔗糖、甘露醇、葡萄糖、乳糖)、醇(山梨醇,甘油丙二醇,乙二醇)、适当的蛋白质(白蛋白)、合适的氨基酸(甘氨酸,谷氨酸)、脂肪酸(乙醇胺)、抗氧化剂(抗坏血酸,半胱氨酸等)、螯合剂(EDTA盐类、组氨酸、天门冬氨酸)或金属离子(钙、镍、镁、锰)。有用的防腐剂有苯甲醇、氯代丁醇、苯扎氯铵,也可使用对羟基苯甲酸酯类。
可以以浓缩的形式或者以粉末的形式以根据需要重构提供根据本发明的药物组合物。这样的粉剂可以使用上述赋形剂。在冷冻干燥的情况下,某些冷冻保护剂是优选的,其包括聚合物(聚乙烯吡咯酮、聚乙二醇、葡聚糖)、糖(蔗糖、葡萄糖、乳糖)、氨基酸(甘氨酸、精氨酸、谷氨酸)和白蛋白。
本发明还提供了一种非诊断目的的检测SFTSV水平的方法,其特征在于,所述方法包括如下步骤:
(1)提取含有SFTSV的样品;
(2)将步骤(1)获取的样品与前面所述的特异性结合SFTSV蛋白的单克隆抗体或其抗原结合片段接触;
(3)检测样品与抗体或其抗原结合片段的免疫反应。
本发明还提供了前面所述的单克隆抗体或其抗原结合片段在制备检测SFTSV水平的产品中的应用。
所述检测产品包括但不限于检测试剂、试剂盒、芯片或试纸。
本发明还提供了前面所述的单克隆抗体或其抗原结合片段的用途,所述用途包括以下任一项:
(1)在制备前面所述的检测产品中的应用;
(2)在制备前面所述的药物组合物中的应用;
(3)在制备调节SFTSV活性或水平的药物中的应用;
(4)在制备中和SFTSV毒力的药物中的应用;
(5)在制备抗SFTSV感染的药物中的应用;
(6)在制备治疗由SFTSV感染导致的疾病的药物中的应用。
本发明还提供了前面所述的药物组合物的用途,所述用途包括以下任一项:
(1)在制备调节SFTSV活性或水平的药物中的应用;
(2)在制备中和SFTSV毒力的药物中的应用;
(3)在制备抗SFTSV感染的药物中的应用;
(4)在制备治疗由SFTSV感染导致的疾病的药物中的应用。
本发明前面所述的单克隆抗体或其抗原结合片段可以以化学方法或者通过基因工程与其他因子缀合。这些因子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。
根据本发明的单克隆抗体可以以化学方法或者通过基因工程标记,以提供可检测的抗体。
本发明的“治疗”意在包括为受治疗者施用特异性结合SFTSV的单克隆抗体,其目的包括改善、治疗疾病。
本发明的“治疗有效量”是指对疾病的有害影响至少有所改善的水平。本领域技术人员能很容易地确定本发明的单克隆抗体的给药量和方案。
本发明的优点和有益效果:
本发明提供了一种新的特异性结合SFTSV的单克隆抗体,该抗体具有亲和力和中和能力高,无副作用,成本低廉,成分清晰,实现生产标准化,质量控制简单等竞争优势。
附图说明
图1显示Vκ基因的PCR鉴定结果图;
图2显示Vλ基因的PCR鉴定结果图;
图3显示VH基因的PCR鉴定结果图;
图4显示scFv基因的PCR鉴定结果图;
图5显示利用Phage-ELISA鉴定抗SFTSV-Gn蛋白单链抗体的结合特异性的结果图;
图6显示利用SDS-PAGE检测抗体表达的结果图;
图7显示抗体与病毒结合的荧光图;
图8显示利用间接免疫荧光检测抗体微量中和的荧光图;
图9显示HRP标记4-5IgG1的最佳稀释倍数测定图;
图10显示利用ELISA实验研究抗体竞争抑制的曲线图。
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述。本领域技术人员将会理解,下面的实施例仅用于说明本发明,而不应视为限定本发明的范围。实施例中未注明具体技术或条件者,按照本领域内的文献所描述的技术或条件(例如参考J.萨姆布鲁克等著,黄培堂等译的《分子克隆实验指南》,第三版,科学出版社)或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。
实施例1抗SFTSV-Gn蛋白单链抗体的筛选
1、JS-2010-014病毒颗粒的纯化
1.1材料
JS-2010-014,为专利申请者于2010年在一名江苏病人急性期外周血中分离获得。
1.2方法和结果
该病毒接种Vero细胞后,于37℃、5%CO2的条件下培养5天,无菌收集上清液并测定50%组织感染量(50%tissue culture infective dose,TCID50)。病毒悬液经1:4000β-丙内酯于4℃灭活24h,低速离心去除细胞碎片,再超离2h后用PBS悬浮,经分子筛层析技术进一步纯化。通过上述步骤可以获得纯度较高的JS-2010-014病毒颗粒,所有的病毒操作均在生物安全2级(BSL-2)实验室中进行。
2、scFv人源抗体文库构建和抗SFTSV-Gn蛋白单链抗体的筛选
2.1材料
引物:根据《Phage Display》一书设计家族特异性轻链(Vκ和Vλ)、IgG重链(VH)和overlap-PCR引物,其中Vκ12对、Vλ24对、VH 6对、overlap-PCR 1对。
Vκ正向引物:
5’-GGGCCCAGGCGGCCGAGCTCCAGATGACCCAGTCTCC-3’;
5’-GGGCCCAGGCGGCCGAGCTCGTGATGACYCAGTCTCC-3’;
5’-GGGCCCAGGCGGCCGAGCTCGTGWTGACRCAGCTCC-3’。
Vκ反向引物:
5’-GGAAGATCTAGAGGAACCACCTTTGATYTCCACCTTGGTCCC-3’;
5’-GGAAGATCTAGAGGAACCACCTTTGATCTCCAGCTTGGTCCC-3’;
5’-GGAAGATCTAGAGGAACCACCTTTAATCTCCAGTCGTGTCCC-3’;
5’-GGAAGATCTAGAGGAACCACCTTTGATATCCACTTTGGTCCC-3’。
Vλ正向引物:
5’-GGGCCCAGGCGGCCGAGCTCGTGBTGACGCAGCCGCCCTC-3’;
5’-GGGCCCAGGCGGCCGAGCTCGTGCTGACTCAGCCACCCTC-3’;
5’-GGGCCCAGGCGGCCGAGCTCGCCCTGACTCAGCCTCCCTCCGT-3’;
5’-GGGCCCAGGCGGCCGAGCTCGTGCTGACTCAATCGCCCTC-3’;
5’-GGGCCCAGGCGGCCGAGCTCATGCTGACTCAGCCCCACTC-3’;
5’-GGGCCCAGGCGGCCGAGCTCGTGGTGACYCAGGAGCCMTC-3’;
5’-GGGCCCAGGCGGCCGAGCTCGTGCTGACTCAGCCACCTTC-3’;
5’-GGGCCCAGGCGGCCGAGCTCGGGCAGACTCAGCAGCTCTC-3’。
Vλ反向引物:
5’-GGAAGATCTAGAGGAACCACCGCCTAGGACGGTCASCTTGGTS-3’;
5’-GGAAGATCTAGAGGAACCACCGCCTAAAATGATCAGCTGGGTT-3’;
5’-GGAAGATCTAGAGGAACCACCGCCGAGGACGGTCAGCTSGGTS-3’。
VH正向引物:
5’-GGTGGTTCCTCTAGATCTTCCTCCTCTGGGGCGGTGGCTCGGGC-3’;
5’-GGTGGTTCCTCTAGATCTTCCTCCTCTGGTGGCGGTGGCTCGGG-3’;
5’-GGTGGTTCCTCTAGATCTTCCTCCTCTGTGGCGGTGGCTCGGGC-3’;
5’-GGTGGTTCCTCTGATCTTCCTCCTCGGTGGCGGTGGCTCGGGCG-3’;
5’-GGTGGTTCCTCTAGATCTTCCTCCTCTGGTGGCGGTGGCTCGGC-3’;
5’-GGTGGTTCCTCTAGATCTTCCTCCTCTGGTGGCGGTGGTCGGGC-3’。
VH反向引物:
5’-CCTGGCCGGCCTGGCCACTAGTGACCGATGGGCCCTTGGTGGAR-3’。
overlap-PCR正向引物:
5’-GAGGAGGAGGAGGAGGAGGCGGGGCCCAGGCGGCCGAGCTC-3’。
overlap-PCR反向引物:
5’-GAGGAGGAGGAGGAGGAGCCTGGCCGGCCTGGCCACTAGTG-3’。
2.2方法
2.2.1外周血淋巴细胞的分离及总RNA提取
将8名SFTS患者恢复期外周血(经专利申请者所在单位伦理委员会、及相关献血者书面同意)分别与等量的生理盐水混合后按照淋巴细胞分离液说明书吸取单个核细胞,生理盐水洗涤三次后参照总RNA抽提试剂盒说明抽提RNA。
2.2.2抗体可变区基因的PCR扩增
将抽提的8份总RNA混合后反转录出cDNA第一链,反转录条件如下:55℃30min,85℃5min,4℃30min;再以cDNA为模板PCR扩增人源性抗体Vκ、Vλ和VH基因,PCR反应条件为:94℃预变性10min,然后94℃20s,57℃45s,72℃1min,25个循环,最后72℃延伸20min,凝胶电泳并切胶纯化回收。
2.2.3scFv基因的拼接
将纯化的Vκ基因片段与Vλ基因片段等摩尔混合后再与VH基因片段等量混合,利用overlap-PCR拼接scFv基因,overlap-PCR反应条件为:94℃预变性10min,然后94℃20s,57℃45s,72℃1min,25个循环,最后72℃延伸20min,凝胶电泳并切胶纯化回收。
2.2.4噬菌体单链抗体文库的构建及质量鉴定
纯化后的scFv基因与pComb3XSS质粒分别经sfiI酶切,连接胶纯化回收后的目的片段,转入感受态大肠杆菌XL1-Blue,加入20mL 2YT培养液中37℃培养45min后离心,沉淀涂于2YT平板30℃过夜培养。次日将平板上生长的菌苔全部收集于2YT培养基中,37℃培养至OD600为0.7。加入终浓度为1×109PFU/mL的辅助噬菌体VCSM13 37℃培养45min。加入终浓度为50μg/mL的卡纳霉素,37℃继续培养7h,900g离心15min弃沉淀,于上清中加入5×PEG/NaCl,混匀后置于冰上3h,900g离心45min,把沉淀重悬于2mL的PBS中,过0.45μm的滤膜,滤液即为人源性噬菌体单链抗体文库,同时计算文库库容和多样性。
2.2.5抗SFTSV-Gn蛋白特异性单链抗体的筛选
取100μL扩增后的噬菌体文库与固相化包被的SFTSV-Gn蛋白共同孵育,进行4轮“吸附-洗脱-扩增”亲和筛选,取第4轮洗脱液感染对数生长期的大肠杆菌XL1-Blue后,涂布2×YT培养板,37℃培养过夜,随机挑取200个单菌落分别接种96孔深孔板(含100μg/mL氨苄青霉素、12.5μg/mL四环素和1g/mL葡萄糖),37℃过夜振摇培养,次日1:10分别接种到新96孔深孔板(含100μg/mL氨苄青霉素和12.5μg/mL四环素)中37℃振摇培养5h,加入辅助噬菌体VCSM13(终浓度为1×109PFU/mL),37℃孵育1h,加入卡纳霉素(终浓度为50μg/mL)30℃过夜振摇培养制备成噬菌体单链抗体,用0.1μg/孔SFTSV-Gn蛋白包被酶标板,二抗是用PBS缓冲液(含5g/mL脱脂奶粉)按1:2000稀释的HRP标记抗M13抗体,进行Phage-ELISA鉴定并测定OD450值,当Positive/Negative≥2.1时定为阳性,阳性克隆的菌液送上海生工公司测序。
2.3结果
2.3.1人源性抗体Vκ、Vλ和VH基因的鉴定
利用12对Vκ基因引物扩增出12条大小约350bp目的片段,结果见图1,其中M:DNAmarker;1-12:PCR产物;13:阴性对照。利用24对Vλ基因引物扩增出24条大小约350bp目的片段,结果见图2,其中M:DNA marker;1:阴性对照;2-25:PCR产物。利用6对VH基因引物扩增出6条大小约400bp目的片段,结果见图3,其中M:DNA marker;1-6:PCR产物;7:阴性对照。结果与预期相符。
2.3.2scFv抗体基因的拼接
利用overlap-PCR随机拼接得到了750bp左右的目的片段,结果见图4,其中M:DNAmarker;1-3:overlap-PCR产物;4:阴性对照。结果与预期相符。
2.3.4抗SFTSV-Gn蛋白单链抗体的筛选
以SFTSV-Gn蛋白为抗原对人源化SFTSV病毒单链抗体文库进行了4轮亲和筛选,抗SFTSV-Gn蛋白特异性单链抗体得到了选择性富集,产出/投入比值提高了30倍(表1)。随机挑取200个噬菌体单克隆进行Phage-ELISA试验并测定OD450值,结果显示有19个单链抗体与SFTSV-Gn蛋白能特异性结合(图5)。19个阳性克隆菌液经测序分析,获得3种不同氨基酸序列的scFv抗体,分别命名为4-6、2F6、1B2;1B2抗体重、轻链可变区核酸和蛋白序列如下所示:
1B2抗体重链可变区的氨基酸序列如SEQ ID NO:7,核酸序列如SEQ ID NO:12所示;重链可变区CDR1的氨基酸序列如SEQ ID NO:1,核酸序列如SEQ ID NO:9所示;重链可变区CDR2的氨基酸序列如SEQ ID NO:2,核酸序列如SEQ ID NO:10所示;重链可变区CDR3的氨基酸序列如SEQ ID NO:3,核酸序列如SEQ ID NO:11所示。
1B2抗体轻链可变区的氨基酸序列如SEQ ID NO:8,核酸序列如SEQ ID NO:16所示;轻链可变区CDR1的氨基酸序列如SEQ ID NO:4,核酸序列如SEQ ID NO:13所示;轻链可变区CDR2的氨基酸序列如SEQ ID NO:5,核酸序列如SEQ ID NO:14所示;轻链可变区CDR3的氨基酸序列如SEQ ID NO:6,核酸序列如SEQ ID NO:15所示。
表1亲和筛选对抗SFTSV-Gn蛋白特异性scFv抗体的富集效应
实施例2scFv抗体全分子化构建和真核表达
1、杆状病毒重组质粒的构建
以4-6、2F6、1B2三种单链抗体质粒为模板分别PCR扩增VH、VL基因,每个扩增体系均包含以下内容:scfv质粒0.1μg,上游引物60pmol,下游引物60pmol,10×PCR缓冲液10μL,dNTP 8μL,MgCl 2 6μL,Ex Taq 0.5μL,加水至100μL。反应条件为:94℃5min;94℃15sec,56℃30sec,72℃1min,30个循环;72℃10min。胶回收试剂盒回收目的条带。将上述PCR产物,分别用XhoI/NheI(VH)、SacI/HindIII(Vk)进行酶切,同时真核杆状病毒表达载体质粒pAc-K-CH3先进行XhoI/NheI酶切,待VH片段插入后,再进行SacI/HindIII酶切,插入VL片段。酶切休系为:DNA 10μg,XhoI/NheI或SacI/HindIII各10u,10×酶切缓冲液10μL,加水至100μL。37℃酶切20h。1%琼脂糖凝胶电泳切下目的条带,进行胶回收。连接胶回收产物16℃过夜。连接产物转化大肠杆菌DH5α,通过PCR鉴定阳性克隆(如上述),对于VH和VL都有插入的克隆,用Qiagen公司的质粒抽提试剂盒制备大约10μg纯化的重组质粒。
2、全抗体分子在昆虫细胞中的表达
采用pharmingen公司的BaculoGold共转染试剂盒,将重组质粒转染293T细胞。27℃培养4-5天后,观察感染情况;5天后收集感染上清,得到重组病毒。噬斑纯化及重组病毒扩增将293T细胞传至24孔板后,用重组病毒感染。27℃培养4-5天后收获上清。2000rpm离心10min,去除细胞碎片。将收获的蛋白表达上清液用0.45μL的微孔滤膜后上样至GEHealthcare的proteinA亲和层析柱;PBS洗至基线。洗脱液(0.1mol/L的Gly-HCl,pH2.7)洗脱,用1mol/L的Tris中和至pH7.0;对纯化的样品行SDS-PAGE检测,观察纯度。结果如图6所示,其中1:4-6单抗非还原,2:4-6单抗还原,3:1B2单抗非还原,4:1B2单抗还原,5:2F6单抗非还原,6:2F6单抗还原,7:蛋白Marker,纯化获得的全分子抗体命名为4-6IgG1、2F6IgG1、1B2IgG1。
实施例3全分子抗体与SFTSV结合实验
1、方法
(1)将Vero细胞接种于24孔板,37℃培养,当融合率达到90%时每孔接种病毒液。
(2)两天后,移除病毒液,每孔加入400μl 4%多聚甲醛,室温固定30min。
(3)弃掉多聚甲醛,用PBS清洗3次,加入400μl 0.2%Triton X-100通透细胞膜,室温15min。
(4)弃掉Triton X-100,用PBS清洗3次,加入5mg/ml的BSA进行封闭,室温30min。
(5)弃掉封闭液,每孔加入300μl纯化后的全分子单抗,设置复孔,同时设置不加入抗体的空白对照组及4-5IgG1单抗(参见专利文献:一种抗SFTSV的人源抗体,授权公告号CN102942629B)阳性对照组,于37℃孵育1h。
(6)移除一抗,加入500μl PBST,清洗3次,500转/分钟,震荡5min。
(7)加入FITC标记的抗人IgG,37℃避光孵育30min。
(8)移除二抗,加入500μl PBST清洗3次,500转/分钟,震荡5min。
(9)荧光显微镜下观察。
2、结果
4-6IgG1、2F6IgG1、1B2IgG1与SFTSV结合具有较高的结合活性(图7)。
实施例4全分子单抗微量中和实验
1、方法
(1)Vero细胞接种于96孔板,待细胞融合率达到90%时接种病毒抗体复合物。
(2)将等量100TCID50病毒与抗体(100μg/ml)混匀,37℃孵育1h。
(3)弃掉培养液,每孔加入100μl复合物,37℃孵育2h,设置复孔,同时设置无抗体组,无病毒组及4-5IgG1阳性对照组。
(4)弃掉复合物,每孔加入100μl维持液,37℃培养48h。
(5)移除维持液,每孔加入200μl 4%多聚甲醛,室温固定30min。
(6)弃掉多聚甲醛,用PBS清洗3次,加入200μl 0.2%Triton X-100通透细胞膜,室温15min。
(7)弃掉Triton X-100,PBS清洗3次,加入5mg/ml的BSA进行封闭,室温30min。
(8)弃掉封闭液,加入抗NP的一抗,每孔100μl,37℃孵育1h。
(9)移除一抗,加入200μl PBST清洗3次,500转/分钟,震荡5min。
(10)加入100μl FITC标记的抗人二抗,37℃避光孵育30min。
(11)移除二抗,加入500μl PBST清洗3次,500转/分钟,震荡5min。
(12)荧光显微镜下观察。
2、结果
微量中和实验结果显示,4-6IgG1、2F6IgG1、1B2IgG1对SFTSV均具有中和作用(图8)。
实施例5单抗抗原表位初步分析
1、HRP标记4-5IgG1最佳稀释倍数测定
用纯化的SFTSV-Gn蛋白包被酶标板(100ng/孔),将HRP标记4-5IgG1从1:100~1:600开始稀释,100μL/孔37℃孵育1h后洗板,TMB显色,测定OD450值,选择OD450值在1.0~1.5之间的那个HRP标记4-5IgG1稀释度为最佳稀释倍数。结果发现按照1:300稀释时,OD450值(1.363)介于1.0~1.5之间(图9),因此选择1:300为HRP标记4-5IgG1最佳稀释倍数。
2、竞争ELISA实验初步分析3种单抗抗原表位
分别用HRP标记4-5IgG1按1:300制成的工作液将4-5IgG1、4-6IgG1、2F6IgG1、1B2IgG1、抗SFTSV-NP单抗(无关抗体作为对照)从100ng开始,倍比稀释进行竞争ELISA实验,并读取OD450绘制竞争抑制曲线,实验结果显示(图10),伴随4-6IgG1、2F6IgG1、1B2IgG1倍比稀释,OD450值并没有明显变化,说明4-6IgG1、2F6IgG1、1B2IgG1与4-5IgG1结合SFTSV-Gn蛋白互不排斥,没有竞争关系,表明4-6IgG1、2F6IgG1、1B2IgG1的抗原表位与4-5IgG1并不相同。
虽然以上仅描述了本发明的具体实施方式范例,但是本领域的技术人员应当理解,这些仅是举例说明,本发明的保护范围是由所附权利要求书限定的。本领域的技术人员在不背离本发明的原理和实质的前提下,可以对这些实施方式做出多种变更或修改,但这些变更或修改均落入本发明的保护范围。
序列表
<110> 江苏省疾病预防控制中心(江苏省公共卫生研究院)
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Claims (10)
1.一种特异性结合SFTSV蛋白的单克隆抗体或其抗原结合片段,其特征在于,所述单克隆抗体或其抗原结合片段包括轻链CDR1、轻链CDR2、轻链CDR3、重链CDR1、重链CDR2和重链CDR3;
重链CDR1具有SEQ ID NO:1所示的氨基酸序列;
重链CDR2具有SEQ ID NO:2所示的氨基酸序列;
重链CDR3具有SEQ ID NO:3所示的氨基酸序列;
轻链CDR1具有SEQ ID NO:4所示的氨基酸序列;
轻链CDR2具有SEQ ID NO:5所示的氨基酸序列;
轻链CDR3具有SEQ ID NO:6所示的氨基酸序列。
优选地,所述单克隆抗体的重链可变区具有SEQ ID NO:7所示的氨基酸序列;所述单克隆抗体的轻链可变区具有SEQ IDNO:8所示的氨基酸序列。
2.编码权利要求1所述的单克隆抗体或其抗原结合片段的核酸分子。
3.根据权利要求2所述的核酸分子,其特征在于,编码重链CDR1的核酸分子序列如SEQID NO:9所示,编码重链CDR2的核酸分子序列如SEQ ID NO:10所示,编码重链CDR3的核酸分子序列如SEQ ID NO:11所示,编码重链可变区的核酸分子序列如SEQ ID NO:12所示;编码轻链CDR1的核酸分子序列如SEQ ID NO:13所示,编码轻链CDR2的核酸分子序列如SEQ IDNO:14所示,编码轻链CDR3的核酸分子序列如SEQ ID NO:15所示,编码轻链可变区的核酸分子序列如SEQ ID NO:16所示。
4.一种重组载体,其特征在于,所述重组载体包括权利要求2或3所述的核酸分子。
5.一种重组细胞,其特征在于,所述重组细胞中导入了权利要求2或3所述的核酸分子,或转染了权利要求4所述的重组载体。
6.一种药物组合物,其特征在于,所述药物组合物包括治疗有效量的权利要求1所述的单克隆抗体或其抗原结合片段。
7.一种SFTSV的检测产品,其特征在于,所述产品包括权利要求1所述的单克隆抗体或其抗原结合片段。
8.一种非诊断目的的检测SFTSV水平的方法,其特征在于,所述方法包括如下步骤:
(1)提取含有SFTSV的样品;
(2)将步骤(1)获取的样品与权利要求1所述的单克隆抗体或其抗原结合片段接触;
(3)检测样品与抗体或其抗原结合片段的免疫反应。
9.权利要求1所述的单克隆抗体或其抗原结合片段的应用,所述应用包括以下任一项:
(1)在制备权利要求7所述的检测产品中的应用;
(2)在制备权利要求6所述的药物组合物中的应用;
(3)在制备调节SFTSV活性或水平的药物中的应用;
(4)在制备中和SFTSV毒力的药物中的应用;
(5)在制备抗SFTSV感染的药物中的应用;
(6)在制备治疗由SFTSV感染导致的疾病的药物中的应用。
10.权利要求6所述的药物组合物的应用,所述应用包括以下任一项:
(1)在制备调节SFTSV活性或水平的药物中的应用;
(2)在制备中和SFTSV毒力的药物中的应用;
(3)在制备抗SFTSV感染的药物中的应用;
(4)在制备治疗由SFTSV感染导致的疾病的药物中的应用。
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CN113980125A (zh) * | 2021-10-15 | 2022-01-28 | 中国科学院武汉病毒研究所 | 一种抗sftsv的中和性单克隆抗体及其应用 |
CN117229413A (zh) * | 2023-09-04 | 2023-12-15 | 中国人民解放军军事科学院军事医学研究院 | 一种针对SFTSV-Gn和CD3的双特异性抗体及其制备方法 |
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