CN110437332B - 一种抗病毒感染的sftsv蛋白结合分子 - Google Patents
一种抗病毒感染的sftsv蛋白结合分子 Download PDFInfo
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- CN110437332B CN110437332B CN201910770025.2A CN201910770025A CN110437332B CN 110437332 B CN110437332 B CN 110437332B CN 201910770025 A CN201910770025 A CN 201910770025A CN 110437332 B CN110437332 B CN 110437332B
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Abstract
本发明公开了一种抗病毒感染的SFTSV蛋白结合分子,该结合分子是SFTSV蛋白的单克隆抗体。它包括重链和轻链,重链可变区的氨基酸序列如SEQ ID NO:7所示,轻链可变区的氨基酸序列如SEQ ID NO:8所示。本发明的结合分子能够与SFTSV蛋白特异性结合并能中和SFTSV从而发挥抗病毒作用。
Description
技术领域
本发明属于抗病毒治疗、分子免疫学领域,涉及一种治疗SFTSV感染的SFTSV蛋白结合分子,具体涉及一种特异性结合SFTSV蛋白的单克隆抗体。
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
本发明的目的在于提供一种SFTSV蛋白的结合分子,所述结合分子通过中和SFTSV发挥抗病毒感染的作用。
为了实现上述目的,本发明采用了如下技术方案:
本发明提供了一种分离的结合分子,所述结合分子包括:
(1)SEQ ID NO:1所示的重链CDR1、SEQ ID NO:2所示的重链CDR2、SEQ ID NO:3所示的重链CDR3;和/或
(2)SEQ ID NO:4所示的轻链CDR1、SEQ ID NO:5所示的轻链CDR2、SEQ ID NO:6所示的轻链CDR3。
作为本发明的一个方面,本发明的结合分子包括:
(1)重链可变区,所述重链可变区具有SEQ ID NO:7所示的氨基酸序列;和/或
(2)轻链可变区,所述轻链可变区具有SEQ ID NO:8所示的氨基酸序列。
本发明的结合分子中的CDR并不局限于上面提到的VH和VL的具体序列,并且可包括保留了特异性结合SFTSV蛋白能力的这些序列的变体。此类变体可由熟练技术人员利用本领域众所周知的技术由上面提到的具体序列得到。例如,可在FRs和/或CDRs中进行氨基酸取代、缺失或添加。尽管FRs中的变化通常被设计用于改善抗体的稳定性和免疫原性,但CDRs中的变化一般被设计用于提高抗体对其靶的亲和力。FRs变体也包括天然存在的免疫球蛋白同种异型。此类提高亲和力的变化可通过常规技术凭经验确定,所述技术包括改变CDR并测试抗体对其靶的亲和力。例如,可在所公开的任一CDR内进行保守氨基酸取代。可依据AntibodyEngineering,第2版,Oxford University Press,Borrebaeck编,1995中描述的方法进行各种改变。这些包括但不限于通过取代序列内编码功能上等同的氨基酸残基的不同密码子而改变了的核苷酸序列,由此产生“沉默”变化。例如,非极性氨基酸包括丙氨酸、亮氨酸、异亮氨酸、缬氨酸、脯氨酸、苯丙氨酸、色氨酸和甲硫氨酸。极性中性氨基酸包括甘氨酸、丝氨酸、苏氨酸、半胱氨酸、酪氨酸、天冬酰胺和谷氨酰胺。带正电荷的(碱性)氨基酸包括精氨酸、赖氨酸和组氨酸。带负电荷的(酸性)氨基酸包括天冬氨酸和谷氨酸。序列内的氨基酸取代可选自该氨基酸所属类别的其它成员。此外,多肽中的任何天然残基也可用丙氨酸取代。
本发明的“结合分子”是指抗体分子以及具有免疫学活性的片段,即含有免疫特异性结合抗原的抗原结合位点的分子。本发明的抗体分子可以是任何类型(例如IgG、IgE、IgM、IgD、IgA)、任何类别(例如IgG1、IgG2、IgG3、IgG4、IgA1、hIgA2)或亚类的抗体分子。抗体分子的免疫学活性部分包括但不限于Fab、Fab’和F(ab’)2、Fd、单链Fv(scFv)、单链抗体、二硫键连接的Fv(sdFv)和包括VL或VH结构域的单域抗体。包括单链抗体在内的结合抗原的抗体片段可以包括单独的可变区或与以下的全部或一部分组合的可变区:绞链区、CH1、CH2和CH3结构域。本发明也包括抗原结合片段,其包括可变区与绞链区、CH1、CH2和CH3结构域的任一组合。
本发明的结合分子还包括与多肽重组融合的或化学缀合的(包括共价和非共价缀合的)结合分子。
多肽重组融合的例子包括将本发明的结合分子与标记物序列融合,标记物序列包括但不限于HA标签、6xHis标签、c-Myc标签、flag标签。
化学缀合的例子包括将本发明的结合分子与可检测物质缀合。通过将抗体偶联到可检测物质可以促进检测。可检测物质的实例包括各种酶、辅基、荧光材料、发光材料、生物发光材料、放射性材料、利用各种正电子发射断层扫描的正电子发射金属、以及非放射性的顺磁性金属离子。利用本领域已知的技术,可检测物质可以直接地与抗体(或其片段)偶联或缀合,或经中间物(例如本领域已知的接头)间接地偶联或缀合。参见例如关于金属离子的美国专利No.4,741,900,其中所述金属离子可以与抗体缀合,用作本发明的诊断剂。合适的酶的实例包括辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶、或乙酰胆碱酯酶;合适的辅基复合物的实例包括链霉抗生物素蛋白/生物素和抗生物素蛋白/生物素;合适的荧光材料的实例包括伞形酮、荧光素、异硫氰酸荧光素、罗丹明、dichlorotriazinylamine荧光素、丹磺酰氯或藻红蛋白;发光材料的实例包括鲁米诺;生物发光材料的实例包括萤光素酶、萤光素、和水母发光蛋白;合适的放射性材料的实例包括125I、131I、111In或99Tc。
本发明的结合分子还包括前面所述的结合分子的功能变体。如果变体能与亲代结合分子竞争特异性结合SFTSV蛋白或其蛋白片段,则认为该变体分子是本发明结合分子的功能变体。换句话说,所述功能变体仍能结合SFTSV蛋白或其蛋白片段。所述功能变体可以具有保守序列修饰,包括氨基酸取代、添加和缺失。这些修饰可以通过本领域己知的标准技术导入,例如定向诱变和随机PCR介导的诱变,并且可包含天然以及非天然氨基酸。此外,功能变体可包含氨基酸序列在氨基末端或者羧基末端或者这两端的截短体。本发明的功能变体与亲代结合分子相比可具有相同或不同的、更高或更低的结合亲和性,但是仍能结合SFTSV蛋白或其片段。此后,当使用术语“结合分子”时,其也涵盖所述结合分子的功能变体。
作为本发明的另一方面,本发明还提供了前面所述的结合分子的多核苷酸。
编码重链CDR1的多核苷酸序列如SEQ ID NO:9所示,编码重链CDR2的多核苷酸序列如SEQ ID NO:10所示,编码重链CDR3的多核苷酸序列如SEQ ID NO:11所示,编码重链可变区的多核苷酸序列如SEQ ID NO:12所示;编码轻链CDR1的多核苷酸序列如SEQ ID NO:13所示,编码轻链CDR2的多核苷酸序列如SEQ ID NO:14所示,编码轻链CDR3的多核苷酸序列如SEQ ID NO:15所示,编码轻链可变区的多核苷酸序列如SEQ ID NO:16所示。
本发明还包括在严格或较低严格的条件下能与编码本发明的结合分子的多核苷酸杂交的多核苷酸。
本领域技术人员将意识到这些多核苷酸的功能变体也是本发明的一部分。功能变体是这样的核甘酸序列,通过使用标准遗传密码可以将其直接翻译以提供与从亲代核酸分子中翻译的序列相同的氨基酸序列。
可以利用本领域任一已知的方法得到多核苷酸序列,并确定出多核苷酸的核苷酸序列。例如,如果已知抗体的核苷酸序列,就可以从化学合成的寡核苷酸装配出编码所述抗体的多核苷酸。
或者,可以从来自合适来源的核酸中产生编码抗体的多核苷酸。如果无法得到含有编码特定抗体的核酸的克隆,但已知所述抗体分子的序列,那么可以通过化学合成得到编码所述免疫球蛋白的核酸,或利用与所述序列的3’和5’末端可杂交的合成引物从合适的来源(例如抗体cDNA文库、或从表达所述抗体的任一组织或细胞例如所选择的用于表达本发明的抗体的杂交瘤细胞中所产生的cDNA文库,或从中所分离到的核酸,优选聚A+RNA)通过PCR扩增得到编码所述免疫球蛋白的核酸,或通过利用特异于所述特定基因序列的寡核苷酸探针通过克隆得到编码所述免疫球蛋白的核酸,以例如从cDNA文库中鉴定出编码所述抗体的cDNA克隆。然后可以利用本领域任一熟知的方法将PCR所产生的扩增核酸克隆到可复制的克隆载体内。
一旦确定了抗体的核苷酸序列以及相应的氨基酸序列,就可以利用本领域熟知的处理核苷酸序列的方法例如重组DNA技术、定位诱变、PCR等处理抗体的核苷酸序列以产生具有不同氨基酸序列的抗体,例如产生氨基酸取代、缺失、和/或插入。
可以用熟知的方法检查重链和/或轻链可变结构域的氨基酸序列以鉴定出CDR的序列,所述方法例如通过与其它重链和轻链可变区的已知氨基酸序列比较以确定出序列的高变异性区域。利用常规重组DNA技术,可以将一个或多个CDR插入到构架区内,例如插入到人构架区内以人源化非人抗体,如上面所述。构架区可以是天然发生的或共有的构架区,以及优选地是人构架区(见例如,293Tthiaeta l.,J.Mol.Biol.278:457-479(1998)中的人构架区的列表)。优选地,通过构架区和CDR的组合所产生的多核苷酸编码与本发明的多肽特异结合的抗体。优选地,如上面所讨论的,在构架区内可以进行一个或多个氨基酸取代,以及优选地所述氨基酸取代提高了抗体与其抗原的结合。另外,可以用这些方法对参与链内二硫键的一个或多个可变区半胱氨酸残基进行取代或删除,以产生缺少一个或多个链内二硫键的抗体分子。本发明包括对多核苷酸的其它改变,这也在本领域人员的技术范围之内。
本发明还提供了一种含有前面所述的多核苷酸的重组载体。
利用熟知的技术可以制备含有编码本发明结合分子的核苷酸序列的重组载体。所述载体包括与合适的转录或翻译调节核苷酸序列可操纵地连接的核苷酸序列,所述转录或翻译调节核苷酸序列例如那些起源于哺乳动物、微生物、病毒、或昆虫基因的序列。调节序列的实例包括转录启动子、操纵子、增强子、mRNA核糖体结合位点、和/或其它控制转录和翻译起始和终止的合适序列。当所述调节序列与合适的多肽的核苷酸序列功能性相关时,核苷酸序列是“可操纵地连接的”。因此,如果一个启动子核苷酸序列控制合适的核苷酸序列的转录,那么所述启动子核苷酸序列就是可操纵地连接于例如抗体重链序列的。
本发明还提供了一种含有前面所述的多核苷酸或前面所述的重组载体的宿主细胞。
可用于本发明的宿主细胞包括但不限于微生物,例如经含有抗体编码序列的重组噬菌体DNA、质粒DNA或粘粒DNA表达载体转化的细菌(例如大肠杆菌(E.coli)、枯草芽孢杆菌(B.subtilis));经含有抗体编码序列的重组酵母表达载体转化的酵母菌例如酵母属(Saccharomyces)、毕赤酵母属(Pichia));经含有抗体编码序列的重组病毒表达载体(例如杆状病毒)感染的昆虫细胞系统;经重组病毒表达载体(例如花椰菜花叶病毒(CaMV);烟草花叶病毒(TMV)感染的或经含有抗体编码序列的重组质粒表达载体(例如Ti质粒)转化的植物细胞系统;或携带含有来源于哺乳动物细胞基因组的启动子(例如金属硫蛋白启动子)或来源于哺乳动物病毒的启动子(例如,腺病毒晚期启动子、痘苗病毒7.5K启动子)的重组表达构建体的哺乳动物细胞系统(例如COS、CHO、BHK、293、3T3细胞)。
在本发明的具体实施方案中,所述宿主细胞是哺乳动物细胞,更优选293细胞。
用重组DNA转化、转染宿主细胞可用本领域技术人员熟知的常规技术进行。一些采用的转化、转染方法包括但并不限于:常规化学方法如磷酸钙共沉淀法、PEI转染法,常规机械方法如显微注射、电穿孔、脂质体包装等。
获得的转化子可以用常规方法培养,以表达本发明的结合分子。根据所用的宿主细胞,培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后,用合适的方法(如温度转换或化学诱导)诱导选择的启动子,将细胞再培养一段时间。
本发明的结合分子优选的是采用哺乳动物细胞来生产,哺乳动物细胞通常需要在含血清的培养基中进行培养。需要对细胞进行无血清的适应过程后,方可让细胞在无血清培养基中正常的生长。
本发明提供了包括治疗有效量的前面所述的结合分子的药物组合物。
优选地,所述药物组合物包括治疗有效量的前面所述的结合分子。
本发明也提供了包括一个或多个装有本发明的药物组合物的一种或多种组分的容器的药物包装或试剂盒。与这些容器任选地伴随的可以是以管理药物或生物产品的生产、使用或销售的政府机构所规定的形式的说明,这些说明反映了管理人体施用的药物的生产、使用或销售的机构的认证。
本发明还提供了一种包括前面所述结合分子的检测产品。
所述检测产品包括但不限于检测试剂、试剂盒、芯片或试纸。凡是包括前面所述结合分子的能够检测出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)获取含有SFTSV的样品;
(2)将步骤(1)获取的样品与前面所述的结合分子接触;
(3)检测样品与结合分子的结合反应。
本发明还提供了制备前面所述的结合分子的方法,其包括在适合于引起从编码本发明的结合分子的DNA表达蛋白质的条件下培养包含本发明的重组载体的宿主细胞,以及分离结合分子。
本发明还提供了前面所述的结合分子的应用,所述应用包括以下任一项:
(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。
本发明的结合分子还可以与其他具有相同或者互补功能的药物联合应用,联合应用的效果可以是结合分子与其他药物功能之和,还可以远远大于结合分子与其他药物功能之和,这种情况表明该结合分子与其他药物之间产生了协同作用。
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
术语解释
本文所用的术语“单克隆抗体”指从一类基本均一的群体获得的抗体,除少数可能存在的天然发生的突变外,该群体中包含的单个抗体是相同的。修饰语“单克隆”仅表示抗体的特性,是从基本均一的抗体群中获得的,这不能解释成需要用任何特殊方法来生产抗体。
本文所用的术语“可变”表示抗体中可变区的某些部分在序列上有所不同,它形成了各种特定抗体对其特定抗原的结合和特异性。可变性集中于轻链和重链可变区中称为互补决定区(CDR)或超变区中的三个片段中。天然重链和轻链的可变区中各自包含四个FR区(可变区中较保守的部分),它们大致上呈β-折叠构型,由形成连接环的三个CDR相连,可形成部分β折叠结构。每条链中的CDR通过FR区紧密地靠在一起并与另一链的CDR一起形成了抗体的抗原结合部位。恒定区不直接参与抗体与抗原的结合,但是它们表现出不同的效应功能,例如参与抗体的依赖于抗体的细胞毒性。
术语“治疗”既指治疗性治疗又指预防/预防性措施。需要治疗的那些个体可包括早已患有特殊医学紊乱的个体,以及可能最终患有所述紊乱的那些个体(即需要预防性措施的那些个体)。
本文所述的“样品”涵盖了多种样品类型,包括生物学来源的血液及其它体液样品,实体组织样品如活检组织样品或者组织培养物,或者衍生自其中的细胞或者其后代。该术语还包括在获得后已经通过任何方式处理的样品,例如用试剂处理、溶解、或者富集某些成分如蛋白质或者多核苷酸。该术语涵盖了得自任何物种的各种临床样品,也包括培养的细胞、细胞上清和细胞溶解产物。
术语“分离的”是指基本上脱离其天然环境的分子。例如,分离的蛋白基本上不含来自得到它的细胞或组织源的细胞材料或其它蛋白。术语“分离的”也指其中分离的蛋白足够纯以至于能够作为药物组合物施用的制剂,或者至少70-80%(w/w)纯,更优选至少80-90%(w/w)纯,甚至更优选90-95%纯,并且最优选至少95%、96%、97%、98%、99%或100%(w/w)纯。
术语“特异性结合”是指两分子形成在生理条件下相对稳定的复合物。特异性结合以高亲和力以及低度到中度的结合量为特征,这有别于通常具有低亲和力与中度到高度的结合量的非特异性结合。一般地,当亲和常数KA高于106M-1或更优选高于108M-1时,认为结合是特异性的。如必要,可通过改变结合条件降低非特异性结合而基本上不影响特异性结合。熟练技术人员可利用常规技术优化合适的结合条件,如抗体的浓度、溶液的离子强度、温度、允许结合的时间、封闭剂(如血清清蛋白、乳酪蛋白)的浓度等。
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;2F6抗体重、轻链可变区核酸和蛋白序列如下所示:
2F6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。
2F6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
筛选轮数 | 投入scFv的滴度(PFU) | 产出scFv的滴度(PFU) | 产出/投入 |
第一轮 | 5×10<sup>12</sup> | 6×10<sup>5</sup> | 1.20×10<sup>-7</sup> |
第二轮 | 3.6×10<sup>12</sup> | 4×10<sup>5</sup> | 1.11×10<sup>-7</sup> |
第三轮 | 2.4×10<sup>12</sup> | 8×10<sup>5</sup> | 3.33×10<sup>-7</sup> |
第四轮 | 2.1×10<sup>12</sup> | 7.5×10<sup>6</sup> | 3.57×10<sup>-6</sup> |
实施例2 scFv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、2F6 IgG1、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、2F6 IgG1、1B2 IgG1倍比稀释,OD450值并没有明显变化,说明4-6 IgG1、2F6 IgG1、1B2 IgG1与4-5IgG1结合SFTSV-Gn蛋白互不排斥,没有竞争关系,表明4-6 IgG1、2F6 IgG1、1B2 IgG1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)
<120> 一种抗病毒感染的SFTSV蛋白结合分子
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Claims (10)
1.一种分离的结合分子,其特征在于,所述结合分子包括:
(1)SEQ ID NO:1所示的重链CDR1、SEQ ID NO:2所示的重链CDR2、SEQ ID NO:3所示的重链CDR3;和
(2)SEQ ID NO:4所示的轻链CDR1、SEQ ID NO:5所示的轻链CDR2、SEQ ID NO:6所示的轻链CDR3。
2.根据权利要求1所述的结合分子,其特征在于,所述结合分子包括:
(1)重链可变区,所述重链可变区的氨基酸序列如SEQ ID NO:7所示;和
(2)轻链可变区,所述轻链可变区的氨基酸序列如SEQ ID NO:8所示。
3.编码权利要求1或2所述的结合分子的多核苷酸。
4.根据权利要求3所述的多核苷酸,其特征在于,编码重链CDR1的多核苷酸序列如SEQID NO:9所示,编码重链CDR2的多核苷酸序列如SEQ ID NO:10所示,编码重链CDR3的多核苷酸序列如SEQ ID NO:11所示,编码重链可变区的多核苷酸序列如SEQ ID NO:12所示;编码轻链CDR1的多核苷酸序列如SEQ ID NO:13所示,编码轻链CDR2的多核苷酸序列如SEQ IDNO:14所示,编码轻链CDR3的多核苷酸序列如SEQ ID NO:15所示,编码轻链可变区的多核苷酸序列如SEQ ID NO:16所示。
5.一种包括权利要求3或4所述的多核苷酸的重组载体。
6.一种宿主细胞,其特征在于,所述宿主细胞含有权利要求3或4所述的多核苷酸或权利要求5所述的重组载体。
7.一种包括权利要求1或2所述的结合分子的药物组合物或SFTSV检测产品。
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)获取含有SFTSV的样品;
(2)将步骤(1)获取的样品与权利要求1或2所述的结合分子接触;
(3)检测样品与权利要求1或2所述的结合分子的结合情况。
9.权利要求1或2所述的结合分子的应用,所述应用包括以下任一项:
(1)在制备权利要求7所述的药物组合物或检测产品中的应用;
(2)在制备调节SFTSV活性或水平的药物中的应用;
(3)在制备中和SFTSV毒力的药物中的应用;
(4)在制备抗SFTSV感染的药物中的应用;
(5)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7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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