CN110590942B - 一种中和肠道病毒71型的全人源单克隆抗体及其用途 - Google Patents
一种中和肠道病毒71型的全人源单克隆抗体及其用途 Download PDFInfo
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Abstract
本发明属于生物免疫学领域，更具体地，涉及一种中和肠道病毒71型的全人源单克隆抗体及其用途，所述重链可变区的氨基酸序列如SEQ ID NO:14‑20任一项所示；所述轻链可变区的氨基酸序列如SEQ ID NO:21‑26任一项所示。本发明还提供编码该抗体的核酸分子、表达载体、用途和组合物。本发明的结合分子能阻止EV71型病毒侵染易感细胞，且是全人源的，与其它动物源性(如鼠源性)抗EV71型病毒的分子相比，免疫原性低，且亲和性良好，不仅治疗效果好，而且副作用低。
Description
技术领域
本发明涉及生物免疫学领域，具体地说，涉及一种中和肠道病毒71型的全人源单克隆抗体及其用途。
背景技术
手足口病(Hand foot mouth disease，HFMD)是由肠道病毒引起的一种常见的婴幼儿急性传染病，发病年龄多为4岁以下，表现为口痛、厌食、低热及手、足、口腔等部位出现小疱疹或小溃疡，多数患儿1周左右自愈，少数患儿可引起心肌炎、肺水肿、无菌性脑膜炎等并发症，重症患儿病情发展快可导致死亡，目前缺乏有效治疗药物。近几十年来，HFMD已在全世界多个地区爆发和流行，在2008年我国将手足口病列入法定报告管理的丙类传染病，要求在24h之内上报卫生部。2009-2015年，我国卫生部门共收到1300多万手足口病病例报告，死亡3216人，平均每年有400-500例儿童因此死亡。手足口病的发病人数和病死人数占我国丙类传染病的首位。引起手足口病症状的病毒主要以肠道病毒71型(enterovirus 71，EV71)和柯萨奇病毒A组16型(coxsachievirus A16，CoxA16)最为常见，其中重症病例的80％以上、死亡病例的90％以上都是EV71型感染所致。EV71型病毒除了引起HFMD以外，更易合并严重并发症。肠道病毒EV71有明显的季节性，多在夏秋季节流行，人是其敏感宿主，除少数灵长动物外，没有其他宿主。
EV71是小单链正义RNA病毒，属于肠道病毒属小RNA病毒科，分子量为2.6×106D，约含7400个核苷酸。EV71毒粒为二十面体形，立体对称，呈球形状，是裸露的核衣壳，直径约为23-30nm，无包膜。EV7的衣壳蛋白由VP1、VP2、VP3和VP4组成。VP1、VP2和VP3裸露于病毒颗粒的表面，VP4位于病毒颗粒衣壳的内表面与RNA核心紧密相连。根据病毒衣壳蛋白VP1或内部蛋白VP4核苷酸序列的不同，可将EV71分为A、B、C、D四个基因型，其中B型分为B1-B5亚型，C型分为C1-C5亚型，不同亚型感染后临床症状不完全相同。从1998年至今在中国多个地区分离的EV71病毒都有较高的同源性，均属于C4亚型，基因型变化较小。VP1蛋白暴露于毒粒表面，且病毒的主要抗原决定簇位于VP1上，它具有良好的抗原性和免疫原性，是诱导机体产生EV71特异性抗体的有效抗原。
从1992年首个抗体药物Orthoclone上市以来，到2016年底共上市了63个抗体药物，平均每年上市2.5个抗体药物，且速度越来越快。抗体药物用于病毒感染性疾病的治疗早有报道，抗血清用于治疗SARS和重症H5N1丙型肝炎病毒感染者的案例已经证明了抗体在治疗病毒感染中发挥的重要作用。具有中和活性的抗肠道病毒71型的全人源单克隆抗体具有以下潜在优势：一方面它可以阻断病毒与靶细胞的结合；另一方面通过补体和T细胞、NK细胞等效应细胞的作用，将被病毒感染的细胞杀死。我们通过采集EV71型手足口病毒感染者抗凝全血，体外分离活化记忆性B细胞，促使其分泌抗体，筛选出阳性B细胞克隆，并提取阳性B细胞的RNA，从中扩增抗体重链和轻链基因并克隆到表达载体中，再用携带重链基因的质粒和携带轻链基因的质粒共转染HEK293T细胞，ELISA筛选出具有VP1抗原特异性的抗体重组质粒，再进行抗体序列分析和抗体蛋白的表达纯化，最后，进一步利用中和效应实验筛选出具有病毒中和效应的抗体。
2016年上半年针对EV71病毒的疫苗已经获批上市，这是目前唯一可用于预防手足口病的疫苗，能在一定程度上遏制EV71病毒所致的手足口病发病率，但也存在部分局限性：5岁以上的人、发热、急慢性疾病期发作患者及过敏体质者都不得接种该疫苗；对疫苗中心活性物质以及硫酸卡那霉素过敏者也不能接种；疫苗接种具有个体差异性，可以会有一些不良反应；不能防止EV71病毒所有的基因亚型，对已经感染EV71病毒的患者无效。目前临床尚无直接靶向EV71病毒的药物，主要采取对症治疗的策略。在治疗病毒感染时，临床上一线药物普遍使用的是类似于利巴韦林、阿昔洛韦等广谱的抗病毒药物，并可以联合清开灵、双黄连等中药使用，起到了抗病毒的功效。但是利巴韦林等核苷类似物药物易诱发溶血性贫血等不良反应，对婴幼儿生长发育具有一定潜在的抑制作用，用于治疗时需慎重权衡获益与风险。使用的中药虽然都有清热解毒的功效,但也都属于广谱的清热解毒药物,针对性不强，其直接作用机制还不明确。综上所述，目前缺乏特效的、专一的、机制明确的抗EV71药物，因此研制针对EV71病毒高特异性的全人源单克隆抗体具有十分重要的意义。本发明即提供一种针对EV71所致的手足口病的高特异性的全人源单克隆抗体序列及用途。
发明内容
本发明人经过深入的研究，获得一种具有广谱中和效应的肠道病毒71型全人源单克隆抗体，本发明的单克隆抗体是全人源的，其重链、轻链可变区以及恒定区均来源于人的基因，因此具有免疫原性低、安全性高的特点，本发明提供的单克隆抗体能具有显著中和肠道病毒71型。
本发明一方面提供一种中和肠道病毒71型的全人源单克隆抗体，其具有重链可变区和轻链可变区：
(I)所述重链可变区的氨基酸序列如SEQ ID NO:14-20任一项所示；或具有SEQ IDNO:14-20任一项所示氨基酸序列经替换、缺失或增加一个或几个氨基酸形成的具有同等功能的氨基酸序列；
(II)所述轻链可变区的氨基酸序列如SEQ ID NO:21-26任一项所示；或具有SEQID NO:21-26任一项所示氨基酸序列经替换、缺失或增加一个或几个氨基酸形成的具有同等功能的氨基酸序列。
优选地，该单克隆抗体具有如下任一组重链可变区和轻链可变区：
(i)重链可变区的氨基酸序列如SEQ ID NO:14所示；轻链可变区的氨基酸序列如SEQ ID NO:21所示；(7H1+23κ8组合)
(ii)重链可变区的氨基酸序列如SEQ ID NO:15所示；轻链可变区的氨基酸序列如SEQ ID NO:22所示；(12H1+19κ8组合)
(iii)重链可变区的氨基酸序列如SEQ ID NO:16所示；轻链可变区的氨基酸序列如SEQ ID NO:22所示；(14H1+19κ8组合)
(iv)重链可变区的氨基酸序列如SEQ ID NO:17所示；轻链可变区的氨基酸序列如SEQ ID NO:22所示；(9H1+19κ8组合)
(v)重链可变区的氨基酸序列如SEQ ID NO:18所示；轻链可变区的氨基酸序列如SEQ ID NO:22所示；(23H1+19κ8组合)
(vi)重链可变区的氨基酸序列如SEQ ID NO:19所示；轻链可变区的氨基酸序列如SEQ ID NO:21所示。(5H1+23κ8组合)
上述重链可变区和轻链可变区组成的单克隆抗体能特异性结合EV71病毒，试验结果显示，在浓度为0.05μg/ml时，7H1+23κ8组合对病毒的抑制率可达到90％，12H1与19κ8，14H1+19κ8，9H1+19κ8，23H1+19κ8和5H1+23κ8组合对病毒的抑制率可达到80％以上。
在本发明的另一方面，提供一种编码上述单克隆抗体的核酸分子。
优选地，该核酸分子具有如SEQ ID NO:1-7任一项所示的重链可变区的核苷酸序列；和如SEQ ID NO:8-13任一项所示的轻链可变区的核苷酸序列。
更优选地，该核酸分子具有如下任一组重链可变区和轻链可变区：
(i)重链可变区的核苷酸序列如SEQ ID NO:1所示；轻链可变区的核苷酸序列如SEQ ID NO:8所示；
(ii)重链可变区的核苷酸序列如SEQ ID NO:2所示；轻链可变区的核苷酸序列如SEQ ID NO:14所示；
(iii)重链可变区的核苷酸序列如SEQ ID NO:3所示；轻链可变区的核苷酸序列如SEQ ID NO:9所示；
(iv)重链可变区的核苷酸序列如SEQ ID NO:4所示；轻链可变区的核苷酸序列如SEQ ID NO:9所示；
(v)重链可变区的核苷酸序列如SEQ ID NO:5所示；轻链可变区的核苷酸序列如SEQ ID NO:9所示；
(vi)重链可变区的核苷酸序列如SEQ ID NO:6所示；轻链可变区的核苷酸序列如SEQ ID NO:8所示。
在本发明的另一方面，提供一种表达载体，所述的表达载体包括上述核酸分子。
该表达载体除包含上述的核酸分子，还可以包含适当启动子或者控制序列。该载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。
在本发明的另一方面，提供一种宿主细胞，所述的宿主细胞包括上述表达载体。
宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞。代表性例子有：细菌细胞如大肠杆菌，链霉菌属；鼠伤寒沙门氏菌；真菌细胞如酵母；植物细胞；昆虫细胞如果蝇S2或Sf9；动物细胞如CHO、COS7、NSO或Bowes黑素瘤细胞等。特别适用于本发明的宿主细胞是真核宿主细胞，尤其是哺乳动物细胞，如293细胞。
在本发明的另一方面，提供所述的单克隆抗体或其片段在制备用于检测、治疗、预防肠道71型病毒感染的药物中的用途。
在本发明的另一方面，提供一种组合物，包括治疗有效量的所述的单克隆抗体中的一种或多种抗体组成的抗体混合物，以及药学上可接受的载体。
本文所用的术语“药学上可接受的”是指当分子本体和组合物适当地给予动物或人时，它们不会产生不利的、过敏的或其它不良反应。本文所用的“药学上可接受的载体”应当与本发明的单克隆抗体或其片段相容，即能与其共混而不会在通常情况下大幅度降低组合物的效果。
可作为药学上可接受的载体或其组分的一些物质的具体例子是糖类，如乳糖、葡萄糖和蔗糖；淀粉，如玉米淀粉和土豆淀粉；纤维素及其衍生物，如羧甲基纤维素钠、乙基纤维素和甲基纤维素；西黄蓍胶粉末；麦芽；明胶；滑石；固体润滑剂，如硬脂酸和硬脂酸镁；硫酸钙；植物油，如花生油、棉籽油、芝麻油、橄榄油、玉米油和可可油；多元醇，如丙二醇、甘油、山梨糖醇、甘露糖醇和聚乙二醇；海藻酸；乳化剂，如Tween；润湿剂，如月桂基硫酸钠；着色剂；调味剂；压片剂、稳定剂；抗氧化剂；防腐剂；无热原水；等渗盐溶液；和磷酸盐缓冲液等。
本发明的组合物可根据需要制成各种剂型，并可由医师根据患者种类、年龄、体重和大致疾病状况、给药方式等因素确定对病人有益的剂量进行施用。给药方式例如可以采用注射或其它治疗方式。
上述药物组合物可包含两或多个对于EV71型病毒具有中和活性的单克隆抗体或其片段。
在本发明的另一方面，提供一种检测肠道病毒71型的试剂盒，其包括所述的单克隆抗体或其片段。
以所述的单克隆抗体或其片段为基础，可制备方便、快速且准确地检测EV71型病毒的试剂盒。作为本发明的一种检测方式，采用间接ELISA法，将待测的抗原包被于固相载体上，利用本发明的单克隆抗体或其片段进行检测。作为本发明的一种优选方式，所述的单克隆抗体或其片段是抗体，可根据双抗夹心法的原理来检测。双抗夹心法常规的做法是将一抗(如本发明的单克隆抗体)固定于载体，然后使一抗与抗原反应，洗涤后再与二抗反应(所述的二抗携带可检测信号，或可与携带可检测信号的物质结合)，最后进行化学发光或酶联显色反应检测信号。双抗夹心法特别适用于具有两个或两个以上表位的抗原的检测。
为了在检测时更方便，所述试剂盒中除了含有本发明的单克隆抗体或其片段以外，还可以包含其它检测试剂或辅助试剂，所述的辅助试剂例如是ELISA试剂盒中常规使用的一些试剂，这些试剂的特性以及它们的配制方法均是本领域技术人员所熟知的，如显色剂、标记物、二抗、抗抗体、增敏剂等。本领域人员应理解，各种变化形式的检测试剂盒均是包含在本发明中的，只要在其中利用了本发明的单克隆抗体或其片段作为识别EV71型病毒的试剂。
此外，在所述试剂盒中还可包含使用说明书，用于说明其中装载的试剂的使用方法。
在获得了本发明提供的单克隆抗体或其片段后，可以利用多种免疫学相关方法来检测样品中VP1蛋白，从而得知待测样品的供体是否感染EV71型病毒，这些方法均被包含在本发明中。较佳地，所述的方法是以非疾病诊断为目的的。
在本发明的另一方面，提供一种非治疗性地抑制肠道病毒71型的方法，所述的方法包括给予患者有效量的所述的单克隆抗体或其片段。
在本发明的另一方面，提供一种非治疗性检测肠道病毒71型的方法，所述的单克隆抗体或其片段与待测样品进行接触，通过检测所述的单克隆抗体或其片段与受试样品的结合情况，获得肠道病毒71型的存在情况以及存在量。
如本文所用，术语“待测样品”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。
与现有技术相比，本发明具有如下有益效果：所述单克隆抗体能特异性结合EV71病毒，试验结果显示，在浓度为0.05μg/ml时，7H1+23κ8组合对病毒的抑制率可达到90％，12H1与19κ8，14H1+19κ8，9H1+19κ8，23H1+19κ8和5H1+23κ8组合对病毒的抑制率可达到80％以上。此外，本发明所述抗体为全人源性单克隆抗体，相比鼠源抗体，全人源抗体的基因完全来源于人的基因，没有其他种属的成分，在人体内不发生抗鼠抗抗体等毒副作用，具有更好的生物相容性，更适合和更有潜力成为治疗肠道病毒71型的大分子药物。
附图说明
图1为抗体重链可变区基因VH、VH’和轻链可变区基因Vκ、Vλ的PCR扩增后的电泳图。
图2为重链可变区基因、轻链可变区基因和载体的电泳验证结果。
图3为重组质粒的电泳验证结果。
图4为EV71-VP1抗原特异性全人单克隆抗体体外表达纯化后SDS电泳结果。
图5为全人源单克隆抗体中和EV71型病毒的细胞状态图。
具体实施方式
以下实施例进一步说明本发明的内容，但不应理解为对本发明的限制。在不背离本发明精神和实质的情况下，对本发明方法、步骤或条件所作的修改或替换，均属于本发明的范围。
实施例中未注明具体条件的实验方法，通常按照常规条件，例如Sambrook等人，分子克隆：实验室手册(New York:Cold Spring HarborLaboratoryPress,1989)中所述的条件，或按照制造厂商所建议的条件。
实施例1：中和肠道病毒71型的全人源单克隆抗体的制备
1.记忆B细胞的分选
1.1外周血单核细胞的获取
从已感染肠道病毒71型的志愿者体内抽取新鲜血液3.5ml，置于抗凝管中。将新鲜抗凝全血与PBS等体积稀释后，缓慢加入到淋巴细胞分离液(国产Solarbio，Cat.NO.P8610)中，离心之后将其小心拿出，吸第二层云雾状的单核细胞层于装有PBS的离心管中洗涤，离心弃上清，再用PBS洗涤1次，收集到的细胞即为外周血单核细胞(periphery bloodmononuclear cell，PBMC)。
1.2记忆B细胞分选
将分离出的志愿者的PBMC离心彻底去除上清，每107个细胞用40μL缓冲液重悬细胞沉淀。加10μL Biotin-Antibody Cocktail，充分混匀，4℃冰箱内孵育5min。加30μL缓冲液，以及20μL Anti-Biotin MicroBeads，充分混匀，4℃冰箱内放置10min。加1～2mL缓冲液洗涤细胞，1000rpm离心10min，去上清。用500μL缓冲液重悬细胞。将分选柱放置到磁铁上，用3mL的缓冲液冲洗分选柱。将细胞悬液加到分选柱上，防止产生气泡，收集流出的未标记细胞。用500μL缓冲液洗柱3次，收集所有的洗脱液，即为含B细胞的悬液。以上实验用的是“人记忆B细胞分选磁珠”试剂盒(德国Miltenyi，Cat.NO.99-130-046-901)。
细胞计数，1000rpm离心10min，彻底去除上清，用80μL缓冲液重悬细胞沉淀。胞加20μL CD27 MicroBeads，充分混匀，4℃冰箱内孵育15min。加1-2mL缓冲液洗涤细胞，1000rpm离心10min，彻底去除上清。用500μL缓冲液重悬≤108细胞。将柱子放置到磁铁上。用适当体积的缓冲液冲洗柱子。将细胞悬液加到柱子上。收集流出的未标记细胞并用适当体积的缓冲液洗柱子3次。收集所有的洗脱液，即为含未标记细胞的悬液，洗涤过程中要等柱中液体流干以后再加洗液。从分离器上取下柱子，放到一个收集管上，吸取缓冲液至柱子上，立即用力推活塞洗出磁珠标记的细胞。此细胞即为记忆性B细胞。计数分选出的记忆性B细胞。以上实验用的是“人B细胞CD27分选磁珠”试剂盒(德国Miltenyi，Cat.NO.99-130-051-601)。
2.记忆B细胞的体外活化培养及阳性孔筛选
将分选所得的记忆B细胞按照0细胞/孔、5细胞/孔、20细胞/孔的密度接种96孔细胞培养板，记忆B细胞在96孔细胞培养板上的布局见表1，采用10％FBS的IMDM作为基础培养液，培养液中含合适浓度的IL-2和IL-21以及经60Co照射灭活的3T3-CD40L细胞作为饲养层细胞，在37℃，5％CO2条件下培养10天，让记忆性B细胞活化成为抗体分泌细胞，收集上清用于下一步检测。
表1：记忆B细胞在96孔细胞培养板上的布局
| 样本 | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
| A | 水 | 水 | 水 | 水 | 水 | 水 | 水 | 水 | 水 | 水 | 水 | 水 |
| B | 水 | B(0) | B(0) | B(0) | B(0) | B(0) | B(0) | B(0) | B(0) | B(0) | B(0) | PBS |
| C | 水 | B(0) | B(0) | B(0) | B(0) | B(0) | B(0) | B(0) | B(0) | B(0) | B(0) | PBS |
| D | 水 | B(5) | B(5) | B(5) | B(5) | B(5) | B(5) | B(5) | B(5) | B(5) | B(5) | PBS |
| E | 水 | B(5) | B(5) | B(5) | B(5) | B(5) | B(5) | B(5) | B(5) | B(5) | B(5) | PBS |
| F | 水 | B(20) | B(20) | B(20) | B(20) | B(20) | B(20) | B(20) | B(20) | B(20) | B(20) | PBS |
| G | 水 | B(20) | B(20) | B(20) | B(20) | B(20) | B(20) | B(20) | B(20) | B(20) | B(20) | PBS |
| H | PBS | PBS | PBS | PBS | PBS | PBS | PBS | PBS | PBS | PBS | PBS | PBS |
使用酶联免疫吸附测定(Enzyme-linked immunosorbent assay，ELISA)方法检测B细胞上清特异性结果如表2所示。
表2：Elisa检测B细胞培养基上清IgG的OD值
通过表2可知，20细胞/孔的密度接种时，培养基上清中IgG的表达量最高(F2-11,G2-11)。随后我们将F2-11和G2-11这20个IgG表达量较高孔的记忆B细胞混合在一起，随机均等分成了1号样品、2号样品和3号样品进行后续实验。
3.抗体可变区基因文库的构建及筛选
3.1阳性孔B细胞RNA提取
用枪头吹打活化培养后的细胞，将其吸到无核酸酶的PCR管中，再用适量的PBS洗一次，然后加入0.25％胰蛋白酶进行消化，FBS终止消化后迅速将细胞放入液氮和98℃的PCR仪中处理，随后放入液氮速冻，每管加入适量蛋白酶K和RNA酶抑制剂，最后置于PCR仪中53℃，1h。
3.2抗体重链可变区和轻链可变区基因的扩增
用两步RT-PCR方法扩增抗体重链可变区基因VH(为了提高每步实验效率，将重链可变区基因VH引物分成了H和H’两大类，再分别进行PCR的扩增及后续的实验，在重组质粒转化时再将两大类混合在一起挑取单克隆)和轻链可变区基因Vκ或Vλ：以反转录后的cDNA作为PCR模板，进行巢式PCR第一轮反应；然后再以巢式PCR第一轮反应产物为模板，进行巢式PCR第二轮反应。具体引物序列、PCR反应程序以及PCR反应体系参考文献(NatProtoc.2009；4(3):372-84.)。
重链VH和轻链Vκ、Vλ的PCR产物用琼脂糖凝胶电泳检测，结果如图1所示，图1中条带1到4分别为1号样品的VH、VH’、Vκ和Vλ；条带5到8分别为2号样品的VH、VH’、Vκ和Vλ；条带9到12分别为3号样品的VH、VH’、Vκ和Vλ。胶回收目的条带，具体操作参照OMEGA胶回收试剂盒说明书。最后使用Nanodrop2000测浓度，-20℃长期保存。结果表明，我们顺利检测到了目的条带，而且筛选到的重链和轻链基因大小在400bp左右。
3.3抗体基因重链可变区和轻链可变区文库的构建
将扩增的抗体重链可变区基因VH和轻链可变区基因Vκ、Vλ分别连接到表达载体pIgH(AbVec-hIgG1for VH)，pIgκ(AbVec-hIgKappa for Vκ)和pIgλ(IG-lambdaexpression vector for Vλ)(NCBI GenBank编号分别为:FJ475055，FJ475056，FJ517647)上。
依次使用AgeI-HF和Sal I-HF酶切VH、pIgH；用AgeI-HF和Xho I酶切Vλ、pIgλ；用AgeI-HF酶切Vκ、pIgκ，再用BsiW I酶切Vκ、pIgκ。将上述酶切产物用1.2％琼脂糖凝胶电泳检测，观察条带，目的基因条带在300bp-500bp位置，实验结果见图2。胶回收目的条带，具体操作参照OMEGA胶回收试剂盒说明书。
图2为重链可变区基因、轻链可变区基因和载体的电泳验证结果。其中VH、VH’和Vλ都为酶切验证，Vκ为PCR验证。条带1到3分别为1号样品的VH、VH’和Vλ；条带4到6分别为2号样品VH、VH’和Vλ；条带7到9分别为3号样品的VH、VH’和Vλ；条带10为阴性对照；条带11为pIgH载体的酶切产物；12为pIgλ载体的酶切产物；条带13、14和15分别为1号、2号和3号样品的Vκ；条带16-18为阴性对照；条带19为pIgκ载体的酶切产物。
通过图2可知，载体和目的基因的酶切产物大小都很正常。
将酶切之后的目的基因片段与相应酶切之后的恒定区载体pIgH(AbVec-hIgG1forVH)、pIgκ(AbVec-hIgKappa for Vκ)或pIgλ(IG-lambda expression vector for Vλ)连接，16℃酶连接过夜。
将连接好的重组质粒转化入TOP10大肠杆菌细胞中。第二天挑取阳性克隆菌落于含Amp+的LB培养基中，200rpm，37℃振荡培养12h后，用50％已灭菌的甘油保菌，抽提质粒(详见OMEGA质粒提取试剂盒说明书)。酶切验证重链VH-pIgH和轻链Vλ-pIgλ的重组质粒，PCR验证轻链Vκ-pIgκ的重组质粒。实验结果见图3。其中VH、VH’和Vλ都为酶切验证，Vκ为PCR验证。条带1到3分别为1号样品的VH、VH’和Vλ；条带4到6分别为2号样品VH、VH’和Vλ；条带7到9分别为3号样品VH、VH’和Vλ；条带10-12分别为1号、2号和3号样品的Vκ；条带13为阴性对照。
通过图3可知，我们成功地将重链基因和轻链基因构建到了其对应的表达载体pIgH，pIgκ和pIgλ上。
4.抗体重链可变区和轻链可变区基因的筛选
将上述重组质粒转化之后挑取单克隆质粒，将抗体重链某个质粒与抗体λ轻链或者抗体κ轻链(轻链有两种，为λ和κ；重链只有H这一种，H与λ或者κ两者的任何一种结合都能组成一种抗体)某个质粒共转染HEK293T细胞，采用酶联免疫吸附测定方法(Elisa法)检测EV71-VP1蛋白的特异性抗体分泌量，我们筛选到了高表达EV71-VP1特异性IgG抗体的抗体重组质粒组合。
细胞转染的具体步骤如下：
(1)接种细胞：将0.25％的胰酶-EDTA消化终止后的细胞，按每孔1×104个细胞接种96孔板，再补加培养液至190μL。同时设置对照组，对照组1为加转染试剂和细胞，但不加质粒；对照组2为只加细胞，不加转染试剂和质粒。
(2)用opti-MEM分别稀释质粒和转染试剂，使得加入每孔的转染试剂为0.15μL，每孔的质粒为100ng。
(3)按1：1的比例将稀释的质粒和稀释的转染试剂混匀，5min后将其加入到接种有细胞的孔中，37℃、5％的CO2培养箱中培养，48h后检测抗体含量。
(4)Elisa检测细胞培养上清IgG分泌量：收集步骤(3)中的转染后细胞培养上清，使用VP1蛋白包被酶标板，采用人IgG作为标准品，标准品对应的二抗为1:5000稀释的HRP标记的Goat Anti-Human IgG，待检测的培养基上清对应的二抗为1:10000稀释的Goat x-human IgG-Fc Fragment HRP Conjugated。最后加入显色液100μL/孔，室温避光显色10min；加入2mol/L硫酸50μL/孔，终止显色反应，检测OD值，参考波长为630nm。
将阳性质粒克隆送南京金斯瑞生物技术有限公司测序，测序引物为5＇GCTTCGTTAGAACGCGGCTAC3＇。通过以上方法，从EV71感染者体内分离到7条与EV71-VP1抗原特异性结合的重链序列和6条与EV71-VP1抗原特异性结合的轻链序列，7条重链可变区的核苷酸序列如SEQ ID NO：1-7所示；6条轻链可变区的核苷酸序列如SEQ ID NO：8-13所示；7条重链可变区的氨基酸序列如SEQ ID NO：14-20所示；6条轻链可变区的核苷酸序列如SEQ IDNO：21-26所示。
5.抗体蛋白表达及纯化
将筛选到的具有EV71-VP1抗原特异性的抗体重组质粒组合进行转染，大量表达抗体，并用Protein A beads(GE公司，Cat.NO.17075601)纯化，实验步骤如下：
(1)样品准备及结合：收集转染后的细胞培养上清，室温900g，离心10min，将上清吸到上述准备好的Beads中，混匀后，置于四维旋转混匀器，室温结合1-2h(或4℃过夜结合)
(2)洗涤：将结合后的beads置于室温离心后吸弃上清，每个管子中加入35mL的1MNaCl，室温2100g，离心10min吸弃上清。再用35mL的PBS冲洗beads，离心后弃上清；空离一次，再弃上清。
(3)洗脱：向管子中加入3-5mL的0.1M的甘氨酸盐酸盐(glycine-HCl)，置于四维旋转混匀器上混匀15min。室温2100g，离心10min，将上清转移至一个新的50mL离心管中。
(4)中和和浓缩：用1M的Tris-HCl调pH至7.4。将中和后的样品吸入超滤蛋白浓缩管中；加PBS至终体积为15mL。室温2100g，离心8-12min，直至浓缩管剩余液体体积为0.5-1mL。
将纯化之后的抗体样品进行SDS电泳检测，结果显示，各抗体样品的检测结果均正常，抗体重链蛋白大小在55KD左右，轻链蛋白大小在25KD左右。
本说明书示例性列举了重链14H1和轻链19k8组合的单克隆抗体体外表达纯化后SDS电泳结果，见图4。
6.抗体中和活性检测
使用微量细胞病变法检测抗体的中和活性。将表达纯化后的抗体进行2倍梯度稀释，置于96孔板中，50μL/孔；然后分别加入100CCID50的EV71-C4型病毒，50μL/孔，混匀后置于37℃，5％CO2培养箱中和2h；再加入100μL消化的RD细胞，10000个/孔；最后置于37℃、5％CO2温箱中孵育，5-7d记录病变情况。同时设置标准品板以及病毒回滴板。实验结果见图5。图中A为阴性对照，正常细胞状态图；B为抗体原液接种EV71感染的细胞状态图；C为抗体按照1:8比例稀释后接种EV71感染的细胞状态图；D为阳性对照，EV71感染细胞状态图。
通过试验发现，我们筛选到的抗体能够明显地抑制EV71病毒的复制，在浓度为0.05μg/ml时，7H1+23κ8组合对病毒的抑制率可达到90％，12H1+19κ8，14H1+19κ8，9H1+19κ8，23H1+19κ8和5H1+23κ8组合对病毒的抑制率可达到80％以上。
SEQUENCE LISTING
<110> 南昌大学
<120> 一种中和肠道病毒71型的全人源单克隆抗体及其用途
<130> GZ2018060244
<160> 26
<170> PatentIn version 3.3
<210> 1
<211> 368
<212> DNA
<213> 7H1重链可变区核苷酸序列
<400> 1
accggtgtac attctgaggt gcagctggtg gagtcggggg gaaccttggt acagcctggg 60
gggtccctga gactctcttg tgaagcctct ggattcacct ttagcaacta tgccatgggc 120
tgggtccgcc agactcaggg aaaggggctg gagtggctgt cggctattcg taaaagtggc 180
actaccacat actacgcgga ctccgtgaag ggccggttca tcatctccag agacaattcc 240
aagaacaccc tgtatctgca aatgaatagg ctgagagtcg gcgacacggc cacttattac 300
tgtgcgactc accccatcgc gggctactgg ggccagggaa ccacggtcac cgtctcctca 360
gcgtcgac 368
<210> 2
<211> 398
<212> DNA
<213> 12H1重链可变区核苷酸序列
<400> 2
accggtgtac attctgaagt gcagctggtg gagtctgggg gaggcgtggt ccagcctggg 60
aggtccctga gactctcctg tgcagcctct ggattcacct tcagtagcta tgctatgcac 120
tgggtccgcc aggctccagg caaggggctg gagtgggtgg cagttatatc atatgatgga 180
agcaataaat actacgcaga ctccgtgaag ggccgattca ccatctccag agacaattcc 240
aagaacacgc tgtatctgca aatgaacagc ctgagagatg aggacacggc tgtgtattac 300
tgtgcgagag ctcccgattg tagtggtggt agctgctcca ctgaatactt ccagcactgg 360
ggccagggca ccacggtcac cgtctcctca gcgtcgac 398
<210> 3
<211> 368
<212> DNA
<213> 14H1重链可变区核苷酸序列
<400> 3
accggtgtac attccgaggt gcagctggtg cagtcggggg gaaccttggt acagcctggg 60
gggtccctga gactctcttg tgaagcctct ggattcacct ttagcaacta tgccatgggc 120
tgggtccgcc agactccagg aaaggggctg gagtggctgt cggctattcg taaaagtggc 180
actaccacat actacgcgga ctccgtgaag ggccggttca tcatctccag agacaactcc 240
aagaacaccc tgtatctgca aatgaatagg ctgagagtcg gcgacacggc cacttattac 300
tgtgcgactc accccatcgc gggctactgg ggccagggaa ccacggtcac cgtctcctca 360
gcgtcgac 368
<210> 4
<211> 368
<212> DNA
<213> 9H1重链可变区核苷酸序列
<400> 4
accggtgtac attcccaggt gcagctgcag gagtcggggg gaaccttggt acagcctggg 60
gggtccctga gactctcttg tgaagcctct ggattcacct ttagcaacta tgccatgggc 120
tgggtccgcc agactccagg aaaggggctg gagtggctgt cggctattcg taaaagtggc 180
actaccacat actacgcgga ctccgtgaag ggccggttca tcatctccag agacaattcc 240
aagaacaccc tgtatctgca aatgaatagg ctgagagtcg gcgacacggc cacctattac 300
tgtgcgactc accccatcgc gggctactgg ggccagggaa ccacggtcac cgtctcctca 360
gcgtcgac 368
<210> 5
<211> 345
<212> DNA
<213> 23H1重链可变区核苷酸序列表
<400> 5
accggtgtac attgtgccat ccggatgacc cagtctccag ccaccctgtc tgtgtctcca 60
ggggaaagag ccaccctctc ctgcagggcc agtcagagtg ttagcagcaa cttagcctgg 120
taccggcaga aacctggcca ggctcccagg ctcctcatct atggtgcatc caccagggcc 180
actggtatcc cagccaggtt cagtggcagt gggtctggga cagagttcac tctcaccatc 240
agcagcctgc agtctgaaga ttttgcagtt tattactgtc agcagtataa taactggcct 300
ccgtggacgt tcggccaagg gaccaaggtg gagatcaaac gtacg 345
<210> 6
<211> 398
<212> DNA
<213> 5H1重链可变区核苷酸序列
<400> 6
accggtgtac attcccaggt ccagctggta cagtctgggg gaggcgtggt ccagcctggg 60
aggtccctga gactctcctg tgcagcctct ggattcacct tcagtagcta tgctatgcac 120
tgggtccgcc aggctccagg caaggggctg gagtgggtgg cagttatatc atatgatgga 180
agcaataaat actacgcaga ctccgtgaag ggccgattca ccatctccag agacaattcc 240
aagaacacgc tgtatctgca aatgaacagc ctgagagatg aggacacggc tgtgtattac 300
tgtgcgagag ctcctgattg tagtggtggt agctgctcca ctgaatactt ccagcactgg 360
ggccagggca ccacggtcac cgtctcctca gcgtcgac 398
<210> 7
<211> 368
<212> DNA
<213> 8H1重链可变区核苷酸序列
<400> 7
accggtgtac attcccaggt ccagctggta cagtcggggg gaaccttggt acagcctggg 60
gggtccctga gactctcttg tgaagcctct ggattcacct ttagcaacta tgccatgggc 120
tgggtccgcc agactccagg aaaggggctg gagtggctgt cggctattcg taaaagtggc 180
actaccacat actacgcgga ctccgtgaag ggccggttca tcatctccag agacaattcc 240
aagaacaccc tgtatctgca aatgaatagg ctgagagtcg gcgacacggc cacttattac 300
tgtgcgactc accccatcgc gggctactgg ggccagggaa ccacggtcac cgtctcctca 360
gcgtcgac 368
<210> 8
<211> 368
<212> DNA
<213> 23K8轻链（κ链）可变区核苷酸序列
<400> 8
accggtgtac attccgaggt gcagctggtg cagtcggggg gaaccttggt acagcctggg 60
gggtccctga gactctcttg tgaagcctct ggattcacct ttagcaacta tgccatgggc 120
tgggtccgcc agactccagg aaaggggctg gagtggctgt cggctattcg taaaagtggc 180
actaccacat actacgcgga ctccgtgaag ggccggttca tcatctccag agacaattcc 240
aagaacaccc tgtatctgca aatgaatagg ctgagggtcg gcgacacggc cacttattac 300
tgtgcgactc accccatcgc gggctactgg ggccagggaa ccacggtcac cgtctcctca 360
gcgtcgac 368
<210> 9
<211> 345
<212> DNA
<213> 19K8轻链（κ链）可变区核苷酸序列
<400> 9
accggtgtac attgtgccat ccggatgacc cagtctccag ccaccctgtc tgtgtctcca 60
ggggaaagag ccaccctctc ctgcagggcc agtcagagtg ttagcagcaa cttagcctgg 120
taccggcaga aacctggcca ggctcccagg ctcctcatct atggtgcatc caccagggcc 180
actggtatcc cagccaggtt cagtggcagt gggtctggga cagagttcac tctcaccatc 240
agcagcctgc agtctgaaga ttttgcagtt tattactgtc agcagtataa taactggcct 300
ccgtggacgt tcggccaagg gaccaaagtg gatatcaaac gtacg 345
<210> 10
<211> 342
<212> DNA
<213> 12K8轻链（κ链）可变区核苷酸序列
<400> 10
accggtgtac atggggatat tgtgatgact cagtctccat cctccctgtc tgcatctgta 60
ggagacagag tcaccatcac ttgccgggtg agtcagggca ttagcagtta tttaaattgg 120
tatcggcaga aaccagggaa agttcctaag ctcctgatct atagtgcatc caatttgcaa 180
tctggagtcc catctcggtt cagtggcagt ggatctggga cagatttcac tctcactatc 240
agcagcctgc agcctgaaga tgttgcaact tattacggtc aacggactta caatgccctg 300
gggactttcg gccctgggac caaggtggaa atcaaacgta cg 342
<210> 11
<211> 342
<212> DNA
<213> 13K8轻链（κ链）可变区核苷酸序列
<400> 11
accggtgtac attctgacat ccagatgacc cagtctccat cttccgtgtc tgcatctgta 60
ggagacagag tcaccatcac ttgtcgggcg agtcagggta ttagcagctg gttagcctgg 120
tatcagcaga aaccagggaa agcccctaag ctcctgatct atgctgcatc cagtttgcaa 180
agtggggtcc catcaaggtt cagcggcagt ggatctggga cagatttcac tctcaccatc 240
agcagcctgc agcctgaaga ttttgcaact tactattgtc aacaggctaa cagtttcctc 300
ggaactttcg gccctgggac caaagtggat atcaaacgta cg 342
<210> 12
<211> 165
<212> DNA
<213> 8K8轻链（κ链）可变区核苷酸序列
<400> 12
accggtgtac attgtgccat ccggaaccca gtctcaaaag aacacagatg atatgcactc 60
actgataagt gaatattagc ccagaaactt agaataccca agatacaatt tgcaaaacac 120
atgaaactca taaagaagga agaccaaagt ggatatcaaa cgtac 165
<210> 13
<211> 144
<212> DNA
<213> 22k8轻链（κ链）可变区核苷酸序列
<400> 13
accggtgtac attcagaaat agtgatgacg cagtctcctc cggaaagaaa agacaccact 60
ctcctgcact ttgcacaaaa tgtatttctg tgacatcctg aagtacagac atttccccag 120
accaaagtgg atatcaaacg tacg 144
<210> 14
<211> 122
<212> PRT
<213> 7H1重链可变区氨基酸序列
<400> 14
Thr Gly Val His Ser Glu Val Gln Leu Val Glu Ser Gly Gly Thr Leu
1 5 10 15
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Phe
20 25 30
Thr Phe Ser Asn Tyr Ala Met Gly Trp Val Arg Gln Thr Gln Gly Lys
35 40 45
Gly Leu Glu Trp Leu Ser Ala Ile Arg Lys Ser Gly Thr Thr Thr Tyr
50 55 60
Tyr Ala Asp Ser Val Lys Gly Arg Phe Ile Ile Ser Arg Asp Asn Ser
65 70 75 80
Lys Asn Thr Leu Tyr Leu Gln Met Asn Arg Leu Arg Val Gly Asp Thr
85 90 95
Ala Thr Tyr Tyr Cys Ala Thr His Pro Ile Ala Gly Tyr Trp Gly Gln
100 105 110
Gly Thr Thr Val Thr Val Ser Ser Ala Ser
115 120
<210> 15
<211> 132
<212> PRT
<213> 12H1重链可变区氨基酸序列
<400> 15
Thr Gly Val His Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val
1 5 10 15
Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
20 25 30
Thr Phe Ser Ser Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Lys
35 40 45
Gly Leu Glu Trp Val Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr
50 55 60
Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser
65 70 75 80
Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr
85 90 95
Ala Val Tyr Tyr Cys Ala Arg Ala Pro Asp Cys Ser Gly Gly Ser Cys
100 105 110
Ser Thr Glu Tyr Phe Gln His Trp Gly Gln Gly Thr Thr Val Thr Val
115 120 125
Ser Ser Ala Ser
130
<210> 16
<211> 122
<212> PRT
<213> 14H1重链可变区氨基酸序列
<400> 16
Thr Gly Val His Ser Glu Val Gln Leu Val Gln Ser Gly Gly Thr Leu
1 5 10 15
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Phe
20 25 30
Thr Phe Ser Asn Tyr Ala Met Gly Trp Val Arg Gln Thr Pro Gly Lys
35 40 45
Gly Leu Glu Trp Leu Ser Ala Ile Arg Lys Ser Gly Thr Thr Thr Tyr
50 55 60
Tyr Ala Asp Ser Val Lys Gly Arg Phe Ile Ile Ser Arg Asp Asn Ser
65 70 75 80
Lys Asn Thr Leu Tyr Leu Gln Met Asn Arg Leu Arg Val Gly Asp Thr
85 90 95
Ala Thr Tyr Tyr Cys Ala Thr His Pro Ile Ala Gly Tyr Trp Gly Gln
100 105 110
Gly Thr Thr Val Thr Val Ser Ser Ala Ser
115 120
<210> 17
<211> 122
<212> PRT
<213> 9H1重链可变区氨基酸序列
<400> 17
Thr Gly Val His Ser Gln Val Gln Leu Gln Glu Ser Gly Gly Thr Leu
1 5 10 15
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Phe
20 25 30
Thr Phe Ser Asn Tyr Ala Met Gly Trp Val Arg Gln Thr Pro Gly Lys
35 40 45
Gly Leu Glu Trp Leu Ser Ala Ile Arg Lys Ser Gly Thr Thr Thr Tyr
50 55 60
Tyr Ala Asp Ser Val Lys Gly Arg Phe Ile Ile Ser Arg Asp Asn Ser
65 70 75 80
Lys Asn Thr Leu Tyr Leu Gln Met Asn Arg Leu Arg Val Gly Asp Thr
85 90 95
Ala Thr Tyr Tyr Cys Ala Thr His Pro Ile Ala Gly Tyr Trp Gly Gln
100 105 110
Gly Thr Thr Val Thr Val Ser Ser Ala Ser
115 120
<210> 18
<211> 115
<212> PRT
<213> 23H1重链可变区氨基酸序列
<400> 18
Thr Gly Val His Cys Ala Ile Arg Met Thr Gln Ser Pro Ala Thr Leu
1 5 10 15
Ser Val Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln
20 25 30
Ser Val Ser Ser Asn Leu Ala Trp Tyr Arg Gln Lys Pro Gly Gln Ala
35 40 45
Pro Arg Leu Leu Ile Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro
50 55 60
Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile
65 70 75 80
Ser Ser Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr
85 90 95
Asn Asn Trp Pro Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
100 105 110
Lys Arg Thr
115
<210> 19
<211> 132
<212> PRT
<213> 5H1重链可变区氨基酸序列
<400> 19
Thr Gly Val His Ser Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val
1 5 10 15
Val Gln Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
20 25 30
Thr Phe Ser Ser Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Lys
35 40 45
Gly Leu Glu Trp Val Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr
50 55 60
Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser
65 70 75 80
Lys Asn Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr
85 90 95
Ala Val Tyr Tyr Cys Ala Arg Ala Pro Asp Cys Ser Gly Gly Ser Cys
100 105 110
Ser Thr Glu Tyr Phe Gln His Trp Gly Gln Gly Thr Thr Val Thr Val
115 120 125
Ser Ser Ala Ser
130
<210> 20
<211> 122
<212> PRT
<213> 8H1重链可变区氨基酸序列
<400> 20
Thr Gly Val His Ser Gln Val Gln Leu Val Gln Ser Gly Gly Thr Leu
1 5 10 15
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Phe
20 25 30
Thr Phe Ser Asn Tyr Ala Met Gly Trp Val Arg Gln Thr Pro Gly Lys
35 40 45
Gly Leu Glu Trp Leu Ser Ala Ile Arg Lys Ser Gly Thr Thr Thr Tyr
50 55 60
Tyr Ala Asp Ser Val Lys Gly Arg Phe Ile Ile Ser Arg Asp Asn Ser
65 70 75 80
Lys Asn Thr Leu Tyr Leu Gln Met Asn Arg Leu Arg Val Gly Asp Thr
85 90 95
Ala Thr Tyr Tyr Cys Ala Thr His Pro Ile Ala Gly Tyr Trp Gly Gln
100 105 110
Gly Thr Thr Val Thr Val Ser Ser Ala Ser
115 120
<210> 21
<211> 122
<212> PRT
<213> 23K8 轻链（κ链）可变区氨基酸序列
<400> 21
Thr Gly Val His Ser Glu Val Gln Leu Val Gln Ser Gly Gly Thr Leu
1 5 10 15
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Glu Ala Ser Gly Phe
20 25 30
Thr Phe Ser Asn Tyr Ala Met Gly Trp Val Arg Gln Thr Pro Gly Lys
35 40 45
Gly Leu Glu Trp Leu Ser Ala Ile Arg Lys Ser Gly Thr Thr Thr Tyr
50 55 60
Tyr Ala Asp Ser Val Lys Gly Arg Phe Ile Ile Ser Arg Asp Asn Ser
65 70 75 80
Lys Asn Thr Leu Tyr Leu Gln Met Asn Arg Leu Arg Val Gly Asp Thr
85 90 95
Ala Thr Tyr Tyr Cys Ala Thr His Pro Ile Ala Gly Tyr Trp Gly Gln
100 105 110
Gly Thr Thr Val Thr Val Ser Ser Ala Ser
115 120
<210> 22
<211> 115
<212> PRT
<213> 19K8轻链（κ链）可变区氨基酸序列
<400> 22
Thr Gly Val His Cys Ala Ile Arg Met Thr Gln Ser Pro Ala Thr Leu
1 5 10 15
Ser Val Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln
20 25 30
Ser Val Ser Ser Asn Leu Ala Trp Tyr Arg Gln Lys Pro Gly Gln Ala
35 40 45
Pro Arg Leu Leu Ile Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro
50 55 60
Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile
65 70 75 80
Ser Ser Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr
85 90 95
Asn Asn Trp Pro Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Asp Ile
100 105 110
Lys Arg Thr
115
<210> 23
<211> 114
<212> PRT
<213> 12k8轻链（κ链）可变区氨基酸序列
<400> 23
Thr Gly Val His Gly Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu
1 5 10 15
Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Val Ser Gln
20 25 30
Gly Ile Ser Ser Tyr Leu Asn Trp Tyr Arg Gln Lys Pro Gly Lys Val
35 40 45
Pro Lys Leu Leu Ile Tyr Ser Ala Ser Asn Leu Gln Ser Gly Val Pro
50 55 60
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
65 70 75 80
Ser Ser Leu Gln Pro Glu Asp Val Ala Thr Tyr Tyr Gly Gln Arg Thr
85 90 95
Tyr Asn Ala Leu Gly Thr Phe Gly Pro Gly Thr Lys Val Glu Ile Lys
100 105 110
Arg Thr
<210> 24
<211> 114
<212> PRT
<213> 13K8轻链（κ链）可变区氨基酸序列
<400> 24
Thr Gly Val His Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val
1 5 10 15
Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln
20 25 30
Gly Ile Ser Ser Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala
35 40 45
Pro Lys Leu Leu Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro
50 55 60
Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
65 70 75 80
Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala
85 90 95
Asn Ser Phe Leu Gly Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys
100 105 110
Arg Thr
<210> 25
<211> 53
<212> PRT
<213> 8K8轻链（κ链）可变区氨基酸序列
<400> 25
Thr Gly Val His Cys Ala Ile Arg Asn Pro Val Ser Lys Glu His Arg
1 5 10 15
Tyr Ala Leu Thr Asp Lys Ile Leu Ala Gln Lys Leu Arg Ile Pro Lys
20 25 30
Ile Gln Phe Ala Lys His Met Lys Leu Ile Lys Lys Glu Asp Gln Ser
35 40 45
Gly Tyr Gln Thr Tyr
50
<210> 26
<211> 47
<212> PRT
<213> 22k8轻链（κ链）可变区氨基酸序列
<400> 26
Thr Gly Val His Ser Glu Ile Val Met Thr Gln Ser Pro Pro Glu Arg
1 5 10 15
Lys Asp Thr Thr Leu Leu His Phe Ala Gln Asn Val Phe Leu His Pro
20 25 30
Glu Val Gln Thr Phe Pro Gln Thr Lys Val Asp Ile Lys Arg Thr
35 40 45
Claims (8)
1.一种中和肠道病毒71型的全人源单克隆抗体，其特征在于，其具有如下任一组重链可变区和轻链可变区：
（i）重链可变区的氨基酸序列如SEQ ID NO:14所示；轻链可变区的氨基酸序列如SEQID NO:21所示；
（ii）重链可变区的氨基酸序列如SEQ ID NO:15所示；轻链可变区的氨基酸序列如SEQID NO:22所示；
（iii）重链可变区的氨基酸序列如SEQ ID NO:16所示；轻链可变区的氨基酸序列如SEQID NO:22所示；
（iv）重链可变区的氨基酸序列如SEQ ID NO:17所示；轻链可变区的氨基酸序列如SEQID NO:22所示；
（v）重链可变区的氨基酸序列如SEQ ID NO:18所示；轻链可变区的氨基酸序列如SEQID NO:22所示；
（vi）重链可变区的氨基酸序列如SEQ ID NO:19所示；轻链可变区的氨基酸序列如SEQID NO:21所示。
2.编码如权利要求1所述单克隆抗体的核酸分子。
3.根据权利要求2所述的核酸分子，其特征在于，其具有如下任一组重链可变区和轻链可变区：
（i）重链可变区的核苷酸序列如SEQ ID NO:1所示；轻链可变区的核苷酸序列如SEQ IDNO:8所示；
（ii）重链可变区的核苷酸序列如SEQ ID NO:2所示；轻链可变区的核苷酸序列如SEQID NO:9所示；
（iii）重链可变区的核苷酸序列如SEQ ID NO:3所示；轻链可变区的核苷酸序列如SEQID NO:9所示；
（iv）重链可变区的核苷酸序列如SEQ ID NO:4所示；轻链可变区的核苷酸序列如SEQID NO:9所示；
（v）重链可变区的核苷酸序列如SEQ ID NO:5所示；轻链可变区的核苷酸序列如SEQ IDNO:9所示；
（vi）重链可变区的核苷酸序列如SEQ ID NO:6所示；轻链可变区的核苷酸序列如SEQID NO:8所示。
4.一种表达载体，其特征在于，包括权利要求2或3所述核酸分子。
5.一种宿主细胞，其特征在于，包括权利要求4所述的表达载体。
6.权利要求1所述的单克隆抗体或其结合片段在制备用于检测、治疗或者预防肠道71型病毒感染的药物中的用途。
7.一种治疗或者预防肠道71型病毒感染的药物组合物，其特征在于，包括治疗有效量的权利要求1所述的单克隆抗体中的一种或多种抗体组成的抗体混合物，以及药学上可接受的载体。
8.一种检测肠道病毒71型的试剂盒，其特征在于，包括权利要求1所述的单克隆抗体。
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