CN114920835B - 一种高亲和力抗狂犬病病毒的全人源单克隆抗体及其用途 - Google Patents
一种高亲和力抗狂犬病病毒的全人源单克隆抗体及其用途 Download PDFInfo
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Abstract
本发明涉及一种高亲和力抗狂犬病病毒的全人源单克隆抗体，属于生物免疫学领域。所述的单克隆抗体或其结合片段的重链可变区具有具有如SEQ ID NO:17所示的重链可变区的氨基酸序列；和如SEQ ID NO:18所示的轻链可变区的氨基酸序列。本发明还提供该抗体的编码基因、表达载体、用途和组合物。狂犬病毒糖蛋白(Glycoprotein，GP)是该病毒表面唯一的保护性抗原蛋白。本发明成功构建了狂犬病毒特异性抗原蛋白的原核表达载体，并利用大肠杆菌原核系统对其进行了表达和纯化，该抗原蛋白已成功用于抗体的筛选。并且已利用我们的筛选平台成功地筛选到了1株针对狂犬病毒GP片段的特异性全人源单克隆抗体序列，为临床上开发治疗狂犬病的抗体药物奠定了基础。
Description
技术领域
本发明涉及生物免疫学领域，具体地说，涉及一种高亲和力抗狂犬病病毒的全人源单克隆抗体及其用途。
背景技术
狂犬病(rabies)主要是由狂犬病毒(Rabies virus，RABV)引起的严重侵害中枢神经系统的急性传染病，在我国传染病分类中为乙类传染病。世界上最早关于狂犬病的资料记载始于4300年前的美索不达米亚，我国关于狂犬病的文字记载最早出现在距今约2500年的《左传》中。许多研究认为狂犬病起源于亚洲或欧洲，18世纪或由欧洲传入西半球，19世首次在南美洲发现。目前，狂犬病存在于除南极洲外的其他各个大洲，覆盖100多个国家。该病毒主要通过破损的皮肤、黏膜或呼吸道感染机体，唾液是其主要传染途径之一。几乎所有的哺乳类温热动物，如浣熊、臭鼬、蝙蝠、狐狸都是RABV宿主，主要传染源是患病或携带病毒的犬类[7]。大多数人类狂犬病主要是被患病动物咬伤所导致，部分患者是被抓挠或伤口、黏膜等污染所引起，鲜有因移植该病患者捐赠的器官或组织而发病的案例。RABV主要作用于中枢神经系统，大量存在于易感动物的脑脊液。人狂犬病主要表现为急性进展性脑脊髓炎，发病时通常有两种形式：以间接性的兴奋及幻觉伴随的狂躁型狂犬病，同时伴有恐风、恐水、恐声等症状，患者通常在7天内死亡，此外还有以肌无力为特征的麻痹型狂犬病，通常14天内导致死亡。该病潜伏期一般为3周至3个月，10天以内或长达1年及以上的报道极为少见[9]。
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。当前国内外狂犬单抗市场空缺较大，国内华北制药研发的重组人源狂犬抗体NM57s/NC08注射液于2018年已进入III期临床试验阶段，并于2020年4月取得III期临床试验报告，该单抗是由两种单抗NM57S、NC08联合所得的组合制剂，需与疫苗联合使用。国外市场方面，Zydus cadila研发的单抗RabiMabs于2019年5月被美国食品药品监督管理局(Food and Drug Administration，FDA)授予预防狂犬病的孤儿药资格，并于同年9月3日获得印度药品管理总局(Drugs Controller General of India，DCGI)批准，可与疫苗联合用于狂犬病暴露后预防。印度血清研究所和Mass Biologics共同研发的重组抗RABV单抗注射液Rabishield已于2016年12月在印度批准上市。全球市场上尚无用于防治狂犬病的全人源单抗，该类产品一旦上市并规模化生产，将极大地降低狂犬病的防治成本，具有传统血液制品和疫苗不可比拟的竞争优势。所以，我们希望通过本实验室特有的人记忆性B细胞体外活化后特异筛选法筛选到一株甚至多株广谱的具有治疗作用的抗狂犬病全人源单克隆抗体，改善狂犬病防治困难的现状。
发明内容
本发明人经过深入的研究，获得一种具有广谱结合狂犬病病毒的全人源单克隆抗体，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。
1861年狂犬病病毒基因首次被报道，其是单股不分节的负链RNA病毒，其外形类似子弹状，直径约为75nm，长度为100～300nm，约为12kb。病毒RNA编码5个蛋白，依次是核衣壳蛋白(N)、磷蛋白(P)、基质蛋白(M)、糖蛋白(G)、聚合酶大蛋白(L)。本发明选用G蛋白作为抗原表位，G蛋白由1575个核苷酸组成，是唯一位于病毒表面的蛋白，能与细胞内受体乙酰胆碱受体结合使病毒具有嗜神经性，也能诱导宿主产生中和抗体，具有保护作用。
本发明的一个方面在于提供一种抗狂犬病病毒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具有SEQ ID NO:11、13、15、17和19任一项所示的重链可变区的核苷酸序列；或具有SEQ ID NO:11、13、15、17和19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具有如SEQ ID NO:12、14、16、18和20任一项所示的轻链可变区的核苷酸序列；或具有SEQ ID NO:12、14、16、18和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。
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:11所示(1H10)；轻链可变区的氨基酸序列如SEQ ID NO:12所示(3λ5)；
(ii)重链可变区的氨基酸序列如SEQ ID NO:13所示(1H16)；轻链可变区的氨基酸序列如SEQ ID NO:14所示(3λ7)；
(iii)重链可变区的氨基酸序列如SEQ ID NO:15所示(1H17)；轻链可变区的氨基酸序列如SEQ ID NO:16所示(3λ8)；
(iv)重链可变区的氨基酸序列如SEQ ID NO:17所示(1H3)；轻链可变区的氨基酸序列如SEQ ID NO:18所示(3λ1)；
(v)重链可变区的氨基酸序列如SEQ ID NO:19所示(1H18)；轻链可变区的氨基酸序列如SEQ ID NO:20所示(3λ10)。
本发明提供的上述的单克隆抗体，能够特异性结合狂犬病病毒G蛋白。
在本发明的另一方面，本发明还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、3、5、7和9任一项所示的重链可变区的核苷酸序列；和如SEQ ID NO:2、4、6、8和10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(1H10)；轻链可变区的核苷酸序列如SEQ ID NO:2所示(3λ5)；
(ii)重链可变区的核苷酸序列如SEQ ID NO:3所示(1H16)；轻链可变区的核苷酸序列如SEQ ID NO:4所示(3λ7)；
(iii)重链可变区的核苷酸序列如SEQ ID NO:5所示(1H17)；轻链可变区的核苷酸序列如SEQ ID NO:6所示(3λ8)；
(iv)重链可变区的核苷酸序列如SEQ ID NO:7所示(1H3)；轻链可变区的核苷酸序列如SEQ ID NO:8所示(3λ1)；
(v)重链可变区的核苷酸序列如SEQ ID NO:9所示(1H18)；轻链可变区的核苷酸序列如SEQ ID NO:10所示(3λ10)。
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，提供上述单克隆抗体的制备方法，包括以下步骤：
(1)记忆B细胞分选：从已感染狂犬病病毒的志愿者的血液样品中分离外周血单核细胞，从外周血单核细胞中分选得到记忆性B细胞；
(2)记忆B细胞的体外活化培养及阳性孔筛选：将记忆性B细胞活化为浆细胞，ELISA检测记忆性B细胞体外活化成浆细胞后分泌性的IgG抗体，筛选获得阳性孔；
(3)抗体可变区基因文库的构建及筛选：对阳性孔细胞进行反转录得到cDNA，扩增抗体重链可变区和轻链可变区基因；
(4)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和轻链可变区基因分别连接到表达载体上；
(5)抗体重链可变区和轻链可变区基因的筛选：将重组质粒共转染HEK293T细胞进行表达。
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、预防狂犬病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狂犬病病毒具有结合活性的单克隆抗体或其片段。
在本发明的另一方面，提供一种检测狂犬病病毒的试剂盒，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、快速且准确地检测狂犬病病毒的试剂盒。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狂犬病病毒的试剂。
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狂犬病毒糖蛋白(Glycoprotein，GP)，从而得知待测样品的供体是否感染狂犬病毒，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狂犬病病毒的方法，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狂犬病病毒的方法，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，获得狂犬病病毒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。
有益效果：与现有技术相比，本发明成功制备了抗狂犬病毒的全人源单克隆抗体，该单克隆抗体具有高亲和力的特点，所述单克隆抗体能特异性结合狂犬病毒，能显著抗狂犬病毒。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狂犬病毒的大分子药物，为此类抗体的标准化生产成药提供了很大的保障。
附图说明
图1阳性孔细胞筛选。A：Elisa筛选记忆B细胞阳性孔，红色为用于质粒构建的阳性孔细胞；B：样本琼脂糖电泳检测，M为DNA分子标准。
图2样本抗体重链VH、VH’与轻链VK、Vλ可变区基因文库扩增电泳结果；M为DNA分子标准。
图3抗体重轻链可变区基因文库的构建。A：样本目的质粒及载体质粒酶切电泳结果；B：K、IgK第二次酶切电泳结果；C：重组质粒TOP10转化结果；D：重组质粒酶切电泳鉴定结果，M为DNA分子标准。
图4为抗体重轻链可变区基因组合筛选结果。
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：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
从已感染狂犬病病毒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
我们采集了2名血清中RABV IgG抗体表达较高的志愿者新鲜全血50ml，利用磁珠分选法分离记忆B细胞，将其与饲养层细胞共同培养于U型96孔板中，培养10天后用Elisa检测上清中抗体的表达，结果见表1(图1A为其量化图)，从中筛选出3个OD值最高的阳性细胞孔依次编号为Ⅰ、Ⅱ、Ⅲ(分别含有记忆B细胞个数为10个、50个、100个)提取RNA，反转录为cDNA后以GAPDH为引物进行验证，结果如图1B，单独的正常3T3细胞无条带，阳性孔Ⅰ、Ⅱ、Ⅲ号样本均在250bp处条带明显，表明我们所筛选的分泌抗体阳性孔细胞RNA提取成功，可进行后期的质粒构建。
表1Elisa筛选分泌抗狂犬病毒抗体阳性孔细胞
表1.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。
3.抗体可变区基因文库的构建及筛选
我们将Ⅰ、Ⅱ、Ⅲ号样本提取每孔细胞RNA，用巢式PCR两轮扩增法扩增抗体的重链VH、轻链VK、Vλ可变区，由于重链可变区分子量大，氨基酸序列较长，我们将其分为VH、VH’两部分分别进行扩增。通过琼脂糖凝胶电泳检测扩增结果，进行胶回收，获得扩增后的抗体重链VH、VH’及轻链VK、Vλ可变区片段的DNA。电泳结果如图2，抗体重链VH、VH’及轻链VK、Vλ可变区在400bp处均有明显条带，符合预期。
3.4.2抗体重轻链可变区基因文库的构建
将扩增的重链VH、VH’可变区基因，轻链VK、Vλ可变区基因及恒定区载体质粒IgH、IgK、Igλ按照酶切试剂盒说明进行酶切，电泳结果如图3A和3B所示，目的基因在400bp处有明显条带，载体质粒在5000bp有明显条带，结果符合预期。随后将目的质粒与载体质粒进行酶连反应，将重组质粒经TOP10细胞转化，结果如图3C，对照板无菌落生长，各实验组长满菌落。小提质粒后将重组质粒酶切进行鉴定，结果如图3D，我们前期筛选的重组质粒Ⅰ、Ⅱ、Ⅲ号样本经酶切后得到分子量大小不同的两条带，其中目的质粒重链VH、VH’及轻链VK、Vλ在400bp条带处均有明显条带，载体质粒IgH、IgK、Igλ在5000bp处有明显条带，结果符合预期，说明抗体重轻链可变区基因文库重组质粒构建成功。
3.5单克隆抗体的筛选
我们从重轻链各自总质粒文库中筛选单克隆抗体序列。①：将筛选的3个样品重轻链组合ⅠH+ⅠK、ⅠH+Ⅰλ、ⅡH+ⅡK、ⅡH+Ⅱλ、ⅢH+ⅢK、ⅢH+Ⅲλ依次转染HEK239T细胞，筛选出分泌特异性抗体的质粒组合②：根据OD值筛选出ⅢH、Ⅲλ，将其转化后平均分为8份，提取质粒依次编号为1H、2H、3H.......8H和1λ、2λ、3λ.......8λ，分别将ⅢH总与1λ、2λ、3λ.......8λ，Ⅲλ总与1H、2H、3H.......8H，这16个组合依次转染HEK239T细胞筛选出分泌特异性抗体的质粒组合。③：我们筛选出ⅢH、Ⅲλ8小份中的1H和3λ，取20ng质粒进行转化，分别挑选单克隆菌落30份，依次编号为1H1、1H2、1H3.......1H30和3λ1、3λ2、3λ3.......3λ30，同样将1H与3λ1、3λ2、3λ3.......3λ30，3λ与1H1、1H2、1H3.......1H30组合转染HEK239T细胞筛选出分泌特异性抗体的质粒组合。④：我们从中挑选出OD值较高的10个重链单克隆质粒：1H3、1H10、1H17、1H18、1H21、1H23、1H24、1H27、1H28、1H9与10个轻链单克隆质粒：3λ2、3λ6、3λ7、3λ8、3λ10、3λ11、3λ14、3λ15、3λ20、3λ29。将这些重轻链单克隆质粒两两组合，并将这100个质粒组合依次转染HEK293T细胞筛选出分泌特异性抗体的单克隆质粒组合，Elisa检测细胞上清中抗体表达情况。表2(图4为量化图)为100个组合中OD较高的前20名质粒组合Elisa检测结果。
表2Elisa筛选单克隆抗体重轻链组合质粒
筛选得到重轻链单克隆序列各挑选5条进行“一对一”配对体外转染表达，5条重链单克隆序列：1H10,1H16,1H17,1H3,1H218和5条轻链单克隆序列：3λ5，3λ7,3λ8,3λ1,3λ10，ELISA筛选后最终得到5株高亲和力的单克隆抗体，抗体组合：1H10+3λ5、1H16+3λ7、1H17+3λ8、1H3+3λ1、1H18+3λ10(结果对应图4)。这五个质粒组合的OD值在1.1以上，具有非常好的亲和力。
前述对本发明的具体示例性实施方案的描述是为了说明和例证的目的。这些描述并非想将本发明限定为所公开的精确形式，并且很显然，根据上述教导，可以进行很多改变和变化。对示例性实施例进行选择和描述的目的在于解释本发明的特定原理及其实际应用，从而使得本领域的技术人员能够实现并利用本发明的各种不同的示例性实施方案以及各种不同的选择和改变。本发明的范围意在由权利要求书及其等同形式所限定。
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gtcactctca cctgtggctc cagtactgga gctgtcacca gtggtcttta tgcctactgg 120
ttccagcaga agcctggcca agtcccccgg acactgattt ataatataag caacaaacag 180
tcctggaccc ctgcccggtt ctcaggctcc ctccttgggg gcaaagctgc cctgaccctt 240
tcgggtcctg aggatgaggc tgactattac tgcttgctct cctatagtcg tactcgggtg 300
ttcggcggag ggaccaagct gaccgtccta ggtcagccca aggctgcccc ctcggtcact 360
ctgttcccgc c 371
<210> 7
<211> 383
<212> DNA
<213> 1H3重链可变区核苷酸序列
<400> 7
gtacattctc aggtgcagct ggtggagtct gggggaggcg tggtccagcc tgggaggtcc 60
ctgagactct cctgtgcagc ctctggattc accttcagta gctatgctat gcactgggtc 120
cgccaggctc caggcaaggg gctggagtgg gtggcagtta tatcatatga tggaaataaa 180
tactacgcag actccgtgaa gggccgattc accatctcca gagacaattc caagaacacg 240
ctgtatctgc aaatgaacag cctgagagat gaggacacgg ctgtgtatta ctgtgcgaga 300
gctcctgatt gtagtggtgg tagctgctcc actgaatact tccagcactg gggccagggc 360
accctggtca ccgtctcctc agc 383
<210> 8
<211> 371
<212> DNA
<213> 3λ1轻链可变区核苷酸序列
<400> 8
tcctgggccc agtctgtgct gacgcaggag ccctcactga ctgtgtcccc aggagggaca 60
gtcactctca cctgtggctc cagtactgga gctgtcacca gtggtcttta tgcctactgg 120
ttccagcaga agcctggcca agtcccccgg acactgattt ataatataag caacaaacag 180
tcctggaccc ctgcccggtt ctcaggctcc ctccttgggg gcaaagctgc cctgaccctt 240
tcgggtgcgc agcctgagga tgaggctgac tattactgct tgctctccta tagtcgtact 300
cgggtgttcg ggaccaagct gaccgtccta ggtcagccca aggctgcccc ctcggtcact 360
ctgttcccac c 371
<210> 9
<211> 368
<212> DNA
<213> 1H18重链可变区核苷酸序列
<400> 9
gtacattccg aggtgcagct ggtgcagtct gggggaggct tggtccagcc tggggggtcc 60
ctgagactct cctgtgcagc ctctggattc acctttagta catattggat gaactgggtc 120
cgcctctccc agactccagg gaaggggctg gagtgggtgg ccaacataaa ccgagatgga 180
agtcagaaat actatgtgga ctctgtggag ggccgattca ccatctccag actctccgac 240
aacgccaaga gctcactgta tctgcaaatg aacagcctga gagccgatga cacggctgtc 300
tattactgta tgagtggcta cgattccggc cactggggcc agggatccac ggtcaccgtc 360
tcctcagc 368
<210> 10
<211> 368
<212> DNA
<213> 3λ10轻链可变区核苷酸序列
<400> 10
tccaattccc agactgtggt gacccaggag ccctcactga ctgtgtcccc aggagggaca 60
gtcactctca cctgtggctc cagtactgga gctgtcacca gtggtcttta tgcccagcag 120
aagcctggcc aagtcccccg gacactgatt tataatataa gcaacaaaca gtcctggacc 180
cctgcccggt tctcaggctc cctccttggg ggcaaagctg ccctgaccct ttcgggtgcg 240
cagcctgagg atgaggctga ctattactgc ttgctctcct atagtcgtac tcgggtgttc 300
ggcggaggga ccaagctgac cgtcctaggt cagcccaagg ctgccccctc ggtcactctg 360
ttcccgcc 368
<210> 11
<211> 118
<212> PRT
<213> 1H10重链可变区氨基酸序列
<400> 11
Val His Ser Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln
1 5 10 15
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Val Glu Ala Phe
20 25 30
Ser Thr Tyr Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
35 40 45
Glu Trp Val Ala Asn Ile Asn Arg Asp Gly Ser Gln Lys Tyr Tyr Val
50 55 60
Asp Ser Val Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser
65 70 75 80
Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Asp Asp Thr Ala Val
85 90 95
Tyr Tyr Cys Met Ser Gly Tyr Asp Ser Gly His Trp Gly Gln Gly Ser
100 105 110
Leu Val Thr Val Ser Ser
115
<210> 12
<211> 130
<212> PRT
<213> 3λ5轻链可变区氨基酸序列
<400> 12
Phe Asn Ser Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser
1 5 10 15
Pro Gly Gly Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val
20 25 30
Thr Ser Gly Leu Tyr Ala Tyr Trp Phe Gln Gln Lys Pro Gly Gln Val
35 40 45
Pro Arg Thr Leu Ser Thr Gly Ala Val Ile Tyr Asn Ile Ser Asn Lys
50 55 60
Gln Ser Trp Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly Lys
65 70 75 80
Ala Ala Leu Thr Leu Ser Gly Ala Gln Pro Glu Asp Glu Ala Asp Tyr
85 90 95
Tyr Cys Leu Leu Ser Tyr Ser Arg Thr Arg Val Phe Gly Gly Gly Thr
100 105 110
Lys Leu Thr Val Leu Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu
115 120 125
Phe Pro
130
<210> 13
<211> 118
<212> PRT
<213> 1H16重链可变区氨基酸序列
<400> 13
Val His Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
1 5 10 15
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
20 25 30
Ser Thr Tyr Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
35 40 45
Glu Trp Val Ala Asn Ile Asn Arg Asp Gly Ser Gln Lys Tyr Tyr Val
50 55 60
Asp Ser Val Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser
65 70 75 80
Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Asp Asp Thr Ala Val
85 90 95
Tyr Tyr Cys Met Ser Gly Tyr Asp Ser Gly His Trp Gly Gln Gly Ser
100 105 110
Leu Val Thr Val Ser Ser
115
<210> 14
<211> 128
<212> PRT
<213> 3λ7轻链可变区氨基酸序列
<400> 14
Ser Asn Ser Gln Ala Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser
1 5 10 15
Pro Gly Gly Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val
20 25 30
Thr Ser Gly Leu Tyr Ala Tyr Trp Thr Val Thr Phe Gln Gln Lys Pro
35 40 45
Gly Gln Val Pro Arg Thr Leu Ile Tyr Asn Ile Ser Asn Lys Gln Ser
50 55 60
Trp Thr Pro Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala
65 70 75 80
Leu Thr Leu Ser Gly Ala Gln Pro Glu Asp Glu Ala Asp Tyr Tyr Cys
85 90 95
Leu Leu Ser Tyr Ser Arg Thr Arg Val Phe Gly Gly Gly Thr Lys Leu
100 105 110
Thr Val Leu Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro
115 120 125
<210> 15
<211> 116
<212> PRT
<213> 1H17重链可变区氨基酸序列
<400> 15
Val His Ser Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln
1 5 10 15
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
20 25 30
Ser Thr Tyr Trp Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
35 40 45
Glu Trp Val Ala Asn Arg Asp Gly Ser Gln Lys Tyr Tyr Val Asp Ser
50 55 60
Val Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Ser Ser Leu
65 70 75 80
Tyr Leu Gln Met Asn Ser Leu Arg Ala Asp Asp Thr Ala Val Tyr Tyr
85 90 95
Cys Met Ser Gly Tyr Asp Ser Gly His Trp Gly Gln Gly Ser Thr Val
100 105 110
Thr Val Ser Ser
115
<210> 16
<211> 123
<212> PRT
<213> 3λ8轻链可变区氨基酸序列
<400> 16
Ser Trp Ala Asn Phe Met Leu Thr Gln Glu Pro Ser Leu Thr Val Ser
1 5 10 15
Pro Gly Gly Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val
20 25 30
Thr Ser Gly Leu Tyr Ala Tyr Trp Phe Gln Gln Lys Pro Gly Gln Val
35 40 45
Pro Arg Thr Leu Ile Tyr Asn Ile Ser Asn Lys Gln Ser Trp Thr Pro
50 55 60
Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu
65 70 75 80
Ser Gly Pro Glu Asp Glu Ala Asp Tyr Tyr Cys Leu Leu Ser Tyr Ser
85 90 95
Arg Thr Arg Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln
100 105 110
Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro
115 120
<210> 17
<211> 127
<212> PRT
<213> 1H3重链可变区氨基酸序列
<400> 17
Val His Ser Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln
1 5 10 15
Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
20 25 30
Ser Ser Tyr Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
35 40 45
Glu Trp Val Ala Val Ile Ser Tyr Asp Gly Asn Lys Tyr Tyr Ala Asp
50 55 60
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
65 70 75 80
Leu Tyr Leu Gln Met Asn Ser Leu Arg Asp Glu Asp Thr Ala Val Tyr
85 90 95
Tyr Cys Ala Arg Ala Pro Asp Cys Ser Gly Gly Ser Cys Ser Thr Glu
100 105 110
Tyr Phe Gln His Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 18
<211> 123
<212> PRT
<213> 3λ1轻链可变区氨基酸序列
<400> 18
Ser Trp Ala Gln Ser Val Leu Thr Gln Glu Pro Ser Leu Thr Val Ser
1 5 10 15
Pro Gly Gly Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val
20 25 30
Thr Ser Gly Leu Tyr Ala Tyr Trp Phe Gln Gln Lys Pro Gly Gln Val
35 40 45
Pro Arg Thr Leu Ile Tyr Asn Ile Ser Asn Lys Gln Ser Trp Thr Pro
50 55 60
Ala Arg Phe Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu
65 70 75 80
Ser Gly Ala Gln Pro Glu Asp Glu Ala Asp Tyr Tyr Cys Leu Leu Ser
85 90 95
Tyr Ser Arg Thr Arg Val Phe Gly Thr Lys Leu Thr Val Leu Gly Gln
100 105 110
Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro
115 120
<210> 19
<211> 122
<212> PRT
<213> 1H18重链可变区氨基酸序列
<400> 19
Val His Ser Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val Gln
1 5 10 15
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe
20 25 30
Ser Thr Tyr Trp Met Asn Trp Val Arg Leu Ser Gln Thr Pro Gly Lys
35 40 45
Gly Leu Glu Trp Val Ala Asn Ile Asn Arg Asp Gly Ser Gln Lys Tyr
50 55 60
Tyr Val Asp Ser Val Glu Gly Arg Phe Thr Ile Ser Arg Leu Ser Asp
65 70 75 80
Asn Ala Lys Ser Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Asp
85 90 95
Asp Thr Ala Val Tyr Tyr Cys Met Ser Gly Tyr Asp Ser Gly His Trp
100 105 110
Gly Gln Gly Ser Thr Val Thr Val Ser Ser
115 120
<210> 20
<211> 122
<212> PRT
<213> 3λ10轻链可变区氨基酸序列
<400> 20
Ser Asn Ser Gln Thr Val Val Thr Gln Glu Pro Ser Leu Thr Val Ser
1 5 10 15
Pro Gly Gly Thr Val Thr Leu Thr Cys Gly Ser Ser Thr Gly Ala Val
20 25 30
Thr Ser Gly Leu Tyr Ala Gln Gln Lys Pro Gly Gln Val Pro Arg Thr
35 40 45
Leu Ile Tyr Asn Ile Ser Asn Lys Gln Ser Trp Thr Pro Ala Arg Phe
50 55 60
Ser Gly Ser Leu Leu Gly Gly Lys Ala Ala Leu Thr Leu Ser Gly Ala
65 70 75 80
Gln Pro Glu Asp Glu Ala Asp Tyr Tyr Cys Leu Leu Ser Tyr Ser Arg
85 90 95
Thr Arg Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Pro
100 105 110
Lys Ala Ala Pro Ser Val Thr Leu Phe Pro
115 120
Claims (4)
1.一种高亲和力抗狂犬病病毒的全人源单克隆抗体，其特征在于，其具有如下重链可变区和轻链可变区：
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 IDNO:18所示。
2.编码如权利要求1所述单克隆抗体的核酸分子，其特征在于，其具有如下重链可变区和轻链可变区：
重链可变区的核苷酸序列如SEQ ID NO:7所示；轻链可变区的核苷酸序列如SEQ IDNO:8所示。
3.一种表达载体，其特征在于，包括权利要求2所述核酸分子。
4.权利要求1所述的单克隆抗体在制备用于检测狂犬病病毒感染的药物中的用途。
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| WO1990011358A1 (fr) * | 1989-03-29 | 1990-10-04 | Institut Pasteur | Procede de detection et/ou d'identification des infections a lyssavirus, clonage et expression de genes codant pour des peptides et/ou des fragments de peptides du lyssavirus mokola, vaccin contre le virus mokola et/ou l'ensemble des lyssavirus ainsi que procede d'obtention dudit vaccin par genie genetique |
| CN103910796A (zh) * | 2013-01-08 | 2014-07-09 | 广州泰诺迪生物科技有限公司 | 一种全人源抗狂犬病毒的中和抗体 |
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| CN101812131B (zh) * | 2010-05-06 | 2012-07-04 | 中国疾病预防控制中心病毒病预防控制所 | 人源抗狂犬病毒糖蛋白中和性抗体(RVFab8) |
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| CN112430264A (zh) * | 2020-11-18 | 2021-03-02 | 南京医科大学 | 抗狂犬病病毒G蛋白scFv20抗体及其应用 |
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| WO1990011358A1 (fr) * | 1989-03-29 | 1990-10-04 | Institut Pasteur | Procede de detection et/ou d'identification des infections a lyssavirus, clonage et expression de genes codant pour des peptides et/ou des fragments de peptides du lyssavirus mokola, vaccin contre le virus mokola et/ou l'ensemble des lyssavirus ainsi que procede d'obtention dudit vaccin par genie genetique |
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| CN114957458A (zh) | 2022-08-30 |
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| CN115260308A (zh) | 2022-11-01 |
| CN115260308B (zh) | 2024-02-09 |
| CN115260307A (zh) | 2022-11-01 |
| CN114920835A (zh) | 2022-08-19 |
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| CN115260307B (zh) | 2024-02-09 |
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