CN115124617B - 一种高亲和力抗丙型肝炎病毒的全人源单克隆抗体及其用途 - Google Patents
一种高亲和力抗丙型肝炎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丙型肝炎病毒的全人源单克隆抗体及其用途，属于生物工程技术领域。所述的单克隆抗体或其片段的重链可变区的氨基酸序列如SEQ ID NO:11所示；所述轻链可变区的氨基酸序列如SEQ ID NO:15所示。本发明还提供该抗体的编码基因、表达载体、用途和组合物。本发明的结合分子能阻止丙型肝炎病毒侵染易感细胞，且是全人源的，与其它动物源性(如鼠源性)抗丙型肝炎病毒的分子相比，免疫原性低，且亲和性良好，不仅治疗效果好，而且副作用低，同时可靠的有效性及安全性也为此类抗体的标准化生产成药提供了很大的保障。
Description
技术领域
本发明涉及生物工程技术领域，具体地说，涉及一种高亲和力抗丙型肝炎病毒的全人源单克隆抗体及其用途。
背景技术
上世纪八十年代，研究人员第一次从黑猩猩的血液中分离出了一种区别于甲乙型的肝炎病毒，该病毒后被命名为丙肝病毒(HCV)。丙肝感染已经成为全球性的健康问题，不同性别、年龄、种族人群均对HCV易感。目前世界上感染HCV的患者据估计已经超过1.84亿人，通过血清阳性率估计的全球HCV感染率约为1.5％-3.5％。
在全球范围内，由于HCV的高度变异和免疫逃逸机制，各地区的流行分布情况存在着较大差异。目前，全世界主要流行的丙肝病毒株主要分为11个基因型，70多种亚型，不同基因型的HCV病毒毒力不同，致病能力也有着很大差异。通过对我国29个省市的32030患者样本结果进行分析发现，我国主要流行的丙肝亚型为1b(n＝16 713，52.18％)，2a(n＝9188，28.69％)，3b(n＝2261，7.06％)，6a(n＝2052，6.41％)和3a(n＝1479，4.62％)，而在其他小部分样本中发现了主要亚型的混合感染，其组合如下：1b-2a，1b-3b，1b-6a，3a-3b，1b-3a和2a-6a，这些HCV基因亚型的分布与人群性别，年龄及所在的地理位置有着密切联系。
依照长期的临床实践经验和实验室技术的检测，丙肝的临床诊断标准可如下：临床表现为全身乏力、食欲减退，右季肋部不适，肝脾肿大等特点；实验室检查谷丙转氨酶、谷草转氨酶多呈轻度和中度升高，抗-HCV抗体阳性，HCV RNA阳性；进一步结合患者病程持续时间长短及对应流行病学史的有无等进行诊断。
丙肝治疗主要目的是清除病毒，使HCV在体内的复制水平长期保持在极低的载量，充分减轻HCV感染造成的肝损伤病变等，提高患者的长期生存率，和生存质量。在长期的丙肝治疗中，临床上经典的治疗方法为干扰素合并利巴韦林，这类方法副作用较大，容易产生耐药性，对不同基因型患者效果差异较大。随着国际新型药物如盐酸达拉他韦片和阿舒瑞韦软胶囊这类直接抗病毒药物(Direct Anti-virial Agents,DAAs)的问世，我国已经批准大部分DAAs在临床使用，DAA药物的主要作用机制为抑制HCV颗粒内的NS3/4A蛋白酶和NS5A/B聚合酶，在使用直接抗病毒药物之前，通过患者所感染的丙肝基因型进行鉴定，结合患者的肝脏损伤其其他情况，选择合适的抗病毒方案能够有效治疗HCV感染。在2018年，吉利德公司生产的“丙通沙”(索磷布韦维帕他韦片)正式进入我国药品目录，标志着丙肝治疗新时代的来临，这款全口服、泛基因型、单一片剂的丙肝治疗新药为患者带来福音，但其不可避免的耐药性和较高的价格令部分患者望而却步。
1975年，两位剑桥大学的研究者Milstein和首次发明杂交瘤技术，该技术原理是将免疫后的鼠脾细胞与骨髓瘤细胞进行融合，在融合后筛选出能够稳定分泌单一型抗体的杂交瘤细胞。同时，单克隆抗体的概念也因此而来，这种抗体是由单一B细胞克隆产生的高度均一，仅针对某一特定抗原表位的抗体,其具有纯度高、特异性强，交叉反应少等先进的优势特点。然而，由于此类方法生产的是鼠源性蛋白，对人体具有相当的免疫原性，无法满足长期治疗。随着，现代分子生物学和蛋白质工程技术的发展，该技术的局限性得到不同程度的解决。最初，研究人员通过人源的可结晶片段(Fragment crystallizable,Fc)序列取代大多数鼠源Fc序列，实现生产新型的“嵌合”抗体。之后，进一步通过生物工程改造，将鼠抗原结合区(Fragment of antigen binding,Fab)移植到人免疫球蛋白(IgG)骨架上，以提高单克隆抗体的人源化程度，如今成熟的技术已经能够生产出全人源的单克隆抗体。
目前，能够制备出全人源的单克隆抗体技术多样。主要有抗体库技术，如噬菌体展示抗体库技术和核糖体展示抗体库技术，单细胞克隆表达技术等。这些技术各有优劣，抗体库技术可在体外直接制备特异、稳定的单抗，然而前期的建库工作量十分庞大，所需的库容量需要囊括某种动物的抗体多样性，高通量的筛选往往只能获得抗体的部分片段，在后期无法保证抗体的高亲和力。单细胞克隆表达的方法是利用流式细胞仪分选得到某种特异性B细胞，通过基因技术分离其同源性抗体的重轻链可变区基因，从而在真核系统中表达。这种方法中，前期B细胞的分选尤为关键，需要高度特异并稳定的抗原作为探针，才能有效分选。
近十年来，单克隆抗体药物成为药物研发的热点。作为新型的大分子蛋白类药物，其具有特异性强，效果显著的优势，同时由于单抗药物的人源化水平较高，免疫原性相对较低，人体不易产生排斥反应，安全性也得到了极大的保障。随着单克隆抗体在肿瘤领域广泛应用，感染性疾病对应的单克隆抗体药物研发也在积极推进中，针对呼吸道合胞病毒感染的帕丽珠单抗，以及抗HIV感染的伊巴珠单抗相继问世，治疗丙肝的单克隆抗体药物虽然尚未问世。
发明内容
本发明人经过深入的研究，选取HCV表面具有良好免疫原性的E2蛋白作为抗原表位，通过从感染丙肝病毒病原体后康复人群的全血中提取记忆性B细胞，体外活化成浆细胞，采用某种特异性抗原筛选分泌有特异性抗体的浆细胞，再利用少量细胞RNA的高效提取和巢式PCR等技术筛选出特异性抗体基因。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，试验结果证明，本发明的单克隆抗体具有高亲和力的特点，本发明提供的单克隆抗体能具有显著抗丙型肝炎病毒，为此类抗体的标准化生产成药提供了很大的保障。
HCV是一种较小有包膜的单链RNA病毒，基因组全长9.6kb。HCV颗粒由一个带5'和3'非翻译区(UTR)的正极性RNA基因组，以及一段编码近3000个氨基酸的蛋白前体的开放阅读框(ORF)组成。UTR构成高度保守的顺式作用RNA元件，可调节病毒基因组的翻译和复制。经由开放阅读框编码的蛋白前体组装加工后，主要形成10种结构及非结构蛋白(core，E1，E2，p7，NS2，NS3，NS4A，NS4B，NS5A和NS5B蛋白)。HCV颗粒的外包膜主要由E1和E2糖蛋白组成，这两种蛋白的编码区域分别位于HCV蛋白结构N端的残基192-383位点(E1)和384-746位点(E2)，作为高度糖基化的I型跨膜蛋白，它们能够结合形成非共价异二聚体结构，对于病毒识别、入侵宿主细胞有着重要作用。HCV颗粒的(core)核心蛋白作为一种多功能蛋白，其主要作用是形成病毒衣壳，在病毒在宿主细胞之间转移时，发挥包裹并保护基因组RNA的作用。其他非结构蛋白如NS2,NS3，有研究表明具有RNA解旋酶活性及丝氨酸蛋白酶活性，能够与其他非结构蛋白共同协助参与病毒基因组的复制、组装。E1与E2蛋白，作为病毒表面的包膜蛋白，是引起保护性免疫反应的主要病毒抗原。这两种蛋白的分子量差异较大，E2蛋白的氨基酸数量远多于E1蛋白，但相对于E1蛋白，E2蛋白的高变区域(High Variable Region,HVR)更少，蛋白结构更加稳定，因此在抗原靶点的选择时，我们选择E2蛋白作为抗原表位，最终获得了具有高安全性，高亲和力的抗丙型肝炎病毒的全人源单克隆抗体。
本发明一方面提供一种高亲和力抗丙型肝炎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如SEQ ID NO:10-13任一项所示；或具有SEQ IDNO:10-13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:14-18任一项所示；或具有SEQID NO14-18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:10所示(5H12)；轻链可变区的氨基酸序列如SEQ ID NO:14所示(7κ1)；
(ii)重链可变区的氨基酸序列如SEQ ID NO:10所示(5H12)；轻链可变区的氨基酸序列如SEQ ID NO:17所示(7κ13)；
(iii)重链可变区的氨基酸序列如SEQ ID NO:11所示(5H17)；轻链可变区的氨基酸序列如SEQ ID NO:15所示(7κ2)；
(iv)重链可变区的氨基酸序列如SEQ ID NO:12所示(5H20)；轻链可变区的氨基酸序列如SEQ ID NO:18所示(7κ30)；
(v)重链可变区的氨基酸序列如SEQ ID NO:13所示(5H24)；轻链可变区的氨基酸序列如SEQ ID NO:16所示(7κ9)。
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丙型肝炎病毒E2蛋白。
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:1-4任一项所示的重链可变区的核苷酸序列；和如SEQ ID NO:5-9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(5H12)；轻链可变区的核苷酸序列如SEQ ID NO:5所示(7κ1)；
(ii)重链可变区的核苷酸序列如SEQ ID NO:1所示(5H12)；轻链可变区的核苷酸序列如SEQ ID NO:8所示(7κ13)；
(iii)重链可变区的核苷酸序列如SEQ ID NO:2所示(5H17)；轻链可变区的核苷酸序列如SEQ ID NO:6所示(7κ2)；
(iv)重链可变区的核苷酸序列如SEQ ID NO:3所示(5H20)；轻链可变区的核苷酸序列如SEQ ID NO:9所示(7κ30)；
(v)重链可变区的核苷酸序列如SEQ ID NO:4所示(5H24)；轻链可变区的核苷酸序列如SEQ ID NO:7所示(7κ9)。
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：从丙肝康复患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结合片段在制备用于检测、治疗、预防丙型肝炎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丙型肝炎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，提供一种检测丙型肝炎病毒的试剂盒，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、快速且准确地检测丙型肝炎病毒的试剂盒。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丙型肝炎病毒的试剂。
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HCV-E2蛋白，从而得知待测样品的供体是否感染丙型肝炎病毒，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丙型肝炎病毒的方法，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丙型肝炎病毒的方法，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，获得丙型肝炎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：相对于现有技术，本发明成功筛选了抗丙型肝炎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丙型肝炎病毒，能显著抗丙型肝炎病毒。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丙型肝炎病毒的大分子药物，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1ELISA筛选阳性记忆B细胞孔。
图2为抗体重链可变区基因H,H’和轻链可变区基因κ,λ链的PCR扩增后的电泳图。
图3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4为重组质粒的电泳验证结果。
图5为抗体重轻链可变区基因的筛选模式。
图6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：丙型肝炎病毒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
由专业人员在无菌环境下采血，将新鲜的丙肝康复志愿者全血抽取至一次性使用真空采血管中，每人所需全血量50ml。将0.22μm滤膜过滤后的预冷PBS溶液与新鲜抗凝全血1:1稀释。将稀释后的血液缓慢加入到等体积的淋巴细胞分离液中离心，离心条件为2000rpm，Acc4，Dec4，室温离心30min。离心完成后缓慢取出离心管，小心地吸取离心后第二层云雾状的单核细胞层于预先装有预冷PBS溶液的15ml离心管中。将吸取的单核细胞进行离心，离心条件为1200rpm室温离心5min,弃去上清后，用预冷的PBS溶液轻轻吹打洗涤。得到的溶液再次离心，收集到的细胞即为外周血单核细胞(periphery blood mononuclearcell，PBMC)并计数。
1.2记忆B细胞分选
(细胞计数后，根据分离得到的PBMC挑选特定的MS或LS柱)细胞离心300g，10min，彻底去除上清。以每1×107数量的细胞40μl缓冲液为单位，按比例吸取适量的缓冲液重悬细胞沉淀。以每1×107数量的细胞加10μl Naive B cell Biotin-Antibody Cocktail为单位，充分混匀细胞沉淀，4℃冰箱内孵育5min。以每1×107数量的细胞30μl缓冲液以及20μlAnti-Biotin Micro Beads为单位，充分混匀细胞沉淀，4℃冰箱内放置10min。加1～2ml缓冲液洗涤细胞，300g离心10min，弃去上清。用500μl缓冲液重悬细胞沉淀。将柱子放置到磁铁上，用适当体积的缓冲液冲洗柱子(LS柱子用3ml缓冲液，MS柱子用500μl缓冲液)。将细胞悬液缓慢加入到柱子中，收集流出的未标记细胞。吸取适量的缓冲溶液洗柱3次(LS柱子中加入3ml体积缓冲液，MS柱子中加入500μl体积缓冲液)，洗涤过程中要等柱中液体流干以后再加洗液。收集所有的洗脱液，即为含B细胞的悬液。从分离器上取下柱子，放到一个收集管上，吸取缓冲液加入到柱子中并立即用力推活塞洗出磁珠标记的细胞。细胞计数，300g离心10min，彻底去除上清，每107总细胞用80μl缓冲液重悬细胞沉淀。以每1×107细胞数量加20μlCD27 MicroBeads为单位加入适量标记磁珠，充分混匀溶液，4℃冰箱内孵育15min。以每1×107细胞数量加1ml缓冲液为单位洗涤细胞，300g离心10min，彻底去除上清。当细胞数量少于1×108时，用500μl缓冲液重悬细胞，(如果细胞较多，缓冲液加倍)。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(LS柱子用3ml缓冲液，MS柱子用500μl缓冲液)。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，立即放置在收集管上，吸取适量体积的缓冲液加入到柱子中，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细胞并冻存。
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细胞按照不同细胞密度梯度接种96孔细胞培养板。每孔加入200μl 10％ FBS的IMDM作为基础培养液，培养液中含合适浓度的的IL-21作为B细胞活化因子。在以上基础上，每孔再加入经Co-60灭活3T3-CD40L细胞稳定细胞系作为饲养层细胞。37℃，5％CO2条件下培养数天，使记忆性B细胞能够活化成为浆细胞，分泌抗体；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：收集细胞培养上清，使用Goat Anti-Human Kappa(羊抗人κ)蛋白和Goat Anti-Human Lambda(羊抗人λ)蛋白包被酶标板，采用ELISA法检测记忆性B细胞的培养基上清，确定IgG抗体的分泌量。
将Goat Anti-Human Kappa蛋白和Goat Anti-Human Lambda蛋白100ng/孔溶于50μl/孔包被液分别包被酶标板，4℃过夜，洗板机洗板5遍，洗液为含0.05％Tween-20的PBS溶液；用封闭液200μl/孔，37℃封闭2h，洗板5遍；分别在对应孔中加入细胞培养上清，或者梯度稀释的IgG标准品，同时用水或PBS作为阴性对照，50μl/孔，37℃放置1h，洗板5遍；分别在对应孔中加入1:5000稀释后的过氧化物辣根酶标记的Goat Anti-Human IgG抗体，以及1:10000稀释的Goat x-human IgG-Fc Fragment HRP Conjugated 50μl/孔，37℃放置45min，洗板5遍；加入显色液100μl/孔，室温避光显色15min；加入2mol/L硫酸50μl/孔，终止显色反应，在波长为450nm条件下检测各孔吸光度的OD值。最终通过比较分析各孔OD值高低及每孔对应记忆B细胞接种数量，确定阳性记忆B细胞孔。由图1可知，其中A1、A2为饲养层细胞作为对照孔，OD值为0.0613，最后确定12个阳性孔进行编号A3-A14，其中编号A3、A6、A7三个阳性孔的OD值明显高于其他孔，OD值分别为0.731，0.4003，0.4179。其余阳性孔也有一定的程度升高，OD值均在0.13左右。
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
显微镜下观察后，先小心吸弃细胞培养上清，用PBS洗一次，再每孔加入40μl的0.25％胰蛋白酶进行消化，镜下观察消化情况，然后每孔用5μl的胎牛血清终止消化，用枪头吹打、刮下消化后的细胞，最后每管再加入2.25μl的RNA酶抑制剂(40U/μl)。将消化后的细胞迅速放入液氮2min以上；再放PCR仪中98℃，3min，立即放入液氮；速冻之后，每管加入0.5μl的蛋白酶K(20mg/ml)和2.25μl的RNA酶抑制剂(40U/μl)，置于PCR仪中53℃，1h。将提取的RNA测定浓度后，使用Promega反转录试剂盒进行反转录后得到cDNA。
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2所示，图2A：抗体重链(H,H’链)可变区序列的巢式PCR两轮扩增结果；B:抗体轻链(κ,λ链)可变区序列的巢式PCR两轮扩增结果；C：A3，A4，A7号样品重链与轻链可变区序列巢式PCR扩增验证结果。M：Marker DL2000。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，其中A3、A4、A7号样品经过两次巢式PCR扩增(A3号样品κ链无扩增)都检测到明显的目的条带，目的条带位置在250bp到500bp之间，即确定选取A3、A4、A7号样品进行后续实验。
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-hIgG1 for 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3。胶回收目的条带，具体操作参照OMEGA胶回收试剂盒说明书。
图3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、Vκ都为酶切验证。A：A3、A4、A7号样重链(H链，H’链)可变区基因酶切结果；B：A3、A4、A7号样轻链(κ链，λ链)可变区基因酶切结果；C：载体质粒(IgH,Igκ,Igλ)的酶切结果，M：MarkerDL2000，M2:Marker DL10000。
根据目的片段大小，即抗体重链和轻链可变区基因片段为400bp，对应重链与轻链载体质粒为5000bp，可以判断出A3、A4、A7号样抗体重轻链可变区基因及载体质粒酶切成功。
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-hIgG1for VH)、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H-IgH，H’-IgH，轻链λ-Igλ的阳性克隆菌抗体载体质粒。实验结果见图4，将A3，A4，A7号重组质粒进行酶切后，可在2000bp条带以上(5000bp)和400bp条带位置观察到两条条带，即重组质粒酶切后形成的质粒基因和抗体重轻链可变区基因两段条带，表明前期A3，A4，A7号样重组质粒酶连成功(M：DL2000)。
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HCV-E2抗原蛋白(自制)的特异性抗体分泌量，我们筛选到了高表达HCV-E2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，24-72h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，用包被液(50μl/孔)将HCV-E2抗原蛋白(自制)按100ng/孔的包被量，包被酶标板；另外用Goat Anti-Human Kappa+Goat Anti-Human Lambda(各100ng/孔)蛋白包被酶标板，4℃过夜。洗板机洗板5遍。洗液为含0.05％ Tween的PBS溶液。用封闭液200μl/孔，37℃封闭2h，洗板5遍。分别在对应孔中加入细胞培养上清，梯度稀释的IgG标准品及抗HCV-E2蛋白抗体(一抗)，同时用水或PBS溶液作为阴性对照，50μl/孔，37℃放置1h，洗板5遍。分别在对应孔中加入1:5000稀释的HRP标记的Goat Anti-Human IgG和Goat Anti-Mouse IgG，以及1:10000稀释的Goat x-human IgG-Fc Fragment HRP Conjugated 50μl/孔，37℃放置45min，洗板5遍。加入TMB显色剂100μl/孔，室温避光显色20min。加入2mol/L硫酸50μl/孔，终止显色反应，检测OD/450nm，参考波长为630nm。
采用图5所示的筛选模式，我们通过ELISA的方法对阳性记忆B细胞孔进行特异性筛选。在不同批次的筛选中以抗体亲和力高低为标准进行排名，排名后选取最佳重轻链序列进行下一批次筛选，从混合序列到单克隆序列层层分级，最后筛选得到的高亲和力的单克隆抗体，鉴定分析后选取有效的单克隆重链与轻链组合进行单克隆抗体的检测(测序由江苏金斯瑞生物科技公司完成)。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：5H12,5H16,5H17,5H20,5H24和5条轻链单克隆序列：7κ1,7κ2,7κ9,7κ13,7κ30，ELISA筛选后最终得到5株高亲和力的单克隆抗体，抗体组合：5H12+7κ1，5H12+7κ13，5H17+7κ2，5H20+7κ30，5H24+7κ9(结果对应图6)。
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。
SEQUENCE LISTING
<110> 南昌大学
<120> 一种高亲和力抗丙型肝炎病毒的全人源单克隆抗体及其用途
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cgccagactc caggaaaggg gctggagtgg ctgtcggcta ttcgtaaaag tggcactacc 180
acatactacg cggactccgt gaagggccgg ttcatcatct ccagagacaa ttccaagaac 240
accctgtatc tgcaaatgaa taggctgagg gtcggcgaca cggccactta ttactgtgcg 300
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cgccagactc caggaaaggg gctggagtgg ctgtcggcta ttcgtaaaag tggcactacc 180
acatactacg cggactccgt gaagggccgg ttcatcatct ccagagacaa ttccaagaac 240
accctgtatc tgcaaatgaa taggctgagg gtcggcgaca cggccactta ttactgtgcg 300
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actaccacat actacgcgga ctccgtgaag ggccggttca tcatctccag agacaattcc 240
aagaacaccc tgtatctgca aatgaatagg ctgagggtcg gcgacacggc ctcggggact 300
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ccagccaggt tcagtggcag tgggtctggg acagagttca ctctcaccat cagcagcctg 240
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aaa 363
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35 40 45
Glu Trp Leu Ser Ala Ile Arg Lys Ser Gly Thr Thr Thr Tyr Tyr Ala
50 55 60
Asp Ser Val Lys Gly Arg Phe Ile Ile Ser Arg Asp Asn Ser Lys Asn
65 70 75 80
Thr Leu Tyr Leu Gln Met Asn Arg Leu Arg Val Gly Asp Thr Ala Thr
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Tyr Tyr Cys Ala Thr His Pro Ile Ala Gly Tyr Trp Gly Gln Gly Thr
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Thr Val Thr Val Ser Ser
115
<210> 12
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<212> PRT
<213> 5H20重链可变区氨基酸序列
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35 40 45
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50 55 60
Tyr Ala Asp Ser Val Lys Gly Arg Phe Ile Ile Ser Arg Asp Asn Ser
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Lys Asn Thr Leu Tyr Leu Gln Met Asn Arg Leu Arg Val Gly Asp Thr
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Gly Thr Thr Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Ile Ile
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<210> 14
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<212> PRT
<213> 7κ1轻链可变区氨基酸序列
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Gly Ala Ser Gln Ser Val Ser Ser Asn Leu Ala Trp Tyr Arg Gln Lys
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Pro Gly Gln Ala Pro Arg Leu Leu Ile Tyr Gly Ala Ser Thr Arg Ala
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Thr Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe
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Thr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Phe Ala Val Tyr Tyr
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<210> 15
<211> 115
<212> PRT
<213> 7κ2轻链可变区氨基酸序列
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Gly Gln Ala Pro Arg Leu Leu Ile Tyr Gly Ala Ser Thr Arg Ala Thr
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Gly Ile Pro Pro Leu Val Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr
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Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Ser Glu Asp Phe Ala Val
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Claims (5)
1.一种高亲和力抗丙型肝炎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:11所示；轻链可变区的氨基酸序列如SEQ IDNO:15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:2所示；轻链可变区的核苷酸序列如SEQ IDNO:6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丙型肝炎病毒感染的试剂盒中的用途。
5.一种组合物，其特征在于，包括权利要求1所述的单克隆抗体，以及药学上可接受的载体。
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