CN101812129B - 手足口ev71病毒单克隆抗体及其应用 - Google Patents
手足口ev71病毒单克隆抗体及其应用 Download PDFInfo
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- CN101812129B CN101812129B CN2009101808062A CN200910180806A CN101812129B CN 101812129 B CN101812129 B CN 101812129B CN 2009101808062 A CN2009101808062 A CN 2009101808062A CN 200910180806 A CN200910180806 A CN 200910180806A CN 101812129 B CN101812129 B CN 101812129B
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Abstract
本发明提供了手足口EV71病毒的一种单克隆抗体，其是以EV71病毒的VP1蛋白为靶蛋白，设计合成多肽序列，与载体蛋白偶联作为免疫原，通过所述免疫原制备获得。本发明提供的单克隆抗体与EV71病毒其他蛋白、CA16病毒以及其他抗原和病原体无交叉反应，用于检测具有高特异性、高敏感性的优点，可以准确检测样品中EV71病毒的水平，在临床检测中将会得到广泛应用。
Description
技术领域
本发明涉及杂交瘤细胞系及其产生的单克隆抗体与应用，特别是涉及针对手足口病病原体之一肠道病毒71的单克隆抗体和分泌该抗体的杂交瘤细胞系以及该抗体的应用。
技术背景
手足口病(Hand，foot，and mouth disease，HFMD)是由肠道病毒引起的传染病，多发生于5岁以下的婴幼儿，可引起发热和手、足、口腔等部位的皮疹、溃疡，个别患者可引起心肌炎、肺水肿、无菌性脑膜脑炎等并发症。引发手足口病的肠道病毒有20多种，其中柯萨奇病毒A16型(Coxsackieviruses 16，CA16)和肠道病毒71型(Enterovirus71，EV71)最常见。肠道病毒71型简称EV71。1957年，新西兰首次爆发大规模疫情并报道。1958年，首次分离出柯萨奇病毒。1959年，有了手足口病的命名。
肠道病毒71型和柯萨奇病毒A16型均属小RNA病毒科肠道病毒属成员，其感染主要引起患者手足口病。通常情况下，EV71感染引起的手足口病在临床症状等方面与CA16感染所引起的手足口病难以区别，但EV71感染除了引起HFMD以外，还能够引起无菌性脑膜炎、脑干脑炎和脊髓灰质炎样麻痹等多种与神经系统相关的疾病。自1974年Schmidt等首次报道从美国加利福尼亚爆发表现为神经系统症状疾病的患者中分离到EV71以来，EV71已在世界范围内引起10多次爆发与流行。EV71的流行在亚太地区呈上升趋势。儿童感染后易引起严重并发症，病死率高，死亡速度快，造成了社会的不安定，快速而特异地诊断EV71感染，及早发出潜在暴发EV71相关神经系统疾病的警报，对指导公共卫生部门的干预和临床决策，控制EV71引起的疾病暴发流行是十分重要的。而CA16则很少会引起中枢神经系统的感染，因此，临床对EV71的快速鉴别诊断需求极为迫切。
目前在国内尚未见到有效检测EV71病毒单克隆抗体的报道，国外已有相关的抗体，但是，该抗体与CA16有交叉反应，无法区分EV71和CA16并且该抗体不适于直接快速检测病人样品。
发明内容
本发明的目的是提供能与EV71专一结合的单克隆抗体以及产生该抗体的杂交瘤细胞系。
本发明提供的单克隆抗体，其是以EV71病毒的VP1蛋白(NCBI上的登陆号NO.AF316321)为靶蛋白，设计合成多肽序列，与载体蛋白偶联作为免疫原，通过所述免疫原制备获得。
具体地说是使用可以诱发机体产生免疫反应并生成具有中和效应抗体的多肽序列，偶联至KLH作为免疫原免疫小鼠，采用杂交瘤技术经过细胞融合并筛选得到能持续、稳定分泌抗EV71单克隆抗体的杂交瘤细胞株，由该细胞株分泌得到单克隆抗体。
优选地，上述多肽序列选自：
SP55：PESRESLAWQTATNPC；
SP70：YPTFGEHKQEKDLEYC。
由这两个多肽序列与载体蛋白偶联作为免疫原制备获得的单克隆抗体能够特异性地识别VP1蛋白的SP55序列或SP70序列，将这两个单克隆抗体分别称之为clone 20D12和Clone 22A12。
clone 20D12和Clone 22A12具有良好的特异性，实验表明，clone 20D12和Clone 22A12与CA16没有交叉反应，间接ELISA表明这两个抗体具有较高的效价，因此可用于EV71的检测。此外，由于clone 20D12和Clone 22A12识别VP1不同的抗原决定簇，因此，可以采用双抗夹心法，利用这两个单克隆抗体对VP1进行检测。从而，本发明提供一种用于EV71检测的试剂盒，其含有上述单克隆抗体，特别是同时含有clone 20D12和Clone 22A12，优选以clone 20A12(特异性地识别VP1蛋白的SP70序列的单克隆抗体)为包被抗体，以酶标记的Clone 22D12(异性地识别VP1蛋白的SP55序列)的单克隆抗体作为检测抗体。
此外本发明还提供一种检测试纸卡，包括反应膜、样本垫、结合物释放垫和吸水垫，所述反应膜上具有包被有上述单克隆抗体的测试区，所述结合物释放垫包被有胶体金标记单克隆抗体或酶标记单克隆抗体，并且检测区包被的单克隆抗体与结合物释放垫包被的单克隆抗体分别特异性地识别VP1蛋白的SP55序列或SP70序列。所述反应膜通常是硝酸纤维素膜或醋酸纤维素膜，结合物释放垫通常是玻璃纤维膜。
本发明还提供产生上述单克隆抗体的杂交瘤细胞。
本发明提供的单克隆抗体与EV71病毒其他蛋白、CA16病毒以及其他抗原和病原体无交叉反应，用于检测具有高特异性、高敏感性的优点，可以准确检测样品中EV71病毒的水平，在临床检测中将会得到广泛应用。同时，本发明的抗体具有中和EV71病毒并阻断其感染的特性，可用于针对患者、患病动物的治疗和对易感人群与动物进行被动免疫，具有良好的应用前景。
附图说明
图1是抗体的SDS-PAGE电泳图，其中M是蛋白分子量标准(kDa)，22A12和20D12分别为本发明获得的两个种单克隆抗体；
图2是抗体免疫印迹分析图，其中M是蛋白分子量标准(kDa)，1：22A12，2：20D12，3：阳性对照(heart bleed asprimary antibodies)，4：NC：阴性对照(5％ milk-PBS as primaryantibodies)。
图3是两个克隆的亚型鉴定图，其中clone 20D12显示IgG1信号最强，clone 22A12显示IgG2b信号最强，依据亚型鉴定试剂盒说明中结果判定标准，clone 20D12的亚型为IgG1，clone 22A12的亚型为IgG2b。
图4所示的是ELISA法灵敏度实验的拟合曲线。
图5是试纸卡检测情况，其中由上至下各样品的浓度分别是50ng/ml、10ng/ml、5ng/ml、5ng/ml、2ng/ml以及0.3ng/ml。
具体实施方法
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。
若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段。
实施例1、杂交瘤细胞系的建立
一、实验材料
1、免疫原：本例以EV71病毒的VP1蛋白作为靶蛋白，从中选取两段多肽序列，序列如下：SP55：PESRESLAWQTATNPC，SP70：YPTFGEHKQEKDLEYC，采用化学合成的方式制备这两条多肽，纯度要求大于90％。这两条多肽分别与KLH偶联制备成免疫原。
2、培养基：DMEM培养基购于Hyclone公司；HAT、HT选择培养基、降植烷购于sigma公司。
3、实验动物：Balb/c小鼠，8-12周龄，雌性，SPF级动物培养。
4、其他材料：福氏完全佐剂、福氏不完全佐剂购于Sigma公司；PEG4000购于Fluka公司；HRP-山羊抗小鼠IgG抗体购于JacksonImmune公司；其余试剂均为国产分析纯产品。
二、杂交瘤细胞系的建立
1、动物免疫
1)基础免疫：将抗原与福氏完全佐剂等体积混合并充分乳化，分点皮下注射，每只Balb/c小鼠每次注射量为100μg。
2)加强免疫：加强免疫采用抗原与福氏不完全佐剂的乳化液。在进行细胞融合前3天，经腹腔注射含150ug抗原的生理盐水溶液。
2、杂交瘤细胞的制备
按常规方法收集小鼠的脾细胞与SP2/0细胞按10∶1的比例以500g/L的PEG4000进行融合。用HAT培养液选择培养，融合后10～15天，取上清采用间接ELISA法筛选分泌抗EV71抗原的杂交瘤细胞株。对所得阳性克隆株采用有限稀释法进行亚克隆。间接ELISA法的操作步骤如下：用200μl的SP55和SP70包板，用免疫小鼠血清1∶2000作为阳性对照，无克隆生长的培养基上清和正常小鼠血清作为阴性对照，每孔加1∶2000HRP-山羊抗小鼠IgG 100μl，最后测定450nm OD值。凡OD450值大于阴性对照2倍以上者，即可初步判定为阳性克隆。
3、杂交瘤细胞系的建立
重复步骤2，进行2次细胞融合，经过4次亚克隆和间接ELISA筛选，得到5株分别针对SP55、2株针对SP70的，稳定分泌单克隆抗体的杂交瘤细胞系。
4、应用上述杂交瘤细胞系所得单抗的效价检测
1)细胞培养液上清效价测定：间接ELISA法检测上述杂交瘤细胞培养上清效价为：1∶50000-1∶100000。
2)小鼠腹水效价测定：间接ELISA法检测上述杂交瘤细胞制备的腹水效价为：1∶500000-1∶1000000。
5、杂交瘤细胞系的传代培养
将上述杂交瘤细胞系在含有10％胎牛血清的DMEM培养基中继续进行培养、传代，培养到10代后，杂交瘤细胞系仍然能够生长良好、稳定传代，培养液上清效价仍然可以达到1∶10000以上。
以上结果表明，所得杂交瘤细胞系能够稳定传代，可以持续、稳定分泌抗EV71的单克隆抗体。
获得产生所需的单克隆抗体的杂交瘤细胞后，必须将一部分杂交瘤细胞保存，否则在连续传代的过程中，可能产生突变或染色体的漂移以至丧失固有特性或丢失产生抗体的特性。另外在长期的培养过程中，难免不发生污染以至于毁灭。所以必须冷藏保存一部分。保存方法如下：
1.材料
(1)细胞：取对数生长期的细胞。
(2)10％二甲基亚砜保护液(二甲基亚砜能损坏滤器，而又被高压所破坏，所以不能过滤或高压消毒。其本身就是毒品，无菌)：含10％二甲基亚砜、20％灭活胎牛血清，70％RPMI-1640液。
(3)20％FCS-1640培养液：含青霉素100U/ml，链霉素100μg/ml。
(4)灭菌的2ml安瓶等
2.操作方法
(1)去掉细胞培养瓶中的旧的培养液，加入10％FCS-1640液，使细胞悬浮。
(3)1000r/min离心10min，去上清。细胞沉淀用10％二甲基亚砜保护液制成悬液，使成1.0×107细胞/ml。
(3)取样，台盼兰染色，计数活细胞，应在95％以上。
(4)用注射器将细胞分装安瓶，每瓶0.5ml～1.0ml，熔封安瓶。
(5)放4℃2h。
(6)放液氮罐气态部分(-70℃)15h。
(7)转入液氮部分。
实施例2抗EV71的单克隆抗体的制备
一抗体制备
选择成年BALB/c小鼠，腹腔接种降植烷，每只小鼠0.5ml。7-10天后腹腔接种第16代杂交瘤细胞，每只小鼠1×106-2×106个。间隔5天后，待腹部明显膨大，以手触摸时，皮肤有紧张感，即可用9号针头采集腹水。
将腹水离心(13000r/min 30分钟)，除去细胞成分和其他的沉淀物，收集上清。用Protein G～Sepharose CL-4B进行纯化，上柱液为20mM的PBS缓冲液，柱层析洗脱液为：pH2.7，20mM的甘氨酸缓冲液，得到抗EV71病毒的单克隆抗体(将由SP55序列产生的单克隆抗体称之为clone 20D12由SP70序列产生的单克隆抗体称之为Clone 22A12)。
二抗体的鉴定
1、抗体纯度鉴定：
SDS-PAGE电泳鉴定，纯度在95％以上。
2、抗体类及亚类鉴定：
采用间接ELISA法，使用抗小鼠各种Ig亚型的抗体鉴定上述杂交瘤细胞产生的抗体的Ig亚型，结果显示，SP55组中的5个抗体均为(IgG1)，SP70组的2个抗体属于(IgG2b)(图三)。
3、抗体免疫印迹检测：
常规Western Blot检测方法检测这两株抗体的特异性，使用100μg灭活病毒进行SDS-PAGE电泳，湿转法转至PVDF膜上，使用纯化的抗体进行Western Blot杂交检测。结果显示，上述方法制得的单克隆抗体均能特异性识别EV71病毒。
4、克隆20D12和克隆22A12的可变区序列测定
将两个克隆的细胞提取mRNA，反转录为cDNA，使用可变区通用引物进行高保真PCR扩增，将PCR产物片段插入到T载体内进行DNA序列测定，并将获得的序列翻译成蛋白质的氨基酸序列。clone 20D12和clone 22A12的抗体的可变区氨基酸序列：clone20D12的氨基酸序列为：轻链如SEQ ID No.1所示，重链如SEQ IDNo.2所示。clone 22A12的氨基酸序列为：轻链如SEQ ID No.3所示，重链如SEQ ID No.4所示。将该序列进行比对后未显示有相同序列，说明所获得的序列为两个克隆各自特异的序列。
实施例3应用纯化抗体制备EV71病毒检测试剂
一、ELISA双抗体夹心法
应用clone 20D12和Clone 22A12抗体做配对实验，确定以clone22A12为包被抗体，用HRP标记Clone 20D12作为检测抗体，确定了ELISA检测方法，试剂盒检测灵敏度可达1ng/mL(图4)。采用改良过碘酸钠法标记抗体。
表1ELISA法检测EV71的检测结果
| 试验组 | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 | 11 | 12 |
| 浓度ng/ml | 100 | 30 | 10 | 3 | 1 | 0.3 | 0.1 | 0.03 | 0.01 | 0.003 | 阴性对照 | 空白对照 |
| 20D124000G | 0.846 | 0.635 | 0.386 | 0.245 | 0.211 | 0.183 | 0.172 | 0.167 | 0.167 | 0.128 | 0.112 | 0.053 |
检测方法：包被抗体clone 22A12用pH 9.60.05mol/L的碳酸盐缓冲溶液稀释成10μg/mL，在酶标板的每孔加100μL，4℃下包被过夜，倾去包被液，用PBST洗涤3次，拍干，然后在每孔中加入200μL 1％的明胶，放入37℃恒温箱中封闭2小时后，用PBST洗涤3次，拍干后干燥保存。用辣根过氧化物酶标记clone 20D12的抗体，得20D12-HRP并保存。向酶标板中分别加入EV71梯度稀释液100μL/孔，37℃孵育1小时，再加入20D12-HRP(1∶4000稀释)100μL/，37℃孵育1小时，加入显色剂A、B各50μL/孔进行显色，37℃温育10min，加入终止液50μL/孔，进行读数。
其中显色剂A液配方为每1000mL水中加入过氧化脲1g，10.3g柠檬酸，35.8g Na2HPO4·12H2O，吐温-20100μL，pH5；B液配方为每1000mL蒸馏水中加入四甲基联苯胺(TMB)700mg(40mLDMSO溶解)，10.3g柠檬酸，pH2.4。试剂盒特异性试验：稀释液、正常人血清、正常人唾液均呈阴性，对CA16无交叉反应。
二、检测试纸卡的开发
应用两株单克隆抗体进行进行金标，确定了试纸卡快速检测方法，检测灵敏度可达0.3ng/mL(EV71)(图5)。具体方法：将纯化的EV71病毒抗体与兔抗鼠二抗分别固定在硝酸纤维素膜及玻璃纤维素膜上，组成EV71病毒免疫胶体金双抗体夹心法检测试纸卡。其中加样孔下设有滤过垫，反应区包被有纯化的EV71病毒抗体胶体金偶联标记物，检测线包被有纯化的EV71病毒抗体，质控线包被有兔抗鼠免疫球蛋白，质控线侧贴有吸水垫。检测时，取待检样品一滴，滴在该试纸卡的加样品孔中，根据检测线和质控线是否出现色带来确定样品内是否存在达标的EV71病毒。
序列表
<110>京天成生物技术(北京)有限公司
<120>手足口EV71病毒单克隆抗体及其应用
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Claims (6)
1.一种单克隆抗体，其是以EV71病毒的VP1蛋白为靶蛋白，设计合成多肽序列，与载体蛋白偶联作为免疫原，通过所述免疫原制备获得，所述多肽序列选自：
SP55：PESRESLAWQTATNPC；
SP70：YPTFGEHKQEKDLEYC；
所述载体蛋白为KLH；
所述单克隆抗体特异性地识别VP1蛋白的SP55序列或SP70序列；
所述单克隆抗体的轻链可变区的氨基酸序列如SEQ ID No.1所示，重链可变区的氨基酸序列如SEQ ID No.2所示；或所述单克隆抗体的轻链可变区的氨基酸序列如SEQ ID No.3所示，重链可变区的氨基酸序列如SEQ ID No.4所示。
2.产生权利要求1所述单克隆抗体的杂交瘤细胞。
3.含有权利要求1所述单克隆抗体的检测试剂盒。
4.如权利要求3所述的检测试剂盒，其为ELISA检测试剂盒，其以权利要求1所述的特异性地识别VP1蛋白的SP70序列的单克隆抗体为包被抗体；以酶标记的权利要求1所述的特异性地识别VP1蛋白的SP55序列的单克隆抗体作为检测抗体。
5.一种检测试纸卡，包括反应膜、样本垫、结合物释放垫和吸水垫，其特征在于所述反应膜上具有包被有权利要求1所述的特异性地识别VP1蛋白的SP55序列的单克隆抗体的检测区，所述结合物释放垫包被有胶体金或酶标记的权利要求1所述的特异性地识别VP1蛋白的SP70序列的单克隆抗体；或所述反应膜上具有包被有权利要求1所述的特异性地识别VP1蛋白的SP70序列的单克隆抗体的检测区，所述结合物释放垫包被有胶体金或酶标记的权利要求1所述的特异性地识别VP1蛋白的SP55序列的单克隆抗体。
6.权利要求1所述的单克隆抗体在制备检测EV71病毒试剂中的应用。
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| CN102062780A (zh) * | 2010-11-23 | 2011-05-18 | 北京正旦国际科技有限责任公司 | 一种多肽免疫试剂盒及其检测方法 |
| CN102229915B (zh) * | 2011-06-22 | 2012-08-22 | 浙江大学 | Ev71病毒单克隆抗体、杂交瘤细胞株及应用 |
| WO2013032404A1 (en) * | 2011-08-26 | 2013-03-07 | Temasek Life Sciences Laboratory Limited | Human enterovirus specific antibodies and their uses in diagnostics |
| CN104220090A (zh) * | 2011-09-20 | 2014-12-17 | 淡马锡生命科学实验室有限公司 | 肠道病毒71型的特异性抗体及其用途 |
| CN102854317A (zh) * | 2011-09-27 | 2013-01-02 | 上海博沃生物科技有限公司 | 一种ev71抗原酶联反应检测试剂盒及其制备方法 |
| CN102517317B (zh) * | 2011-12-23 | 2014-03-26 | 中国科学院武汉病毒研究所 | 带有标签的肠道病毒71型全长感染性克隆的制备方法及应用 |
| CN102558313A (zh) * | 2012-01-12 | 2012-07-11 | 东南大学 | 肠道病毒71型特异性重组蛋白抗原及其应用 |
| CN102650635A (zh) * | 2012-05-16 | 2012-08-29 | 湖南康润药业有限公司 | 肠道病毒71型胶体金检测试纸条及其制备方法和应用 |
| CN103421112B (zh) * | 2012-05-24 | 2015-04-29 | 中国科学院上海巴斯德研究所 | 一种抗肠道病毒的结合分子及其用途 |
| CN103833830B (zh) * | 2012-11-22 | 2018-09-04 | 中国科学院上海巴斯德研究所 | 柯萨奇病毒a16型的保守中和表位多肽及其用途 |
| CN104031118B (zh) | 2014-06-19 | 2016-04-13 | 天津大学 | 鼠多瘤病毒衣壳粒的新型亲和肽配基及其设计筛选方法 |
| CN111139233B (zh) * | 2020-01-19 | 2023-04-25 | 中国医学科学院医学生物学研究所 | 广谱中和抗EV71、CA16、CA10和CA6人-鼠嵌合IgM型单克隆抗体及应用 |
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