CN110938140B - 柯萨奇病毒a10型实心病毒的单克隆抗体及其应用 - Google Patents
柯萨奇病毒a10型实心病毒的单克隆抗体及其应用 Download PDFInfo
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- CN110938140B CN110938140B CN201911380626.9A CN201911380626A CN110938140B CN 110938140 B CN110938140 B CN 110938140B CN 201911380626 A CN201911380626 A CN 201911380626A CN 110938140 B CN110938140 B CN 110938140B
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Abstract
本发明公开了一种能与柯萨奇病毒A10型实心病毒反应的单克隆抗体及检测试剂盒。本发明采用纯化的CA10病毒液免疫小鼠制备单克隆抗体。本发明还公开了所述单克隆抗体在检测CA10病毒或诊断手足口病中的应用。所述单克隆抗体对于快速检测试剂盒的制备和疫苗的开发研究,具有广泛的应用。
Description
技术领域
本发明涉及免疫学技术领域,具体涉及柯萨奇病毒A10型实心病毒的单克隆抗体及其应用。
背景技术
手足口病是由多种肠道病毒感染引起的急性传染病,夏季流行,学龄前儿童高发,成人可为间接传染源。手足口病临床主要表现为口腔、手、足部位皮疹,可并发脑膜炎、脑炎、肺水肿、循环衰竭等导致死亡的重症。目前肠道病毒71型(enterovirus 71,EV71)和柯萨奇病毒A16(coxsackievirus,CA16)是中国大陆地区引起手足口病最常见的病原体。但随着检测技术及病毒分型手段的进步,近年来发现肠道病毒中的柯萨奇病毒A6(coxsackievirus,CA6)和柯萨奇病毒A10(coxsackievirus,CA10)的流行率呈现逐年增高的趋势,并成为部分地区的主要流行血清型。针对近几年病毒血清型的流行趋势,国内外众多研究机构及企业均在着力开发能够有效预防由CA6或CA10引起的手足口疾病发生的疫苗或药物。
临床治疗方面为能提供最快速的方案,通常需要一种快速的检测方法,ELISA快速检测试剂盒是最常用的方法,能够在最短的时间内检测出患者所感染的病毒血清型。筛选一株型别特异性的单克隆抗体是建立检测试剂盒的前提。此外,在疫苗的研究过程中,疫苗中抗原含量是指导工艺研究和评价体外有效性的关键指标。建立抗原评价方法最关键的技术也在于筛选到合适的单克隆抗体,继而可利用该单克隆抗体建立一套有效的ELISA评价系统。
柯萨奇病毒A6(coxsackievirus,CA6)和柯萨奇病毒A10(coxsackievirus,CA10)均为肠道病毒,肠道病毒在体外培养时容易形成两种不同的结构状态,一种为包含核酸的完整实心病毒颗粒,一种为不含核酸的空心病毒颗粒,通常这两种状态的病毒颗粒是共同存在的。经文献资料及多次的实验研究表明,实心病毒颗粒的免疫原性明显强于空心病毒颗粒。在疫苗的工艺研究过程中,若能够客观真实的评价每一步工序段产品的实心病毒颗粒和空心病毒颗粒的含量及比例,对指导工艺参数的选择和成品的配比计量具有重要的意义。
抗原评价系统(含ELISA快速检测试剂盒)通常采用双抗夹心法,例如多克隆抗体(简称:多抗)—多抗、多抗—单克隆抗体(简称:单抗)、单抗—多抗、单抗—单抗的形式,多抗的使用,通常容易引起不同型别肠道病毒之间的交叉结合,对病毒的检测特异性不强;单抗的使用容易出现抗原表位结合能力不足,不能够客观反映抗原真实含量。
目前没有文献报道一种能够特异的检测CA10实心病毒颗粒的抗原评价系统。现有的工艺研究中确定病毒液中空心和实心病毒的比例主要是依靠电镜观察的方式,电镜观察的方法对样品的浓度和缓冲体系的成分要求比较高,试验耗时长,对实验室的仪器设备要求比较高,具有一定的局限性。
发明内容
为了解决上述技术问题,本发明提供如下技术方案:
第一方面,本发明提供一种针对CA10病毒的单克隆抗体,所述单克隆抗体具有SEQID NO:5所示的重链互补决定区CDR1、SEQ ID NO:6所示的重链互补决定区CDR2、SEQ IDNO:7所示的重链互补决定区CDR3,以及SEQ ID NO:13所示的轻链互补决定区CDR1、SEQ IDNO:14所示的轻链互补决定区CDR2、SEQ ID NO:15所示的轻链互补决定区CDR3;优选地,所述单克隆抗体具有SEQ ID NO:8所示的氨基酸序列的重链;和/或,优选地,所述单克隆抗体具有SEQ ID NO:16所示的氨基酸序列的轻链;
优选地,所述CA10病毒为CA10实心病毒。
第二方面,本发明提供一种编码所述针对CA10病毒的单克隆抗体的多核苷酸序列,所述多核苷酸序列具有SEQ ID NO:1所示的编码重链互补决定区CDR1的序列、SEQ IDNO:2所示的编码重链互补决定区CDR2的序列、SEQ ID NO:3所示的编码重链互补决定区CDR3的序列,以及SEQ ID NO:9所示的编码轻链互补决定区CDR1的序列、SEQ ID NO:10所示的编码轻链互补决定区CDR2的序列、SEQ ID NO:11所示的编码轻链互补决定区CDR3的序列;优选地,所述多核苷酸序列具有SEQ ID NO:4所示的核苷酸序列;和/或,优选地,所述多核苷酸序列具有SEQ ID NO:12所示的核苷酸序列;
优选地,所述CA10病毒为CA10实心病毒。
第三方面,本发明提供一种用于检测CA10病毒的试剂盒,所述试剂盒包括所述的单克隆抗体或所述的多核苷酸序列编码的单克隆抗体;优选地,所述试剂盒还包括多抗;更优选地,所述多抗为CA10兔多抗;
优选地,所述CA10病毒为CA10实心病毒。
第四方面,本发明提供一种用于诊断手足口病的试剂盒,所述试剂盒包括所述的单克隆抗体或所述的多核苷酸序列编码的单克隆抗体;优选地,所述试剂盒还包括多抗;更优选地,所述多抗为CA10兔多抗;
优选地,所述CA10病毒为CA10实心病毒。
第五方面,本发明提供一种所述的针对CA10病毒的单克隆抗体或所述的多核苷酸序列编码的单克隆抗体在制备用于检测CA10病毒或诊断手足口病的试剂盒中的应用;
优选地,所述CA10病毒为CA10实心病毒。
第六方面,本发明提供一种所述的针对CA10病毒的单克隆抗体或所述的多核苷酸序列编码的单克隆抗体在对含CA10病毒的疫苗的生产进行质量控制中的应用;
优选地,所述CA10病毒为CA10实心病毒。
第七方面,本发明提供一种对含CA10病毒的疫苗的生产进行质量控制的方法,所述方法包括用所述的针对CA10病毒的单克隆抗体或所述的多核苷酸序列编码的单克隆抗体检测CA10病毒的步骤;
优选地,所述CA10病毒为CA10实心病毒。
第八方面,本发明提供一种用于治疗或预防由CA10病毒感染引起的疾病的药物,所述药物含有所述的针对CA10病毒的单克隆抗体或所述的多核苷酸序列编码的单克隆抗体;
优选地,所述CA10病毒为CA10实心病毒。
第九方面,本发明提供一种所述的针对CA10病毒的单克隆抗体或所述的多核苷酸序列编码的单克隆抗体用于检测手足口疫苗制备过程中或疫苗成品中CA10病毒的含量的应用。
优选地,所述应用为用于检测手足口疫苗制备过程中或疫苗成品中CA10实心病毒的含量。
本发明的有益效果在于:
本发明提供了一种主要检测CA10实心病毒颗粒的抗原评价系统。采用本发明提供的由单克隆抗体制备的抗原评价系统,能够快速检测实心病毒,其对空心病毒的结合能力较弱。实心病毒颗粒的比例是疫苗研究工艺中重点关注的指标,与产品的有效性成正相关,在病毒的培养阶段,采用实心系统实时监测实心病毒颗粒的含量,为病毒培养的收获时间提供数据支持。在病毒超速离心纯化阶段,通过实心系统可以监测出收集后的实心病毒管,为后续的超离合并提供指导。采用本发明所述的抗原评价系统不仅能够对疫苗的工艺研究提供指导,而且能够快速的评价产品的有效性。
当然,还可以将抗原评价系统运用于快速检测试剂盒的制备和疫苗的研究开发中。
附图说明
图1为实施例1蔗糖密度梯度离心分离后的离心管照片;
图2为实施例1电镜鉴定的实心病毒颗粒和空心病毒颗粒电镜比较图,图中:左图为22号管实心病毒颗粒,右图为19号管空心病毒颗粒;
图3为实施例3单抗纯化后SDS-PAGE电泳图,图中:2-纯化前,3-纯化洗脱液(重链+轻链),4-纯化流穿液;
图4为实施例5的抗原评价系统线性关系图;
图5为实施例7的抗原评价系统用于超离分段工序样品的评价曲线图。
序列表说明
注:加下划线字体部分为CDR区,加粗字体部分为CH1端引物。
具体实施方式
以下实施例用于说明本发明,但不用来限制本发明的范围。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,实施例中所用的化学试剂均为常规市售试剂,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1CA10空心病毒颗粒和实心病毒颗粒的制备
病毒的培养:采用CA10病毒毒株,培养用细胞为Vero细胞(来源于WHO世界卫生组织)。采用发酵罐微载体的模式培养细胞,将病毒按照MOI=0.0001~0.001接种至发酵罐,培养温度为36.0℃±0.5℃,病毒培养1~5天后收获得到CA10病毒液,采用100KD~300KD的膜包澄清超滤初步纯化。
蔗糖密度梯度离心分离获得不同性质的病毒颗粒:采用蔗糖密度梯度离心的方法对初步纯化后的CA10病毒液进行空空心分离和去除杂质蛋白。蔗糖梯度15%~60%,100000转离心3~15小时。蔗糖密度梯度离心分离出实心病毒条带和空心病毒条带,分别取出两条病毒带,用电镜鉴定空空心分离结果。
蔗糖密度梯度离心分离后的离心管照片见图1所示,从图中可以看出分离出的实心病毒条带和空心病毒条带。电镜鉴定的空心病毒颗粒、实心病毒颗粒电镜比较图见图2。
实施例2CA10实心病毒单克隆抗体的制备
杂交瘤细胞株制备:采用CA10实心病毒纯化液(实心病毒纯化液制备方式:Vero细胞培养CA10病毒,收获病毒液后经澄清超滤初步纯化和浓缩后,经蔗糖梯度密度离心,可获得实心病毒颗粒管和空心病毒颗粒管,将实心病毒颗粒管收集后脱糖处理,即得到CA10实心病毒纯化液。)免疫BALB/c小鼠,每型免疫5只;在0、2、4、6周共4针背部皮下多点免疫;免疫剂量:0.2ml/针/只;佐剂:第1针为弗氏完全佐剂,第2、3针为弗氏不完全佐剂,第4针不加佐剂;采血检测:免疫第3针后1周采血检测间接酶联免疫法效价,对抗体效价达到104以上的小鼠进行第5针腹腔注射进行加强免疫。
细胞融合:腹腔注射加强免疫3天后,处死小鼠取脾融合,两次单克隆化获得阳性杂交瘤细胞株(细胞上清的OD450值>1.0)后,免疫小鼠制备腹水。
从液氮中取出冻存的CA10实心病毒鼠单克隆杂交瘤细胞株进行复苏、扩大培养,106以上数量时提取总核酸,委托北京六合华大基因科技有限公司经PCR扩增单抗的重链和轻链序列并进行测序,然后通过核苷酸序列确定对应的氨基酸序列,测定的多核苷酸序列和氨基酸序列参见序列表。
实施例3CA10实心病毒单克隆抗体的纯化
抗体纯化:将实施例2中免疫小鼠制备的腹水2~8℃、4000~8000r/min离心5~15分钟,取上清经定性滤纸过滤后,采用0.45μm滤膜过滤,经亲和层析获得纯化的单克隆抗体。纯化后检测蛋白质含量为3334μg/ml。对纯化后的单克隆抗体进行纯度检测和效价的测定。
(1)单抗纯度的检测
对纯化后的单抗进行SDS-PAGE电泳,检测IgG重链和轻链所占的比例,采用凝胶成像扫描仪分析单抗的纯度,单抗纯化后SDS-PAGE电泳图见图3。
(2)单抗效价的测定
预包被:CA10纯化液用0.01M磷酸盐缓冲液稀释至0.5~2.0μg/ml,包被96孔酶标板于4℃过夜或37℃2小时。加入终浓度0.05%吐温20的0.01M磷酸盐缓冲液洗2~5遍,再加入含有5~20%小牛血清的0.01M磷酸盐缓冲液于37℃封闭1~2小时,使用时甩去封闭液并拍板去除孔中残留封闭液。
效价测定:待检血清和阴性血清对照均按照10倍梯度法进行系列梯度稀释至108倍,依次加入102至108稀释度样品于上述96孔板,每孔加入100μl,37℃孵育0.5~2小时,0.01M PBST20洗液洗涤2~5遍,加抗小鼠IgG HRP(市售:KPL厂家,购买后直接稀释12000倍使用),于37℃孵育0.5-2小时,洗板2-5遍后,拍干,加入显色底物于37℃显色8~15分钟,2MH2SO4终止,波长450nm处读数。标准:同等稀释倍数下,样品OD450值≥阴性对照×2.1,若阴性对照吸光值<0.05,以0.05计算,判定阳性标准。采用ELISA间接法检测得效价为107。
图3中标记物(Marker)蛋白条带占该泳道总蛋白的比例见下表。
1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | |
标记物 | 0.1339 | 0.1102 | 0.1239 | 0.1945 | 0.0882 | 0.2443 | 0.0363 | 0.0722 | 0.0040 |
检测得到的纯化洗脱液的重链和轻链所占比例分别为:重链占0.6880,轻链占0.3120。从该结果来看,纯化后的单抗洗脱液中重链与轻链的比例之和为100%,说明纯化的效果很好,无杂质蛋白。
实施例4CA10实心病毒单克隆抗体的标酶
将纯化后的单克隆抗体置于透析袋内,在0.05M碳酸盐缓冲液体系中透析2~8小时,每1~2小时换透析液一次;采用高碘酸钠活化HRP,20%乙二醇终止活化。活化后的HRP加至抗体中,继续透析过夜;取出抗体,得到HRP标记的CA10实心病毒单克隆抗体,加入硼氢化钠还原;硫酸铵沉淀后采用0.01MPBS复溶,-20℃以下保存。
实施例5抗原评价系统匹配
将CA10兔多抗(北京科兴生物制品有限公司制备,简称北京科兴)采用碳酸盐缓冲溶液按照一定比例稀释后,包被96孔酶标板于4℃过夜或37℃2小时。加入终浓度0.05%吐温20的0.01M磷酸盐缓冲液洗2~5遍,再加入含有5~20%小牛血清的0.01M磷酸盐缓冲液于37℃封闭1~2小时,使用时甩去封闭液并拍板去除孔中残留封闭液。本发明涉及的技术方案在制成试剂盒时,可提前制成酶标干板,于2-8℃培育。
抗原稀释:将CA10病毒液按照一定浓度进行系列梯度稀释,80U/ml、40U/ml、20U/ml、10U/ml、5U/ml依次加入上述96孔板,每孔加入100μl,37℃孵育0.5-2小时,0.01MPBST20洗液洗涤2~5遍,加实施例4制得的HRP标记后的CA10实心病毒单克隆抗体,于37℃孵育0.5-2小时,洗板2-5遍后,拍干,加入显色底物于37℃显色8~15分钟,2M H2SO4终止,波长450-630nm处读数。考察该抗原评价系统的灵敏度、线性关系。
该抗原评价系统对CA10病毒的检测灵敏度为5U/ml,线性相关性R2≥0.98,结果具体见表1,抗原评价系统线性关系图见图4。
表1灵敏度检测结果
病毒抗原(U/ml) | OD值1 | OD值2 | OD均值 |
空白孔 | 0.069 | 0.073 | 0.071 |
5 | 0.175 | 0.172 | 0.174 |
10 | 0.301 | 0.277 | 0.289 |
20 | 0.562 | 0.525 | 0.543 |
40 | 1.042 | 0.967 | 1.005 |
80 | 1.944 | 1.838 | 1.891 |
实施例6抗原评价系统对空心病毒颗粒或实心病毒颗粒的结合能力评价
参照实施例5中的方法,分别将CA10空心病毒颗粒(来自北京科兴)和CA10实心病毒颗粒(来自北京科兴)按照一定的浓度系列稀释后加入预先包被好的酶标板(CA10兔多抗包被的96孔酶标板)中,检测上述抗原评价系统对空心病毒颗粒和实心病毒颗粒的结合能力差异。按照达到相同OD值的条件下,加入的空心病毒、实心病毒蛋白浓度比值的倒数即为该抗原评价系统对空心病毒颗粒和实心病毒颗粒的反应能力比值。实验结果显示,该抗原评价系统对实心病毒颗粒的反应能力是与空心病毒颗粒反应能力的80倍,结果见表2。
表2对实心病毒和空心病毒反应能力的比较结果
实施例7抗原评价系统在疫苗生产中的应用
在疫苗的生产阶段,可采用该抗原评价系统对整个工艺流程的样品进行抗原含量评价。本实施例重点介绍该系统对蔗糖密度梯度离心各管样品的检测。经电镜观察,19号管为实心病毒管,22号管为空心病毒管。将抗原标准品和样品分别稀释至一定的浓度,加入预先包被好多抗的酶标板,参照实施例5步骤执行,将参比品的OD值与抗原标示量制作标准曲线,样品OD值带入标准曲线计算样品的抗原含量,检测结果见表3,图5所示为抗原评价系统用于超离分段工序样品的评价曲线图。
表3抗原评价系统应用于超离分段样品的评价结果
病毒液经蔗糖密度梯度离心后,实心病毒颗粒与空心病毒颗粒分别分布在不同糖度区域中,19号管为空心病毒区域,22号管为实心病毒颗粒。表3为采用本发明所建立的抗原系统进行抗原检测的ELISA结果,22号管为抗原的检测峰值,说明本系统主要与实心病毒颗粒反应。
实施例8抗原评价系统专属性的验证
参照实施例5方法,分别加入EV71病毒液、CA16病毒液、CA10病毒液、甲肝病毒液、脊髓灰质炎病毒Ⅰ型原液、脊髓灰质炎病毒Ⅱ型原液、脊髓灰质炎病毒Ⅲ型原液、样品稀释液、CA10病毒纯化液、CA10感染小鼠粪便样品(粪便样品采用PBS缓冲液稀释后离心取上清)和CA10感染小鼠血清样品(眼球采血、离心分离血清),验证该抗原评价系统对肠道病毒检测的专属性。
结果显示,该抗原评价系统具有很好的专属性,对其它型别的肠道病毒均不反应。结果见表4。表4结果说明,本抗原评价系统对CA10病毒检测具有专属特异性。
表4抗原评价系统专属性验证结果
虽然,上文中已经用一般性说明、具体实施方式及试验对本发明作了详尽的描述,但在本发明基础上,可以对之作一些修改或改进,这对本领域技术人员而言是显而易见的。因此,在不偏离本发明精神的基础上所做的这些修改或改进,均属于本发明要求保护的范围。
SEQUENCE LISTING
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<120> 柯萨奇病毒A10型实心病毒反应的单克隆抗体及抗原检测系统
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Claims (16)
1.针对CA10病毒的单克隆抗体,其特征在于,所述单克隆抗体具有SEQ ID NO:5所示的重链互补决定区CDR1、SEQ ID NO:6所示的重链互补决定区CDR2、SEQ ID NO:7所示的重链互补决定区CDR3,以及SEQ ID NO:13所示的轻链互补决定区CDR1、SEQ ID NO:14所示的轻链互补决定区CDR2、SEQ ID NO:15所示的轻链互补决定区CDR3。
2.根据权利要求1所述的单克隆抗体,其特征在于,所述单克隆抗体具有SEQ ID NO:8所示的氨基酸序列的重链;和/或,所述单克隆抗体具有SEQ ID NO:16所示的氨基酸序列的轻链。
3.根据权利要求1或2所述的单克隆抗体,其特征在于,所述CA10病毒为CA10实心病毒。
4.编码权利要求1-3中任一项所述的针对CA10病毒的单克隆抗体的多核苷酸序列,其特征在于,所述多核苷酸序列具有SEQ ID NO:1所示的编码重链互补决定区CDR1的序列、SEQ ID NO:2所示的编码重链互补决定区CDR2的序列、SEQ ID NO:3所示的编码重链互补决定区CDR3的序列,以及SEQ ID NO:9所示的编码轻链互补决定区CDR1的序列、SEQ ID NO:10所示的编码轻链互补决定区CDR2的序列、SEQ ID NO:11所示的编码轻链互补决定区CDR3的序列。
5.根据权利要求4所述的多核苷酸序列,其特征在于,所述多核苷酸序列具有SEQ IDNO:4所示的核苷酸序列;和/或,所述多核苷酸序列具有SEQ ID NO:12所示的核苷酸序列。
6.根据权利要求4或5所述的多核苷酸序列,其特征在于,所述CA10病毒为CA10实心病毒。
7.用于检测CA10病毒的试剂盒,其特征在于,所述试剂盒包括权利要求1-3中任一项所述的单克隆抗体;其中,所述CA10病毒为CA10实心病毒。
8.根据权利要求7所述的试剂盒,其特征在于,所述试剂盒还包括多抗。
9.根据权利要求8所述的试剂盒,其特征在于,所述多抗为CA10兔多抗。
10.用于诊断手足口病的试剂盒,其特征在于,所述试剂盒包括权利要求1-3中任一项所述的单克隆抗体。
11.根据权利要求10所述的试剂盒,其特征在于,所述试剂盒还包括多抗。
12.根据权利要求11所述的试剂盒,其特征在于,所述多抗为CA10兔多抗。
13.权利要求1-3中任一项所述的针对CA10病毒的单克隆抗体在制备用于检测CA10病毒或诊断手足口病的试剂盒中的应用;
其中,所述CA10病毒为CA10实心病毒。
14.权利要求1-3中任一项所述的针对CA10病毒的单克隆抗体在对含CA10病毒的疫苗的生产进行质量控制中的应用;
其中,所述CA10病毒为CA10实心病毒。
15.对含CA10病毒的疫苗的生产进行质量控制的方法,其特征在于,所述方法包括用权利要求1-3中任一项所述的针对CA10病毒的单克隆抗体检测CA10病毒的步骤;其中,所述CA10病毒为CA10实心病毒。
16.权利要求1-3中任一项所述的针对CA10病毒的单克隆抗体用于检测手足口疫苗制备过程中或疫苗成品中CA10病毒的含量的应用,其中所述CA10病毒为CA10实心病毒。
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