CN116284349B - 一种猴痘抗体检测试剂盒 - Google Patents
一种猴痘抗体检测试剂盒 Download PDFInfo
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Abstract
本发明涉及一种猴痘抗体检测试剂盒。本发明以重组猴痘A29蛋白作为免疫原，通过小鼠杂交瘤技术制备并获得了相应的单克隆抗体，以和A29同源性达到95％以上的痘苗病毒A27蛋白作为反筛蛋白，筛选获得了特异性针对A29的单克隆抗体。所述单克隆抗体通过与市售的A29单克隆抗体相比具有同样的特异性并且具有更好的亲和活性，可以用于制备相应的检测是结合并用于相应的猴痘检测，应用市场价值较高。
Description
技术领域
本申请涉及生物领域，更具体的涉及生物检测方面，主要涉及一种猴痘抗体检测试剂盒。
背景技术
猴痘通常是一种自限性疾病，持续14～21d多可自愈。猴痘病毒主要包括西非和刚果盆地两个分支，其中西非分支的致死率约1％，刚果盆地分支的致死率约10％，儿童、青年和免疫缺陷者感染后致死率较高。
猴痘病毒属于痘病毒科(Poxviridae)，痘病毒为病毒颗粒最大的一类DNA双链线性病毒，病毒颗粒呈砖形或椭圆形，大小为200～400nm，病毒在感染细胞的细胞质中复制。痘病毒科包括两个病毒亚科:脊椎动物痘病毒亚科(Chordopoxvirinae)和昆虫痘病毒亚科(Entomopoxvirinae)。脊椎动物痘病毒亚科包含9个属:正痘病毒属(Orthopoxvirus)、副痘病毒属(Parapoxvirus)、猪痘病毒属(Suipoxvirus)、山羊痘病毒属(Capripoxvirus)、鹿痘病毒属(Cervidopoxvirus)、兔痘病毒属(Leporipoxvirus)、鸡痘病毒属(Avipoxvirus)、雅塔痘病毒属(Yatapoxvirus)和软疣病毒属(Molluscipoxvirus)，猴痘病毒、牛痘病毒(Cowpoxvirus)与著名的天花病毒(Smallpox)同属于正痘病毒属。其中，可导致人类疾病的主要是正痘病毒和副痘病毒，包括天花病毒、猴痘病毒、牛痘病毒、骆驼痘病毒(Camelpoxvirus)和痘苗病毒(Vacciniavirus)。由于亲缘关系相近，接种天花疫苗可有效抵抗猴痘病毒感染，但自1980年停止接种天花疫苗后，猴痘病毒感染病例呈上升趋势。
猴痘病毒的主要传播方式是通过直接接触到受感染的动物的血液、体液、皮肤或黏膜伤口。人类之间的二次传播主要由于密切接触了感染者的呼吸道分泌物、皮肤损伤，或被患者体液或病变组织污染的物品(如患者皮疹破裂后流出的液体沾染的衣物、床单等)。猴痘病毒能在呼吸道飞沫中存在，但通常需要长时间面对面接触才能传播。从病毒总体的传播力来看，猴痘病毒是一种弱病毒，主要通过近距离接触传播，对健康人的致病性不强，因此不必恐慌。但是有效的检测仍然是必不可少的防控手段。
猴痘的最佳诊断样本来自皮肤病变—囊泡和脓疱液体，以及干燥的结痂。病变样本必须储存在干燥、无菌的试管中(无病毒运输介质)并保持低温。猴痘的临床诊断必须考虑其他皮疹疾病如：水痘、麻疹、细菌性皮肤感染、疥疮、梅毒和药物相关过敏。发病前期的淋巴结病可能是区分猴痘与水痘或天花的唯一临床特征。电镜活检：电镜下MPXV呈胞浆内砖状，有侧体，中心核约200-300nm。由于OPV物种在形态上无法区分，该方法不能确定诊断，但可以提示该病毒属于痘病毒科。基因检测：MPXV检测推荐标本类型是皮肤病变部位，用力擦拭病变部位，也可采集口咽拭子以确保收集到足够的病毒DNA。MPXV检测是基于核酸扩增测试(NAAT)。NAAT可以通用于正痘病毒(OPXV)也可特异性用于猴痘病毒(MPXV)。使用实时或常规的聚合酶链式反应(PCR)来检测病毒DNA的独特序列。聚合酶链式反应可以单独使用，也可以与测序结合使用。实时荧光定量PCR：对临床、MPXV感染细胞培养物的MPXVDNA进行常规检测，使用PCR或real-time PCR，建议在生物安全三级设施中进行。采用real-timePCR方法针对细胞外包膜蛋白基因(B6R)、DNA聚合酶基因、E9L的保守区，DNA依赖RNA聚合酶亚基18、rpo18和F3L基因进行检测。限制性内切酶片段长度多态性(RFLP)：PCR扩增基因或基因片段的限制性长度片段多态性(Restriction length fragment polymorphism,RFLP)也可用于MPXV DNA的检测，但RFLP费时且需要病毒培养。PCR产物的RFLP也需要酶切后进行凝胶电泳，在快速、敏感和特异性至关重要的临床环境中可能不是最佳的方法。高通量测序技术
(NGS)：采用NGS技术的全基因组测序仍然是区分MPXV和其他正痘病毒(OPV)表征的金标准，但该技术成本较高，测序数据的下游处理需要巨大的计算能力。因此，在撒哈拉以南非洲资源贫乏的国家NGS可能不是合适方法。基因检测技术中real-time PCR仍是MPXV常规诊断的首选方法，但必须辅以现场基因组测序技术，如OxfordNanopore MinION，才能提供实时的病毒基因组数据，这对于循证流行病学干预是必不可少。免疫检测方法：猴痘病毒免疫检测方法主要包括酶联免疫吸附试验(ELISA)检测IgG、IgM抗体和免疫组化检测病毒抗原。用MPXV抗体进行免疫化学分析，可区分痘病毒感染和疱疹病毒。已经证实，抗病毒抗体和T细胞反应在疾病发作前后增加，在皮疹发生后5天左右和8天以上的血清中分别检测到猴痘病毒IgM和IgG。如果有皮疹和严重疾病史的未接种疫苗的个体血清中存在IgM和IgG抗体，则诊断可能存在MPXV感染。IgM捕获ELISA阳性表明最近接触过MPXV，而IgG捕获ELISA阳性表明该个体以前通过接种疫苗或自然感染接触过MPXV。在一个样本中同时存在IgM和IgG，表明在以前接种过疫苗或接触过自然感染MPXV的个体。然而，正痘病毒具有血清学交叉反应性，抗原和抗体检测方法不能提供猴痘特异性确认，但对于MPXV流行地区的血清学检测也许是可行的方法。
目前，针对猴痘检测已经有多种方法。CN102839224A中根据genbank数据库，分析猴痘各亚型基因组序列，经过序列比对，选取片段在猴痘病毒基因组genbank编码HQ857563.1中的位置为48332-48780bp之间，共448个碱基，设计了特异性的PCR引物，实现了对猴痘病毒的检测。CN101407850B公开了一种猴痘病毒的PCR快速检测试剂盒及其检测方法，包括试剂A、B、PCR试剂管、TaqDNA聚合酶、含有猴痘病毒特异的F3L基因片断的pUC57质粒的阳性对照和灭菌的双蒸水阴性对照组成的试剂盒。采用简单快速敏感的PCR检测试剂盒，根据猴痘病毒所特有的一段保守序列设计特异引物并进行扩增比较，在3-4小时即可得知猴痘病毒检测结果，灵敏度较高，检测下限低至0.3pg的DNA浓度，确保了检测的准确性与特异性。特别适用于出入境检验检疫中，对来自美国和非洲的实验鼠等饲养啮齿动物以及猴等灵长类动物、家兔及其产品中猴痘病毒进行高灵敏快速检测。
但是目前，由于PCR扩增需要特定的仪器并不适宜灵活使用，因此，开发特异性的基于ELISA的检测方法是研究的重要方向。
发明内容
本发明克服现有技术的缺陷，提供一种特异性针对猴痘病毒的单克隆抗体。
本发明以重组猴痘A29蛋白作为免疫原，通过小鼠杂交瘤技术制备并获得了相应的单克隆抗体，以和A29同源性达到95％以上的痘苗病毒A27蛋白作为反筛蛋白，筛选获得了特异性针对A29的单克隆抗体。
具体的，所述的猴痘A29单克隆抗体是7H11。
进一步的，所述猴痘A29单克隆抗体的轻链可变区如SEQ ID NO：1所示，重链可变区如SEQ ID NO：2所示。
进一步的，所述的单克隆抗体在轻链和重链分别被同源性肽序列替换。两个肽序列共有至少80％序列同一性，优选至少90％序列同一性，更优选至少95％序列同一性和最优选至少99％序列同一性。优选地，不相同的残基位置由于保守性氨基酸取代不同。保守性氨基酸取代指具有相似侧链的残基的可交换性。例如，一组具有脂肪族侧链的氨基酸为甘氨酸、丙胺酸、缬氨酸、亮氨酸和异亮氨酸；一组具有脂肪族羟基侧链的氨基酸为丝氨酸和苏氨酸；一组具有含酰胺的侧链的氨基酸为天冬酰胺和谷氨酰胺；一组具有芳香族侧链的氨基酸为苯丙氨酸、酪氨酸和色氨酸；一组具有碱性侧链的氨基酸为赖氨酸、精氨酸和组氨酸；以及一组具有含硫侧链的氨基酸为半胱氨酸和甲硫氨酸。优选的保守性氨基酸取代基团为：缬氨酸-亮氨酸-异亮氨酸、苯丙氨酸-酪氨酸、赖氨酸-精氨酸、丙胺酸-缬氨酸、谷氨酸-天冬氨酸和天冬酰胺-谷氨酰胺。
进一步的，认为抗体的氨基酸序列的变化涵盖于本发明中，条件是氨基酸序列的变化保持至少75％，更优选至少80％、90％、95％和最优选99％。包括其间的某些百分比，例如75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％和99％序列同一性。尤其，包括保守性氨基酸置换。保守性置换是在与侧链相关的氨基酸家族中发生的置换。遗传编码的氨基酸通常分为以下家族：(1)酸性氨基酸为天冬氨酸、谷氨酸；(2)碱性氨基酸为赖氨酸、精氨酸、组氨酸；(3)非极性氨基酸为丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸、色氨酸，和(4)不带电的极性氨基酸为甘氨酸、天冬酰胺、谷氨酰胺、半胱氨酸、丝氨酸、苏氨酸、酪氨酸。亲水性氨基酸包括：精氨酸、天冬酰胺、天冬氨酸、谷氨酰胺、谷氨酸、组氨酸、赖氨酸、丝氨酸和苏氨酸。疏水性氨基酸包括丙氨酸、半胱氨酸、异亮氨酸、亮氨酸、甲硫氨酸、苯丙氨酸、脯氨酸、色氨酸、酪氨酸和缬氨酸。其它氨基酸家族包括(i)为脂肪族羟基家族的丝氨酸和苏氨酸；(ii)为含有酰胺的家族的天冬酰胺和谷氨酰胺；(iii)为脂肪族的丙氨酸、缬氨酸、亮氨酸和异亮氨酸；和(iv)为芳香族的苯丙氨酸、色氨酸和酪氨酸。例如，有理由期望用异亮氨酸或缬氨酸单独置换亮氨酸，用谷氨酸单独置换天冬氨酸，用丝氨酸单独置换苏氨酸，或用结构相关的氨基酸对氨基酸进行相似置换对所得分子的结合或性质无较大影响，特别是如果置换不牵涉骨架位点内的氨基酸时。通过检测多肽衍生物的特异性活性可易于确定氨基酸变化是否产生功能性肽。本文对检测进行了详细描述。本领域的普通技术人员可易于制备抗体或免疫球蛋白分子的片段或类似物。片段或类似物的优选氨基端和羧基端出现在功能结构域边界附近。可通过比较核苷酸和/或氨基酸序列数据和公共或私有序列数据库鉴定结构和功能结构域。优选地，使用计算机化比较法鉴定其它已知结构和/或功能的蛋白质中出现的序列基序或预测蛋白质构象结构域。鉴定折叠成已知三维结构的蛋白质序列的方法已知。
进一步的，本发明提供一种用于猴痘检测的试剂盒，其特征在于含有本发明制备的猴痘A29单克隆抗体。
进一步的，本发明提供一种采用免疫测定检测猴痘的方法。
本发明的免疫测定定法，可举出：酶免疫测定法(EIA)、酶免疫测定法(ELISA)、荧光免疫测定法(FIA)、放射线免疫测定法(RIA)、发光免疫测定法、免疫印迹法、蛋白质印迹法等，从能够简便且灵敏度良好地检测抗体的角度出发，优选ELISA法。
进一步的，本发明的单克隆抗体还可以被可检测的标记所标记。
进一步的，具体的标记是采用辣根过氧化物酶标记。
有益效果
本发明以重组猴痘A29蛋白作为免疫原，通过小鼠杂交瘤技术制备并获得了相应的单克隆抗体，以和A29同源性达到95％以上的痘苗病毒A27蛋白作为反筛蛋白，筛选获得了特异性针对A29的单克隆抗体。所述单克隆抗体通过与市售的A29单克隆抗体相比具有同样的特异性并且具有更好的亲和活性，可以用于制备相应的检测是结合并用于相应的猴痘检测，应用价值广阔，市场价值较高。
附图说明
图1抗体亚型鉴定结果图
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述，实施例中未注明具体条件，按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者，均为可以通过市场购买获得的常规产品。所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例，基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
实施例1猴痘A29单克隆抗体的制备
取猴痘A29重组蛋白VAP0003，VAPOL作为免疫原(80μg)，与等体积弗氏完全佐剂充分乳化，取乳化液背部皮下多点注射免疫7-8周龄雌性BALB/c小鼠，0.3mL/只，一共免疫3只。间隔2周，用弗氏不完全佐剂与免疫原1:1乳化充分，按上述方法进行第二次免疫。间隔2周再进行第三次免疫，同样方法进行第四次免疫，第四次免疫后5d，通过间接ELISA方法测定免疫小鼠血清抗体效价，取血清效价大于1:10000的2号小鼠进行腹腔注射未加佐剂的免疫原100μg加强免疫。加强免疫3d后，采取小鼠脾脏进行细胞融合。
经加强免疫的BALB/c小鼠3天后摘除眼球放血致死，浸泡于75％乙醇中5min消毒体表，放入解剖台板上固定四肢。无菌手术开腹取出脾脏，除去被膜并用培养基冲洗。将脾脏置于200目带柄不锈钢细胞筛网中，将筛网置于加有DMEM基础培养基的10cm培养皿中，用研磨棒轻轻挤压脾脏，并用少量培养基冲洗筛网。用滴管将脾细胞移入离心管中，1000r/min，离心10min，用DMEM基础培养基重悬、计数，备用。
取对数生长期的SP2/0细胞与上述的脾细胞按1:9的比例混合于50mL离心管中充分混匀，1000r/min离心10min，弃上清。用手掌轻击离心管底，将沉淀的细胞打散将离心管底部放入37℃水浴中，在1min内边旋转边加入融合用的PEG40001mL，然后继续旋转并加入DMEM基础培养基15mL。室温静置10min后，1000r/min离心10min，弃上清，将HAT选择培养液加入到细胞中，悬浮细胞，分配到己加有饲养细胞的96孔细胞培养板，置于37℃，5％二氧化碳培养箱中培养，5d后用新鲜预热的HAT培养基半换液，10d后用HT培养液全换液。
观察杂交瘤细胞的生长情况，待其生长至孔底面积的1/3以上时，吸取适量细胞上清利用间接ELISA法对杂交瘤细胞进行筛选包被，其中包被的抗原分别是免疫原A29重组蛋白，以及反筛作用的痘苗病毒A27蛋白(PT18098，科瑞思搏)。共筛选获得对A29高阳性，对A27阴性的杂交瘤细胞13株。将13株杂交瘤细胞进行四次亚克隆，直至每个克隆上清均为阳性时定株，共获得2株阳性杂交瘤细胞5D16和7H11。
将二株杂交瘤细胞分别制备小鼠腹水，并用Protein G亲和层析介质进行纯化，鉴定都为纯化的抗体后，对蛋白浓度进行定量备用。
实施例27H11单克隆抗体亚类鉴定
按照单克隆抗体亚类鉴定试剂盒说明书操作，具体方法如下：用PBST将单抗进行1:2000倍稀释，加入酶标板，100μl/孔，37℃孵育1h，PBST洗涤3次，每次5min拍干；分别加入工作浓度的羊抗鼠亚类血清(IgG1、IgG2a、IgG2b、IgG3、IgM和IgA)每孔100μl，37℃作用30min，PBST洗涤3次，每次5min，拍干；每孔加入用PBST稀释8000倍的兔抗羊辣根过氧化物酶结合物100μl，37℃作用15min，PBST洗涤3次，每次5min，拍干；每孔加入TMB显色液100μl，避光作用15min；每孔加入2mol/L浓硫酸50μl，终止反应。用酶标检测仪测定各孔的OD450值，读数最高的对应亚类血清即为单抗亚类。
经单克隆抗体亚类鉴定试剂盒鉴定腹水单抗亚类，结果如图1显示，单抗7H11为Ig2a亚类。
实施例27H11单克隆抗体特异性鉴定
用PBST将7H11单抗进行1:2000倍稀释，加入酶标板，100μl/孔，37℃孵育1h，PBST洗涤3次，每次5min拍干；分别加入不同的抗原蛋白(A29重组蛋白、痘苗病毒A27蛋白、猴痘A30L重组蛋白、BSA、正常人血清)每孔100μl，37℃作用30min，PBST洗涤3次，每次5min，拍干；每孔加入用PBST稀释8000倍的兔抗羊辣根过氧化物酶结合物100μl，37℃作用15min，PBST洗涤3次，每次5min，拍干；每孔加入TMB显色液100μl，避光作用15min；每孔加入2mol/L浓硫酸50μl，终止反应。用酶标检测仪测定各孔的OD450值。同样操作条件下，使用市售的A29单克隆抗体RM3386作为对照单克隆抗体。结果见表2所示。
表1各孔OD450值
| 蛋白类型 | 7H11单抗 | 对照抗体 |
| A29 | 5.26±0.13 | 3.89±0.10 |
| A27 | 0.38±0.04 | 0.38±0.06 |
| A30L | 0.26±0.02 | 0.20±0.03 |
| BSA | 0.22±0.05 | 0.19±0.04 |
| 正常人血清 | 0.21±0.03 | 0.24±0.03 |
从表1的反应结果来看本发明的抗体具有较好的特异性，只能特异性的结合A29重组蛋白，而不能与其他的常见的抗原进行反应，具有较好的特异性。而且本发明的单克隆抗体与对照抗体相比，也具有相似的特异性效果。
实施例37H11单克隆抗体亲和力鉴定
利用Fortibio测定抗体结合及解离常数。利用OctetRED(Fortebio,USA)仪器，测定7H11单克隆抗体亲和力。PBS稀释抗体浓度为10μg/mL，包被AHC传感器；PBS为对照，稀释7H11单克隆抗体浓度为0.0001μg/mL、0.001μg/mL、0.01μg/mL、0.1μg/mL、1μg/mL、10μg/mL、100μg/mL、测定7H11单克隆抗体与A29蛋白相互作用的结合、解离曲线；利用GlobeFitting拟合曲线，计算两者相互作用的解离常数。以Vazyme市售的A29单克隆抗体RM3386为对照单克隆抗体。结果如表2所示。
表2各抗体解离常数
| 抗体名称 | 解离常数 |
| 7H11单克隆抗体 | (0.36±0.05)nM |
| 对照抗体 | (8.36±0.11)nM |
从表2的结果可以看出，本发明获得的单克隆抗体其解离常数达到了(0.36±0.05)nM。与对照抗体的(8.36±0.11)nM相比，具有更好的结合A29蛋白的能力，效果提高显著。
实施例47H11单克隆抗体可变区序列的鉴定
收集7H11杂交瘤细胞，Trizol提取杂交瘤细胞总RNA，将mRNA逆转录为cDNA，以cDNA为模板，通过NCBI数据库查找鼠源重链FR1区及铰链区基因序列，根据序列设计重链PCR引物，上游序列为：GCGCTCGAGCAGKTCCAGCTGAAGCAGTC，下游序列为：GCGACTAGTGCATTTGCATGGAGGACAG；同样通过NCBI数据库查找鼠源轻链FR1区及恒定区基因序列(NC000072.6)，设计轻链PCR引物，上游序列为：GCGGAGCTCGATRTTGTGATGACCCARAC，下游序列为：
GCGTCTAGACTCATTCCTGTTGAAGCTCT，分别PCR获得抗体的轻链和重链片段。对PCR产物进行胶回收，进行测序分析，获得7H11杂交瘤细胞的轻链可变区氨基酸序列为SEQ.ID.NO.1，重链可变区序列氨基酸序列为SEQ.ID.NO.2。
应当理解的是，本发明在其应用上并不一定局限于在以下说明中所描述和/或在附图中所说明的组件的构造和布置的细节。本发明能够具有除所述和以不同方式实践或进行的那些实施方案之外的实施方案。而且，应理解本文所采用的短语和术语以及摘要出于描述目的并且不应视为限制性的。
虽然已描述并详细举例说明本发明至足以使本领域技术人员制备和使用它，但是在不背离本发明的精神和范围的情况应清楚各种替代方案、修改和改进。本文所提供的实施例代表优选的实施方案，为示例性的，并且不旨在为对本发明的范围的限制。其中的修改和其他用途将是本领域技术人员能够想到的。在本发明的精神内涵盖这些修改并且由权利要求书的范围限定这些修改。
Claims (7)
1.一种特异性结合猴痘A29蛋白的单克隆抗体7H11，其特征在于轻链可变区如SEQ IDNO：1所示，重链可变区如SEQ ID NO：2所示。
2.一种特异性检测猴痘的试剂盒，其特征在于含有权利要求1所述的单克隆抗体。
3.如权利要求2所述的试剂盒，其特征在于所述的单克隆抗体被可检测的标记所标记。
4.如权利要求3所述的试剂盒，其特征在于标记是采用辣根过氧化物酶标记。
5.权利要求1所述的特异性结合猴痘A29蛋白的单克隆抗体7H11在制备用于检测猴痘病毒的试剂盒中的用途。
6.如权利要求5所述的用途，其特征在于所述的单克隆抗体被可检测的标记所标记。
7.如权利要求6所述的用途，其特征在于标记是采用辣根过氧化物酶标记。
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