CN112321706B - 可用于诊断小反刍兽疫的单克隆抗体及诊断试剂盒 - Google Patents
可用于诊断小反刍兽疫的单克隆抗体及诊断试剂盒 Download PDFInfo
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- CN112321706B CN112321706B CN202011011141.5A CN202011011141A CN112321706B CN 112321706 B CN112321706 B CN 112321706B CN 202011011141 A CN202011011141 A CN 202011011141A CN 112321706 B CN112321706 B CN 112321706B
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Abstract
本发明涉及可用于诊断小反刍兽疫的单克隆抗体及诊断试剂盒。本发明的单克隆抗体能够特异性结合小反刍兽疫病毒。本发明的小反刍兽疫诊断试剂盒包括基底、以及固定于所述基底上的本发明的单克隆抗体，其可用于检测对象生物来源的生物样本中是否存在小反刍兽疫病毒。
Description
本发明专利申请为申请日2019年04月08日,申请号201910275169.0,发明创造名称为《可用于检测小反刍兽疫病毒的单克隆抗体及试剂盒》的发明专利申请的分案申请。
技术领域
本发明属于生物检测及医学检测技术领域，具体涉及一种可用于诊断小反刍兽疫的单克隆抗体及诊断试剂盒。
背景技术
小反刍兽疫(peste des petits ruminants，PPR)俗称羊瘟，又名假性牛瘟(pseudorinderpest)、肺肠炎(pneumoenteritis)、口炎肺肠炎复合症(stomatitis-pneumoenteritis complex)，是由小反刍兽疫病毒引起的一种急性病毒性传染病，主要感染小反刍动物，以发热、口炎、腹泻、肺炎为特征。
1942年本病首次在象牙海岸发生，其后，非洲的塞内加尔、加纳、多哥、贝宁等有本病报道，尼日利亚的绵羊和山羊中也发生了本病，并造成了重大损失。亚洲的一些国家也报道了本病，根据世界动物卫生组织(OIE)1993年《世界动物卫生》报道，孟加拉国的山羊有本病发生，印度德拉邦和马哈拉施特拉邦的部分地区绵羊中发生了类似牛瘟的疾病，最后确诊为小反刍兽疫，此后，泰米尔拉德邦也有受到感染报道。1993年，以色列第一次报道有小反刍兽疫发生，传染来源不明，为防止本病传播，以色列对其北部地区的绵羊和山羊接种了牛瘟疫苗。1992年，约旦的绵羊和山羊中发现了本病特异性抗体，1993年，有11个农场出现临诊病例，100多只绵羊和山羊死亡。1993年，沙特阿拉伯首次发现133个病例。2007年7月，该疫病首次传入我国西藏地区。
小反刍兽疫病毒属副黏病毒科麻疹病毒属。与牛瘟病毒有相似的物理化学及免疫学特性。病毒呈多形性，通常为粗糙的球形。病毒颗粒较牛瘟病毒大，核衣壳为螺旋中空杆状并有特征性的亚单位，有囊膜。病毒可在胎绵羊肾、胎羊及新生羊的睾丸细胞、Vero细胞上增殖，并产生细胞病变(CPE)，形成合胞体。
本病主要感染山羊、绵羊、美国白尾鹿等小反刍动物，流行于非洲西部、中部和亚洲的部分地区。在疫区，本病为零星发生，当易感动物增加时，即可发生流行。本病主要通过直接接触传染，病畜的分泌物和排泄物是传染源，处于亚临诊型的病羊尤为危险。小反刍兽疫潜伏期为4-5天，最长21天。自然发病仅见于山羊和绵羊。山羊发病严重，绵羊也偶有严重病例发生。一些康复山羊的唇部形成口疮样病变。感染动物临诊症状与牛瘟病牛相似。急性型体温可上升至41℃，并持续3～5天。感染动物烦躁不安，背毛无光，口鼻干燥，食欲减退。流黏液脓性鼻漏，呼出恶臭气体。在发热的前4天，口腔黏膜充血，颊黏膜进行性广泛性损害、导致多涎，随后出现坏死性病灶，开始口腔黏膜出现小的粗糙的红色浅表坏死病灶，以后变成粉红色，感染部位包括下唇、下齿龈等处。严重病例可见坏死病灶波及齿垫、腭、颊部及其乳头、舌头等处。后期出现带血水样腹泻，严重脱水，消瘦，随之体温下降。出现咳嗽、呼吸异常。发病率高达100％，在严重暴发时，死亡率为100％，在轻度发生时，死亡率不超过50％。幼年动物发病严重发病率和死亡都很高，为我国划定的一类疾病。
因本病毒对胃肠道淋巴细胞及上皮细胞具有特殊的亲和力，故能引起特征性病变。一般在感染细胞中出现嗜酸性胞浆包涵体及多核巨细胞。在淋巴组织中，小反刍兽疫病毒可引起淋巴细胞坏死。脾脏、扁桃体、淋巴结细胞被破坏。含嗜酸性胞浆包涵体的多核巨细胞出现，极少有核内包涵体。在消化系统，病毒引起马尔基氏层深部的上皮细胞发生坏死，感染细胞产生核固缩和核破裂，在表皮生发层形成含有嗜酸性胞浆包涵体的多核巨细胞。
目前，尚无有效方法治疗小反刍兽疫，只能采取疫苗接种、疫情发生后扑杀及定期血清监测的方法来进行控制。
目前，小反刍兽疫主要是通过检测生物样本中的小反刍兽疫病毒抗体来进行诊断。世界动物卫生组织推荐采用的小反刍兽疫病毒抗体检测方法主要有病毒中和试验(VNT)和酶联免疫测定(ELISA)。其中，病毒中和试验方法的检测结果准确，是检测小反刍兽疫病毒的金标准，但该方法检测时间长且不适于检测大量样本。与此相对，酶联免疫测定的特异性和敏感性较高、检测时间比病毒中和试验短且适合检测大量样品，因此，被广泛应用于小反刍兽疫病毒抗体的检测。
但是，生物样本中存在针对小反刍兽疫病毒抗体并非目标生物罹患小反刍兽疫的直接证据，这是通过检测抗体来诊断小反刍兽疫的诊断方法的固有不足。因此，希望有直接检测生物样本中的小反刍兽疫病毒的ELISA方法。
但是，ELISA方法均需使用单克隆抗体，而正如前述，小反刍兽疫病毒是一种囊膜病毒，可用于ELISA方法检测小反刍兽疫病毒的单克隆抗体不容易获得。
发明内容
鉴于上述现有技术中存在的问题，本发明的目的在于提供一种能够特异性与小反刍兽疫病毒结合的单克隆抗体，以及利用了该单克隆抗体的特异性和敏感性高的小反刍兽疫诊断试剂盒。
发明人为解决上述技术问题进行了深入研究，用经纯化的小反刍兽疫病毒免疫家兔制备了兔抗小反刍兽疫病毒单克隆抗体(代号：NMS-1-17H6L3)，该兔抗小反刍兽疫病毒单克隆抗体能够特异性地结合小反刍兽疫病毒，并能够用于制备基于ELISA原理的小反刍兽疫诊断试剂盒。
即，本发明包括：
一种可用于诊断小反刍兽疫的单克隆抗体，其包含三个重链互补决定区(CDR-H1、CDR-H2和CDR-H3)和三个轻链互补决定区(CDR-L1、CDR-L2和CDR-L3)，其中：
(a)CDR-H1的氨基酸序列如SEQ ID NO：1所示；
(b)CDR-H2的氨基酸序列如SEQ ID NO：2所示；
(c)CDR-H3的氨基酸序列如SEQ ID NO：3所示；
(d)CDR-L1的氨基酸序列如SEQ ID NO：4所示；
(e)CDR-L2的氨基酸序列如SEQ ID NO：5所示；且
(f)CDR-L3的氨基酸序列如SEQ ID NO：6所示。
其中，
SEQ ID NO：1：GIDLGGYA(氨基酸的单字母符号，下同)
SEQ ID NO：2：IDTTDS
SEQ ID NO：3：ARYAGDGGGGYFFDY
SEQ ID NO：4：QSVYNNNC
SEQ ID NO：5：GAS
SEQ ID NO：6：VGAYIGSNYA
上述的单克隆抗体，其包含重链和轻链，其中，
所述重链的氨基酸序列如SEQ ID NO：7所示；且，
所述轻链的氨基酸序列如SEQ ID NO：8所示。
其中，
SEQ ID NO：7：
CQSLEESGGRLVTPGGSLTLTCTVSGIDLGGYAVGWVRQAPGKGLEYIGIIDTTDSTYYASWAKGRFTSSKTSSTTVDLKMTSLTTEDTATYFCARYAGDGGGGYFFDYWGSGTLVTITSGQPKAPSVFPLAPCCGDTPSSTVTLGCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKPTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVSTLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEWEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK
SEQ ID NO：8：
AAVLTQTPSPISAAVGGTVTINCQSSQSVYNNNCLSWYQQKPGQPPKFLIYGASTLASGVPSRFKGSGSGTEFTLTISDVQCADAATYYCVGAYIGSNYAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC
一种检测器件，其包括基底、以及固定于所述基底上的上述单克隆抗体。
上述的检测器件，其用于检测对象生物来源的生物样本中是否存在小反刍兽疫病毒。
上述的检测器件，其中，所述对象生物是山羊或绵羊。
上述的检测器件，其中，所述生物样本是全血、血浆或血清。
一种包括上述的单克隆抗体的小反刍兽疫诊断试剂盒，其用于通过检测对象生物来源的生物样本中是否存在小反刍兽疫病毒，诊断对象生物是否罹患小反刍兽疫。
上述的小反刍兽疫诊断试剂盒，其中，所述对象生物是山羊或绵羊。
上述的小反刍兽疫诊断试剂盒，其中，所述生物样本是全血、血浆或血清。
具体实施方式
实施例1用作抗原的小反刍兽疫病毒的纯化
1.1纯化浓缩系统：台式膜过滤系统，AKTA Pure蛋白纯化层析系统、高速冷冻离心机，超速冷冻离心机、500K中空纤维柱、100kd超滤离心管。
1.2主要试剂：0.02mol/L PBS，0.5N NaOH溶液sephrose 6FF层析介质、蔗糖。
1.3抗原制备及澄清
无菌条件下，取2000mL小反刍兽疫病毒抗原，4℃、5000rpm离心30min。弃沉淀，取上清用于超滤浓缩。
1.4浓缩纯化
1.4.1安装500K中空纤维柱及管路，再用2％NaOH碱液循环清洗500K的中空纤维柱并浸泡过夜，然后用纯化水冲洗中空纤维柱内残留碱液至中性并测水通量。用无菌PBS液平衡中空纤维柱。
1.4.2无菌连接中空纤维柱的进液和回液后进行浓缩处理，处理时保持泵转数在10％～15％之间。每批样品分别浓缩倍数至6倍时取样检测，用无菌PBS连续洗滤6倍浓缩液4次，以洗滤出液体计算洗滤次数(即洗滤出液体体积为5倍浓缩液2倍时结束洗滤)，分别取每次洗滤后的样品至少5个。
1.4.3凝胶过滤层析纯化
取24mL PPR超滤浓缩液，按照5mL/min体积流速进行上样，使用0.02mol/L PBS(pH＝7.4，Cond＝18.0ms/cm)进行洗脱，按照UV 280紫外吸收峰进行收集。
1.4.4 100KD超滤膜浓缩
使用默克-密理博100KD超滤离心管，对1#吸收峰进行5倍浓缩。
1.4.5蔗糖密度梯度离心
将1mL样品加到不连续蔗糖密度梯度(15％-80％)w/v溶液中,35000rpm/min、4℃离心3h,每0.5mL收集1管，共23管，依次编号为1-23。每管取70uL进行SDS-PAGE检测。
1.5检测
对于1-23号管，每管取70uL进行SDS-PAGE蛋白电泳检测。检测结果表明，第11-14号管中蛋白浓度最高。因此，合并第11-14号管中的样品，作为免疫用抗原。
实施例2抗原免疫、B细胞筛选及单克隆抗体的制备
抗原免疫家兔、B细胞筛选及单克隆抗体的制备委托杭州百凌生物科技有限公司完成。用上述实施例1中制备的免疫用抗原免疫家兔，从完成免疫后的家兔的外周血中筛选分泌抗小反刍兽疫病毒单克隆抗体的单个B细胞，并通过测序获得该单克隆抗体的编码核苷酸序列(从而能够获知该单克隆抗体的氨基酸序列，参见SEQ ID NO：1-8)。通过将该编码核苷酸序列克隆至适当的表达载体，并将该表达载体导入合适的宿主细胞进行表达和纯化，从而得到了单克隆抗体NMS-1-17H6L3。
实施例3小反刍兽疫诊断试剂盒(检测器件)的制备及应用
采用常规方法在常规的ELISA用固相载体上点样上述单克隆抗体NMS-1-17H6L3的溶液，另外点样一个PB点作为阴性质控点，制备了检测器件(诊断试剂盒)。
检测步骤(抗体夹心法)
(1)、用纯化水将20×浓缩清洗液(TBST：0.4M Tris-HCl，2.74M NaCl，2％Tween20，pH7.2±0.2)按1:20进行稀释，得到清洗液。为使检测器件表面完全浸润，用移液枪将约200μL清洗液加到检测器件表面，并浸泡检测器件一定时间。
(2)、用样本稀释液(0.05MPBS，1％BSA，0.2％PVP，0.5％Tween20，pH7.2±0.2)将待测血清样本按照1:50稀释。
(3)、对于弃去了清洗液的检测器件，在表面完全湿润的状态下，吸取200μL稀释后的血清样本加入到检测器件上。
(4)、在恒温摇床中以150转/分钟将检测器件孵育30分钟，温度为室温。
(5)、弃去血清样本，用清洗液清洗检测器件的表面。
(6)、清洗后，在检测器件上加入200μL的HRP标记的单克隆抗体NMS-1-17H6L3的溶液，在恒温摇床中以150转/分钟将检测器件孵育30分钟，温度为室温。
(7)、弃去酶标抗体溶液，用清洗液将检测器件表面清洗干净。
(8)、清洗完成后，在检测器件表面上均匀铺展20μL的发光底物液(Thermo，Prod#37074)。
(9)、将用凝胶成像仪对检测器件进行化学发光成像，并判定结果。
(10)、结果判定：对于每一份血清，分别统计该检测器件上的单克隆抗体NMS-1-17H6L3是否有响应(即，信噪比(SNR)大于等于2)，并进行判定。即，
当检测到单克隆抗体NMS-1-17H6L3有响应时，判定为小反刍兽疫病毒阳性；反之，判定为阴性。
其中，信噪比＝(多肽点信号值-阴性对照点信号值)/阴性对照点信号值。多肽点信号值是指软件读取的多肽点的化学发光强度值，阴性对照点信号值是指软件读取的阴性对照点的化学发光强度值。
对于800份来自中国各地区的山羊和绵羊血清样本，分别采用上述诊断试剂盒以及病毒核酸扩增进行检测，检测结果如下所示。
表1 800份山羊和绵羊血清样本检测结果
敏感性＝505/560*100％＝90％
特异性＝226/240＝94％
由以上可知，该诊断试剂盒的敏感性和特异性均在85％以上(且是采用核酸扩增方法验证，不是其他对照试剂盒方法验证)，完全满足疫病诊断的要求。
序列表
<110> 中国科学院苏州纳米技术与纳米仿生研究所
<120> 可用于诊断小反刍兽疫的单克隆抗体及诊断试剂盒
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Claims (9)
1.一种结合小反刍兽疫病毒的单克隆抗体，其包含三个重链互补决定区(CDR-H1、CDR-H2和CDR-H3)和三个轻链互补决定区(CDR-L1、CDR-L2和CDR-L3)，其中：
(a)CDR-H1的氨基酸序列如SEQ ID NO：1所示；
(b)CDR-H2的氨基酸序列如SEQ ID NO：2所示；
(c)CDR-H3的氨基酸序列如SEQ ID NO：3所示；
(d)CDR-L1的氨基酸序列如SEQ ID NO：4所示；
(e)CDR-L2的氨基酸序列如SEQ ID NO：5所示；且
(f)CDR-L3的氨基酸序列如SEQ ID NO：6所示。
2.根据权利要求1所述的单克隆抗体，其包含重链和轻链，其中，
所述重链的氨基酸序列如SEQ ID NO：7所示；且，
所述轻链的氨基酸序列如SEQ ID NO：8所示。
3.一种检测器件，其包括基底、以及固定于所述基底上的权利要求1或2所述的单克隆抗体。
4.根据权利要求3所述的检测器件，其用于检测对象生物来源的生物样本中是否存在小反刍兽疫病毒。
5.根据权利要求4所述的检测器件，其中，所述对象生物是山羊或绵羊。
6.根据权利要求4所述的检测器件，其中，所述生物样本是全血、血浆或血清。
7.一种包括权利要求1或2所述的单克隆抗体的小反刍兽疫诊断试剂盒，其用于通过检测对象生物来源的生物样本中是否存在小反刍兽疫病毒，诊断对象生物是否罹患小反刍兽疫。
8.根据权利要求7所述的小反刍兽疫诊断试剂盒，其中，所述对象生物是山羊或绵羊。
9.根据权利要求7所述的小反刍兽疫诊断试剂盒，其中，所述生物样本是全血、血浆或血清。
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