CN113354716B - 一种鼠诺如病毒重组抗原、单克隆抗体和病毒检测试纸条 - Google Patents
一种鼠诺如病毒重组抗原、单克隆抗体和病毒检测试纸条 Download PDFInfo
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Abstract
本发明提供了一种鼠诺如病毒重组抗原、单克隆抗体和病毒检测试纸条，涉及病毒检测技术领域。本发明所述重组抗原具有较强的免疫原性和抗原性，单克隆抗体特异性好、快速检测试纸灵敏准确。本发明所述乳胶微球检测试纸可检测MNV 1/3/4型，并具有与荧光RT‑PCR接近的灵敏度。本发明所述乳胶微球试纸，对小鼠诺如病毒不仅具有较高的灵敏度，且快速准确，节约成本，对小鼠诺如病毒感染的流行病学调查、日常监测和预警都有重要意义。
Description
技术领域
本发明属于病毒检测技术领域，具体涉及一种鼠诺如病毒重组抗原、单克隆抗体和病毒检测试纸条。
背景技术
诺如病毒（Norovirus）原名诺瓦克病毒，属杯状病毒科诺如病毒属，为无包膜的单股正链RNA病毒。诺如病毒具有多个基因型，根据基因结构特征，诺如病毒又被划分为7个基因群（Genogroup，GI-GVII），其中GI、GII和GIV可感染人，GI和GII是引起人类感染性腹泻的两个主要基因型，GIII、GV和GVI则分别主要感染牛、小鼠和狗。诺如病毒各基因群之间的核苷酸差异很大，每个基因群又有许多不同的变异株。
小鼠诺如病毒（murine norovirus，MNV）是2002年首次在RAG2/STAT1-/-免疫缺陷小鼠分离到，主要攻击小鼠免疫系统，引起小鼠神经系统、消化系统等症状甚至死亡。MNV发现时间短，但感染率是细小病毒的10倍。MNV感染实验小鼠后，不仅造成相关系统的症状，其对免疫系统的影响，也会对动物实验结果的准确性和稳定性造成干扰，因此包括美国查尔斯河实验室（Charles River）、国际实验动物科学学会（ICLAS）等许多国家和组织将其列为实验小鼠的必检项目。
目前，对小鼠诺如病毒的检测，包括病毒的分离培养鉴定、间接免疫荧光实验（IFA）、酶联免疫吸附试验（ELISA）、逆转录PCR和实时荧光定量PCR（q-PCR）等。IFA和ELISA是检测MNV抗体的主要方法，而某些免疫系统改变的基因编辑小鼠会出现免疫反应的缺失或抑制，导致抗体检测难以适用。分子生物学方法如实时荧光定量PCR则成为主要的监测技术，但其耗时较长，且需专业的技术人员和仪器设备，使其难以在非实验室环境下开展，非常不利于小鼠诺如病毒的控制。以上检测技术中抗体检测存在一定的缺陷，病毒培养和荧光PCR虽然准确灵敏，但操作复杂耗时长。
发明内容
有鉴于此，本发明的目的在于提供一种鼠诺如病毒重组抗原、单克隆抗体和病毒检测试纸条，所述重组抗原具有较强的免疫原性和抗原性，单克隆抗体特异性好，病毒检测试纸条灵敏准确。
为了实现上述发明目的，本发明提供以下技术方案：
本发明提供了一种鼠诺如病毒重组抗原，所述重组抗原的氨基酸序列如SEQ IDNO.1所示。
本发明还提供了一种编码上述重组抗原的重组核酸，所述重组核酸的核苷酸序列如SEQ ID NO.2所示。
本发明还提供了一种与上述重组抗原对应的单克隆抗体，所述单克隆抗体包括6D3和10H6；
所述6D3的重链可变区的氨基酸序列如SEQ ID NO.18所示，所述6D3的轻链可变区的氨基酸序列如SEQ ID NO.20所示；
所述10H6的重链可变区包括三个互补决定区，所述互补决定区的氨基酸序列分别如SEQ ID NO.12~SEQ ID NO.14所示；
所述10H6的轻链可变区包括三个互补决定区的氨基酸序列分别如SEQ ID NO.15~SEQ ID NO.17所示。
优选的，编码所述6D3的重链可变区的核苷酸序列如SEQ ID NO.19所示，编码所述6D3的轻链可变区的核苷酸序列如SEQ ID NO.21所示。
优选的，编码所述10H6的重链可变区的氨基酸序列如SEQ ID NO.22所示；编码所述10H6的轻链可变区的氨基酸序列如SEQ ID NO.24所示。
本发明还提供了上述单克隆抗体在制备检测鼠诺如病毒的工具中的应用。
优选的，所述鼠诺如病毒包括诺如病毒GV群。
优选的，所述工具包括试纸条。
本发明还提供了一种快速检测鼠诺如病毒的试纸条，所述试纸条包括检测线和质控线；
所述检测线的制备方法包括在硝酸纤维膜上喷涂上述单克隆抗体；
所述质控线的制备方法包括在硝酸纤维膜上喷涂羊抗鼠IgG抗体。
本发明还提供了一种快速检测鼠诺如病毒的乳胶微球检测试纸，所述乳胶微球检测试纸包括自左到右，依次粘贴在背板上的：样品垫、乳胶微球垫、硝酸纤维膜和吸水纸；
在所述硝酸纤维膜上喷涂上述单克隆抗体作为检测线，喷涂羊抗鼠IgG抗体作为质控线。
有益效果：本发明提供了一种鼠诺如病毒重组抗原、表达单克隆抗体的杂交瘤细胞和病毒检测试纸条，重组抗原具有较强的免疫原性和抗原性，单克隆抗体特异性好、快速检测试纸灵敏准确。本发明所述乳胶微球试纸，对小鼠诺如病毒不仅具有较高的灵敏度，且快速准确，节约成本，对小鼠诺如病毒感染的流行病学调查、日常监测和预警都有重要意义。
附图说明
图1为乳胶微球检测试纸组装示意图；
图2为IPTG诱导并纯化后蛋白的SDS-PAGE分析；
图3为重组蛋白与阳性血清的反应活性；
图4为单克隆抗体的特异性鉴定。
具体实施方式
本发明提供了一种鼠诺如病毒重组抗原，所述重组抗原的氨基酸序列如SEQ IDNO.1所示。
本发明所述所述重组抗原优选来源于小鼠诺如病毒，本发明称所述重组抗原为MNV-VP1，根据MNV基因序列（Genbank: EF014462），利用Bepipred Linear EpitopePrediction软件进行抗原表位预测，设计出1段VP1截短的序列，并进行基因重组，其具体的氨基酸序列如SEQ ID NO.1所示。
本发明还提供了一种编码上述重组抗原的重组核酸，所述重组核酸的核苷酸序列如SEQ ID NO.2所示。
本发明还提供了一种与上述重组抗原对应的单克隆抗体，所述单克隆抗体包括6D3和10H6；所述6D3的重链可变区的氨基酸序列如SEQ ID NO.18所示，所述6D3的轻链可变区的氨基酸序列如SEQ ID NO.20所示；
所述10H6的重链可变区包括三个互补决定区，所述互补决定区的氨基酸序列分别如SEQ ID NO.12~SEQ ID NO.14所示；
所述10H6的轻链可变区包括三个互补决定区的氨基酸序列分别如SEQ ID NO.15~SEQ ID NO.17所示。
本发明所述单克隆抗体的重链可变区优选包括以下三个互补决定区：CDR1、CDR2和 CDR3；所述单克隆抗体的轻链可变区优选包括以下三个互补决定区：CDR1’、CDR2’和CDR3’。在本发明中，所述6D3的重链可变区的氨基酸序列如SEQ ID NO.18所示，其核苷酸序列如SEQ ID NO.19所示，所述互补决定区的氨基酸序列分别如SEQ ID NO.6~SEQ ID NO.8所示；所述6D3的轻链可变区的氨基酸序列如SEQ ID NO.20所示，其核苷酸序列如SEQ IDNO.21所示，三个互补决定区的氨基酸序列分别如SEQ ID NO.9~SEQ ID NO.11所示。
本发明所述10H6的重链可变区的氨基酸序列优选如SEQ ID NO.22所示，其核苷酸序列如SEQ ID NO.23所示，并且三个互补决定区的氨基酸序列分别如SEQ ID NO.12~SEQID NO.14所示；所述10H6的轻链可变区的氨基酸序列如SEQ ID NO.24所示，核苷酸序列如SEQ ID NO.25所示，三个互补决定区的氨基酸序列分别如SEQ ID NO.15~SEQ ID NO.17所示。
本发明对所述单克隆抗体的制备方法并没有特殊限定，优选通过杂交瘤细胞和腹水制备的方法制备所述单克隆抗体。在本发明实施例中，所述杂交瘤细胞的制备方法，包括以下步骤：（1）利用上述重组抗原免疫若干只小鼠，在第6周时检测血清抗体滴度，筛选抗体滴度最高的小鼠进行加强免疫，加强免疫后3天摘取脾脏；
（2）将所述脾脏细胞与骨髓瘤细胞融合，筛选产鼠诺如病毒重组抗原特异性抗体的细胞株，得所述杂交瘤细胞。
本发明利用上述重组抗原免疫若干只小鼠，在第6周时检测血清抗体滴度，筛选抗体滴度最高的小鼠进行加强免疫，加强免疫后3天摘取脾脏。本发明所述重组抗原优选为利用原核表达系统进行表达并纯化后得到的重组抗原，更优选为将SEQ ID NO.2所示的核苷酸序列连接pGEX-4T-1载体的，BamHI和SalI之间，得重组质粒Pgex-4T-1-VP1，转化Rosetta（DE3）感受态细胞，并利用IPTG诱导，而后利用GE公司的AKTA Start和GST层析柱进行纯化，得重组蛋白Pgex-4T-1-VP1。本发明优选利用所述重组蛋白Pgex-4T-1-VP1（50μg）与等体积的铝佐剂（500μg）混合后腹腔免疫6周雌性Balb/c小鼠，在2周、4周按以上方法再各免疫一次，在第6周时，取小鼠血清检测抗体滴度，选取滴度最高的小鼠通过尾静脉加强免疫20μg所述重组蛋白，3天后取小鼠脾脏用于制备杂交瘤细胞。
本发明将所述脾脏细胞与骨髓瘤细胞融合，筛选产鼠诺如病毒重组抗原特异性抗体的细胞株，得所述杂交瘤细胞。本发明优选取小鼠脾脏细胞与骨髓瘤细胞SP2/0通过PEG1450融合，制备杂交瘤细胞，10天之后，以重组蛋白MNV-VP1、GST蛋白、小鼠肝炎病毒（MHV）、小鼠脑脊髓炎病毒（TMEV）、仙台病毒（SV）等进行ELISA检测，筛选出仅与MNV-VP1反应而不与其他反应的细胞株，稀释至单克隆状态后对细胞株进行扩大培养和冻存，得所述杂交瘤细胞。
本发明优选利用腹水制备的方法制备所述单克隆抗体，本发明对所述腹水制备的方法并没有特殊限定，利用本领域的常规方法即可。本发明所述单克隆抗体与6D3、6H11、7A7、10H6发生特异性反应，其OD450>1.8，表明单克隆抗体具有良好的特异性；并且6D3和10H6配对单抗检测的灵敏度最高，确定最佳配对单抗为6D3和10H6，可用作本发明鼠诺如病毒检测试纸条的标记抗体和捕获抗体。
本发明还提供了上述单克隆抗体在制备检测鼠诺如病毒的工具中的应用。
本发明所述鼠诺如病毒优选包括诺如病毒GV群，在本发明实施例中，所用鼠诺如病毒更优选为鼠诺如病毒较为流行的MNV1/3/4型。本发明所述工具优选包括试纸条。
本发明还提供了一种快速检测鼠诺如病毒的试纸条，所述试纸条包括检测线和质控线；
所述检测线的制备方法包括在硝酸纤维膜上喷涂上述单克隆抗体；
所述质控线的制备方法包括在硝酸纤维膜上喷涂羊抗鼠IgG抗体。
本发明对所述试纸条的结构并没有特殊限定，优选包括本领域的常规试纸条结构。
本发明还提供了一种快速检测鼠诺如病毒的乳胶微球检测试纸，所述乳胶微球检测试纸包括自左到右，依次粘贴在背板上的：样品垫、乳胶微球垫、硝酸纤维膜和吸水纸；
在所述硝酸纤维膜上喷涂上述单克隆抗体作为检测线，喷涂羊抗鼠IgG抗体作为质控线。
本发明所述乳胶微球检测试纸的示意图优选如图1所示，所述乳胶微球垫的制备方法，优选包括乳胶微球的清洗、活化、标记和制备。本发明所述清洗优选包括：量取一定体积粒径为300mm的红色乳胶微球（苏州为度生物技有限公司，DRO5C），倒入洁净的离心管，按照1:9的体积比加入标记缓冲液 （50 mM MES，pH 6.0）。17000 r/min离心10 min，去掉上清，再加1mL的标记缓冲液（50 mM MES，pH 6.0），离心后加入1mL的标记缓冲液重悬微球。
本发明所述活化优选包括：分别称取20 mg NHS和EDC，用1mL标记缓冲液溶解，分别得到20 mg/mL NHS溶液和EDC溶液；取20μL配制好NHS溶液加入到清洗后的微球中，快速混匀；然后再取5μL配制好的EDC溶液加入到微球中，快速混匀，室温孵育20~30min。
本发明所述标记优选包括：将经所述活化后的乳胶微球17000r/min离心10 min，加入1mL的标记缓冲液重悬微球，再次离心后加入1mL的标记缓冲液重悬微球。取一定量的单克隆抗体，按每100μg抗体加入100μL微球缓冲液的比例加入活化后的微球缓冲液，快速混匀后，室温孵育3h。再加入封闭液（20 mg/mL的BSA，用终浓度100 mM乙醇胺溶液溶解），室温孵育1h，17000 r/min离心10 min，弃上清，再重复洗涤两次，去除未结合的抗体。最后一次用1mL的标记缓冲液重悬微球，即为抗体-微球标记复合物，4℃放置备用。
本发明所述制备，优选包括：采用6mm×300mm玻璃纤维膜，经含有1% BSA和1% 吐温-20的PBS处理后，将制备好的抗体-微球标记复合物按照1mL/条喷涂，40℃烘干2h备用。
本发明优选以60mm×300mm PVC背板为支撑，在其上分别贴有硝酸纤维膜、乳胶微球垫、样品垫、吸水纸，硝酸纤维膜上包被有两条线，40℃烘干12h备用。组装好的大板用切条机切成4.05mm的裸条，用塑料卡壳包裹，使样品垫暴露于塑料卡壳的加样孔位置，质控线、检测线暴露于结果观察孔的位置，乳胶微球检测试纸条组装完毕。本发明所述检测线和质控线的制备方法，优选包括：分别在不同的硝酸纤维（20mm×300mm）膜上，用划膜仪横向线状以1.0μL/cm分别喷涂纯化的单克隆抗体1.5mg/mL形成检测线。间隔6mm，以1.0μL/cm横向线状喷涂羊抗鼠IgG，浓度为1mg/mL，形成质控线。
下面结合实施例对本发明提供的一种鼠诺如病毒重组抗原、表达单克隆抗体的杂交瘤细胞和病毒检测试纸条进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
本发明所采用的试剂和材料如下所示，如未特殊说明，均为本领域的常规市售产品。
毒株、血清：MNV1毒株（ATCC，CW1）、MNV3 毒株（K4）、MNV4毒株（SH1603）、小鼠肝炎病毒（MHV，ATCC VR-246）、小鼠脑脊髓炎病毒（TMEV，ATCC VR-995）、仙台病毒（SV，ATCC VR-105）及阳性血清由中国疾控中心病毒病所鉴定保存；
培养基：LB琼脂平板（含100 μg/mL 氨苄青霉素）和LB培养基（含100 μg/mL 氨苄青霉素），均由岭创生物科技（北京）有限公司配制。
试剂：氯化钠、磷酸二氢钠、磷酸氢二钠、Tris、还原型谷胱甘肽等常规化学试剂为国产分析纯。
实施例1
1、表达小鼠诺如病毒重组蛋白MNV-VP1的基因设计及合成（220-469aa）
根据MNV基因序列（Genbank：EF014462），利用Bepipred Linear EpitopePrediction软件进行抗原表位预测，设计出1段VP1截短的序列（SEQ ID NO.2），基因克隆至pGEX-4T-1载体，由苏州泓讯生物科技有限公司合成。
2、重组蛋白Pgex-4T-1-VP1的表达
合成的重组质粒Pgex-4T-1-VP1按常规方法转化Rosetta（DE3）感受态细胞（《分子克隆》第三版，科学出版社），转化菌涂布于LB琼脂平板（含100μg/mL氨苄青霉素），37℃过夜培养。挑取单菌落接入5 mL LB培养基（含100μg/mL氨苄青霉素）中，37℃，220转/分振荡培养过夜。再次测序鉴定正确后冻存甘油菌待用。取生产用菌种，按培养基总体积的1%的量接种到LB培养基（含100 μg/mL氨苄青霉素）中，37℃，220转/分振荡培养3小时左右，至OD600为0.5，于16℃降温1.5小时，加终浓度为0.1 mM IPTG，16℃，220转/分诱导16小时后收集菌体。
3、重组蛋白的纯化
因表达的重组蛋白均带有谷胱甘肽转移酶（GST）标签，用GE公司的AKTA Start和GST层析柱进行纯化。破菌缓冲液为50mM Tris-HCl，300 mM NaCl，PH 8.0，洗脱缓冲液为50mM Tris-HCl，300 mM NaCl，10 mM GSH，pH 8.0。层析柱用破菌缓冲液平衡，然后将发酵好的菌液8000rpm离心20min，沉淀用破菌缓冲液液重悬，在冰水中超声破碎20min，间隔5秒超声5秒，12000rpm离心30min，上清用0.22微米滤器过滤，GST层析柱用破菌缓冲液平衡后上样，再洗涤，最后用洗脱缓冲液洗脱，通过SDS-PAGE蛋白凝胶电泳观察纯化情况。用0.01MPBS透析换液，Nanodrop 测定蛋白浓度，分装成1 ml/管，-20℃保存。
4、重组蛋白的鉴定
4.1用SDS-PAGE电泳鉴定蛋白纯度：
a）蛋白样品前处理：分别留样加同等体积的2×SDS loading buffer，置于沸水浴中煮10min，12000rpm离心3min，取上清加至加样孔。
b）SDS-PAGE电泳胶的配制：垂直电泳玻璃板洗净晾干，垂直放于制胶架上，按照要求配好分离胶体系，加入玻璃板间隙，至胶面达到所要求的高度后，加入无水乙醇密封；室温凝固30min~1h后，弃去胶上层密封液体，并用吸水纸吸干，按比例配制浓缩胶，并插入适合的梳子，室温凝固30min~1h。
c）SDS-PAGE电泳：将5×甘氨酸缓冲液稀释至工作浓度，加入电泳槽至合适液面高度，将梳子从凝固凝胶中轻轻拔出。在加样孔内加入蛋白marker和10μL处理过的蛋白样品。接通电源，调整电压80V恒压电泳至分离胶，再改为120V直至溴酚蓝至凝胶底部。
d）染色及脱色：将凝胶浸泡于考马斯亮蓝染液中，振荡染色4 h，后脱色液脱色至条带清晰。
结果如图2所示，SDS-PAGE鉴定表达蛋白的纯度，显示纯度在90%以上，可以满足后续的免疫和筛选实验。
4.2用ELISA 鉴定重组蛋白的活性
用纯化的重组蛋白包被，ELISA方法鉴定其与MNV1阳性小鼠血清的反应。
先在微孔板中分别包被重组蛋白（包被缓冲液碳酸盐缓冲液：碳酸钠1.59g、碳酸氢钠2.93g定容至1L纯水中），包被浓度为1μg/mL，4℃过夜；1%明胶封闭，每孔150 微升，37℃ 2小时，用洗涤液洗板1次，拍干；将MNV1阳性血清按1:100、1:1000、1:10000、1:10000的梯度进行（PBS）稀释，加入50μL到已包被抗原的微孔板中，37℃反应30min。甩出孔中液体，PBST洗液洗板4次，拍干后加入HRP标记的羊抗鼠二抗（以PBS按照1:10000稀释）50μL/孔，37℃反应30min，再洗板4次，拍干后加入TMB显色液100μL/孔室温显色10min，最后加入0.5M硫酸，终止反应，用酶标仪测定OD450值。
结果如图3所示，用纯化的重组蛋白包被，ELISA方法鉴定其与MNV1阳性小鼠血清的反应，结果显示重组蛋白具有良好的活性。
5、重组蛋白Pgex-4T-1-VP1单克隆抗体的制备
5.1小鼠免疫
将50μg的重组蛋白与等体积的铝佐剂（500μg）混合后腹腔免疫6周雌性Balb/c小鼠，在0周、2周、4周按以上方法再各免疫一次。在第6周时，取小鼠血清检测抗体滴度。选取滴度最高的小鼠通过尾静脉加强免疫20μg重组蛋白。3天后取小鼠脾脏用于制备杂交瘤细胞。
5.2杂交瘤细胞株的制备和筛选
小鼠加强免疫3天后，取小鼠脾脏细胞与骨髓瘤细胞SP2/0通过PEG1450融合，制备杂交瘤细胞。10天之后，按上述ELISA方法，以重组蛋白MNV-VP1、GST蛋白、小鼠肝炎病毒（MHV）、小鼠脑脊髓炎病毒（TMEV）、仙台病毒（SV）等进行检测，筛选出仅与MNV-VP1反应而不与其他反应的细胞株，，有限稀释至单克隆状态后对细胞株进行扩大培养和冻存。
5.3腹水制备和抗体纯化
将筛选到的杂交瘤细胞株进行扩大培养后，于腹腔注射0.2mL （约2×106个细胞）至雌性BALB/c小鼠，约10~15日后，待小鼠腹部明显肿大时，使用无菌注射器针头采集腹水。将收集到腹水以12000 r/min离心10min，去除上层油脂和下层沉淀，收集上清。将腹水上清液用结合缓冲液按照1:10进行稀释，用滤膜过滤，然后结合至Protein G柱（先用结合缓冲液平衡）。再用结合缓冲液冲洗大约5个柱体积，用洗脱缓冲液（0.1M甘氨酸，pH 2.7）洗脱，收集洗脱峰。然后迅速使用1M Tris-HC1 （pH 9.0）溶液中和，使处干中性范围。将纯化抗体多次换液后透析至1x PBS中，测定浓度并分装保存。
5.4单克隆抗体的特异性鉴定
将MNV1、小鼠肝炎病毒（MHV）、小鼠脑脊髓炎病毒（TMEV）、仙台病毒（SV）分别与单克隆抗体进行反应，结果如图4所示，单克隆抗体6D3、6H11、7A7、10H6与MNV1发生特异性反应，其OD450值>1.8，而与其他病毒不发生反应，表明单克隆抗体具有良好的特异性。
5.5双抗体夹心ELISA方法确定配对抗体
将这4株单克隆抗体进行HRP标记。用pH7.4 PBS将单克隆抗体稀释至0.5µg/ml，4℃包被过夜，用 PBST洗板1次，然后使用含有2%马血清+1%蔗糖的封闭液37℃封闭2小时。分别加入稀释的MNV1病毒、鼠肝炎病毒（MHV）、小鼠脑脊髓炎病毒（TMEV）、仙台病毒（SV）进行检测，50μl/孔， 37℃反应30min，洗板4次；加入HRP标记的单抗（1:3000倍稀释），37℃反应30min，洗板4次；加入TMB显色液37℃显色10min，加入终止液，读取OD450值。选择OD450nm值大于1.8的4株单克隆抗体，分别为6D3和10H6、7A7和6H11，用不同梯度稀释的MNV1进行检测，结果如表1和表2所示，6D3和10H6配对单抗检测的灵敏度最高，确定最佳配对单抗为6D3和10H6，用作本发明鼠诺如病毒检测试纸条的标记抗体和捕获抗体。
6、乳胶微球检测试纸条的制备
划膜：分别在不同的硝酸纤维（20mm×300mm）膜上，用划膜仪横向线状以1.0μL/cm分别喷涂纯化的单克隆抗体6D3 1.5mg/ml形成检测线。间隔6mm，以1.0μL/cm横向线状喷涂羊抗鼠IgG，浓度为1mg/ml，形成质控线。
抗体-微球标记复合物制备：
乳胶微球的清洗：量取一定体积粒径为300mm的红色乳胶微球（苏州为度生物技有限公司，DRO5C），倒入洁净的离心管，按照1:9的比例加入标记缓冲液 （50 mM MES，pH6.0）。17000 r/min离心10 min，去掉上清，再加1mL的标记缓冲液，离心后加入1mL的标记缓冲液重悬微球。
乳胶微球的活化：各称取20 mg NHS和EDC，用1mL标记缓冲液溶解，分别得到20mg/ml NHS溶液和EDC溶液：取20μL配制好NHS溶液加入到清洗后的微球中，快速混匀；然后再取5μL配制好的EDC溶液加入到微球中，快速混匀，室温孵育20~30min。
乳胶微球的标记：将得到的活化后的乳胶微球17000 /min离心10 min，加入1mL的标记缓冲液重悬微球，再次离心后加入1mL的标记缓冲液重悬微球。取一定量的单克隆抗体10D6，按每100μg抗体加入100μL微球缓冲液的比例加入活化后的微球缓冲液，快速混匀后，室温孵育3h。再加入封闭液（20 mg/mL的BSA，用终浓度100 mM乙醇胺溶液溶解），室温孵育1h，17000 r/min离心10 min，弃上清，再重复洗涤两次，去除未结合的抗体。最后一次用1mL的标记缓冲液重悬微球，即为抗体-微球标记复合物，4℃放置备用。
乳胶微球垫的制备：采用6mm×300mm玻璃纤维膜，经含有1% BSA和1%吐温-20的PBS处理后，将制备好的乳胶微球标记蛋白按照1ml/条喷涂，40℃烘干2h备用；
乳胶微球试纸条的组装：以60mm×300mm PVC背板为支撑，在其上分别贴有硝酸纤维膜、乳胶微球垫、样品垫、吸水纸，硝酸纤维膜上包被有两条线，40℃烘干12h备用。组装好的大板用切条机切成4.05mm的裸条，用塑料卡壳包裹，使样品垫暴露于塑料卡壳的加样孔位置，质控线、检测线暴露于结果观察孔的位置，乳胶微球检测试纸条组装完毕。
7、乳胶微球检测试纸条的灵敏度、特异性测试：
阳性样本为：MNV1/3/4病毒；
阴性样本为：小鼠肝炎病毒（MHV）、小鼠脑脊髓炎病毒（TMEV）、仙台病毒（SV）；
样本稀释液为：0.01mol/L pH7.4 PBS，含有0.1% Tween 20。
检测方法为：将阴阳性样本用稀释液梯度稀释，混匀后滴加80μL到加样孔，20min内观察结果。
结果判定：T检测线和C质控线都出现清晰可见的红色条带，判为阳性，T线颜色越深，阳性越强；只有C质控线出现清晰可见的红色条带，判为阴性；若C质控线不出现红色条带，判为无效。
8、与实时荧光RT-PCR方法比较
参照2017版中国实验动物学会鼠诺如病毒检测方法推荐的实时荧光RT-PCR探针法进行操作和结果判定，样本为不同稀释度的MNV1、MNV3、MNV4病毒（1×104、1×103、1×102、1×101、1×100 copies/μL）及小鼠肝炎病毒（MHV）、小鼠脑脊髓炎病毒（TMEV）、仙台病毒（SV）阴性样本。
实时荧光RT-PCR引物探针：
Primer-F（SEQ ID NO.3）：TCTGTYCTGCGCTGGGTGC
Primer-R（SEQ ID NO.4）：GCTGCGCCATCACTCATCC
Probe（FAM-SEQ ID NO.5-BHQ1）：FAM-ATGCTGAGACCCCGCAGGAACG-BHQ1
反应参数：
逆转录：42℃ 15min
预变性：95℃ 30s
变性：95℃ 5s
退火延伸：60℃ 34s（采集荧光）， 40个循环。
结果判定：
阴性：无荧光扩增曲线，判定为未检出小鼠诺如病毒；
阳性：有明显扩增曲线，Ct值≤35，判定为检出小鼠诺如病毒；
可疑：Ct值介于35和40之间，应复测，若仍在35到40之间则为阳性，弱Ct值≥40，则为阴性。
结果如表3所示，用组装好的试纸条和推荐的荧光RT-PCR检测方法进行比较，结果发现：试纸条可检测出MNV 1/3/4型，且检测灵敏度与荧光RT-PCR方法接近，具有较好的检测性能。
表 3乳胶微球检测试纸与荧光PCR检测比较
| 样本 | 荧光RT-PCR | 乳胶微球试纸 |
| MNV1 1X 10<sup>4 </sup>copies/μL | + | + |
| MNV1 1X 10<sup>3</sup> copies/μL | + | + |
| MNV1 1X 10<sup>2</sup> copies/μL | — | — |
| MNV1 1X 10<sup>1</sup> copies/μL | — | — |
| MNV3 1X 10<sup>4 </sup>copies/μL | + | + |
| MNV3 1X 10<sup>3</sup> copies/μL | + | + |
| MNV3 1X 10<sup>2</sup> copies/μL | — | — |
| MNV3 1X 10<sup>1</sup> copies/μL | — | — |
| MNV4 1X 10<sup>4 </sup>copies/μL | + | + |
| MNV4 1X 10<sup>3</sup> copies/μL | + | + |
| MNV4 1X 10<sup>2</sup> copies/μL | — | — |
| MNV4 1X 10<sup>1</sup> copies/μL | — | — |
| MHV | — | — |
| TMEV | — | — |
| SV | — | — |
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
序列表
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Claims (7)
1.一种与鼠诺如病毒重组抗原对应的单克隆抗体的组合，其特征在于，所述重组抗原的氨基酸序列如SEQ ID NO.1所示；
所述单克隆抗体的组合包括6D3和10H6；
所述6D3的重链可变区的氨基酸序列如SEQ ID NO.18所示，所述6D3的轻链可变区的氨基酸序列如SEQ ID NO.20所示；
所述10H6的重链可变区包括三个互补决定区，所述互补决定区的氨基酸序列分别如SEQ ID NO.12~SEQ ID NO.14所示；
所述10H6的轻链可变区包括三个互补决定区，所述互补决定区的氨基酸序列分别如SEQ ID NO.15~SEQ ID NO.17所示。
2.根据权利要求1所述单克隆抗体的组合，其特征在于，编码所述6D3的轻链可变区的核苷酸序列如SEQ ID NO.21所示。
3.根据权利要求1所述单克隆抗体的组合，其特征在于，所述10H6的重链可变区的氨基酸序列如SEQ ID NO.22所示；所述10H6的轻链可变区的氨基酸序列如SEQ ID NO.24所示。
4.权利要求1~3任一项所述单克隆抗体的组合在制备检测鼠诺如病毒的工具中的应用。
5.根据权利要求4所述的应用，其特征在于，所述鼠诺如病毒包括诺如病毒GV群。
6.根据权利要求4所述的应用，其特征在于，所述工具包括试纸条。
7.一种快速检测鼠诺如病毒的乳胶微球检测试纸，其特征在于，所述乳胶微球检测试纸包括自左到右，依次粘贴在背板上的：样品垫、乳胶微球垫、硝酸纤维膜和吸水纸；
所述乳胶微球垫上标记权利要求1~3任一项中所述的10H6；
在所述硝酸纤维膜上喷涂权利要求1~3任一项中所述的6D3作为检测线，喷涂羊抗鼠IgG抗体作为质控线。
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