CN105838682A - 牛病毒病性腹泻病毒特异性单克隆抗体细胞株及其应用 - Google Patents
牛病毒病性腹泻病毒特异性单克隆抗体细胞株及其应用 Download PDFInfo
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- CN105838682A CN105838682A CN201610225476.4A CN201610225476A CN105838682A CN 105838682 A CN105838682 A CN 105838682A CN 201610225476 A CN201610225476 A CN 201610225476A CN 105838682 A CN105838682 A CN 105838682A
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- monoclonal antibody
- bovine viral
- diarrhea virus
- cell strain
- specific monoclonal
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Abstract
一种牛病毒病性腹泻病毒特异性单克隆抗体细胞株，保藏于中国微生物菌种保藏管理委员会普通微生物中心登记入册的编号为CGMCC No. 12033。具有制备成本低，所得原料纯度高，可重复获得等特点，可以用于牛病毒性腹泻的免疫检测等技术领域，为奶牛、肉牛养殖该病防控提供方便的检测手段。
Description
技术领域
本发明涉及生物技术领域，涉及到一种牛病毒病性腹泻病毒特异性单克隆抗体细胞株及其应用。
背景技术
牛病毒性腹泻病毒(BVDV)属于黄病毒科，瘟病毒属。BVDV感染牛后，其临床症状可表现为粘膜病、母畜流产，产死胎、畸形胎等，给养牛业造成了很大的经济损失。感染BVDV的怀孕母牛可能产生持续感染（Persistent Infection， PI）的犊牛，PI牛可以正常发育，但其生产性能低下，同时继续还向牛群散毒，导致牛场持续损失。据统计北京地区牛群中BVDV阳性率高达94.6%，而PI牛的阳性率也达到1.02%，每年由BVDV引起的牛群淘汰数量占据总体淘汰数量的30%。全国有超过22个省区报道过BVDV感染，而全世界每年因BVDV感染死亡的牛不少于500万头，由于持续感染导致的间接损失达数百亿美元。目前我国尚无有效应用的BVDV疫苗，该病在我国处于不断扩大的趋势。而BVDV除了在牛群中传播外，还会导致羊、猪、鹿等经济动物感染，整体防控形势非常严峻。
传统的BVDV抗原检测主要依赖于分子检测方法，比如各种类型的PCR方法，其检测耗时，操作复杂，主要应用于出入境检验检疫、疾病控制预防中心等国家级实验室，无法在牧场和基层检测领域应用，因此有必要开发出一种能够现场使用的快速检测方法，满足实际生产需要。
免疫胶体金技术和酶联免疫吸附试剂盒是当前应用最为广泛的快速方法，在食品检测、动物诊断、环境监测和水质分析等领域均有应用，尤其是胶体金免疫检测法检测速度快、灵敏度高、操作方便，无需特殊仪器设备，非常适合我国当前的动物养殖生产模式。而要建立这两种免疫检测方法，必须制备特异性好、灵敏度高的单克隆或多克隆抗体。
发明内容
本申请的目的在于提供一种制备成本低，所得原料纯度高，可重复获得的牛病毒病性腹泻病毒特异性单克隆抗体细胞株。
本发明还要解决技术问题为提供上述牛病毒病性腹泻病毒特异性单克隆抗体细胞株的应用
为解决上述技术问题，本发明通过如下技术方案实现：
一种牛病毒病性腹泻病毒特异性单克隆抗体细胞株，保藏于中国微生物菌种保藏管理委员会普通微生物中心登记入册的编号为CGMCC No. 12033。
本发明的另外一个目的在于提供一种牛病毒病性腹泻病毒特异性单克隆抗体细胞株的应用。
一种牛病毒病性腹泻病毒特异性单克隆抗体，其特征在于：所述单克隆抗体是由权利要求1所述的牛病毒病性腹泻病毒特异性单克隆抗体细胞株分泌产生的。
进一步，牛病毒病性腹泻病毒特异性单克隆抗体，其特征在于，制备该单克隆抗体所用基因片段的核苷酸序列如SEQ ID No.1所示，免疫蛋白的序列如SEQ ID No.2所示。
进一步，本发明的目的还在于提供一种所述的牛病毒病性腹泻病毒特异性单克隆抗体在牛病毒病性腹泻病毒抗原检测中的应用。
进一步，本发明的目的还在于提供所述的牛病毒病性腹泻病毒特异性单克隆抗体细胞株或所述的牛病毒病性腹泻病毒特异性单克隆抗体在制备检测牛病毒病性腹泻病毒试剂盒中的应用。
此外，本发明还提供了一种利用牛病毒病性腹泻病毒特异性单克隆抗体建立的牛病毒病性腹泻病毒抗原的检测方法，方法操作简易，适合于基层应用。
为了达到上述目的，本发明采取了以下的技术措施：
（1）通过重组表达的方式获得牛病毒病性腹泻病毒E0蛋白并纯化：根据GenBank公开的BVDV的E0基因序列设计引物，PCR扩增E0基因片段并与表达载体pFastBac HT-B连接。将阳性重组质粒pFastBac HT-E0转座DH10Bac感受态细胞，通过用KTG抗生素和蓝白斑筛选阳性克隆，提取Bacmid，转染SF9昆虫细胞，观察转染后的细胞病变并收集重组杆状病毒。将重组杆状病毒以MOI 0.1感染SF9昆虫细胞，4d后5000r/min离心10min收集上清。目的蛋白的纯化参照Ni-NTA纯化系统说明书进行。
（2）利用重组E0蛋白免疫小鼠，筛选单抗：以纯化的E0蛋白为免疫抗原，以常规方法免疫Balb/C小鼠，多次免疫后测定血清效价，并加强免疫。随后取免疫小鼠的脾细胞与SP2/0细胞融合，经过筛选得到了能分泌抗E0蛋白特异性抗体的杂交瘤细胞株，命名为MAB106，抗体亚型鉴定为IgG2b，分别以Western和ELISA鉴定抗体的特异性和灵敏度。
（3）利用制备的单克隆抗体检测牛血清中的BVDV抗原：以双抗体夹心胶体金层析免疫为基础，将MAB106单抗用胶体金标记后，建立牛血清中BVDV病毒抗原的快速检测方法，为BVDV的临床诊断提供方法和手段。
本发明的有益效果为：牛病毒病性腹泻病毒特异性单克隆抗体细胞株。利用该细胞株培养或者免疫小鼠可以获得抗牛病毒性腹泻病毒的特异性单克隆抗体，具有制备成本低，所得原料纯度高，可重复获得等特点，可以用于牛病毒性腹泻的免疫检测等技术领域，为奶牛、肉牛养殖该病防控提供方便的检测手段。
保藏说明
参据的生物材料（株）：MAB106
科学描述：
保藏机构：中国微生物菌种保藏管理委员会普通微生物中心
保藏机构简称：CGMCC
地址：北京市朝阳区北辰西路1号院3号中国科学院微生物研究所
保藏日期：2016年1月27日
分类命名：杂交瘤细胞株
保藏中心登记入册编号：CGMCC No. 12033。
附图说明
图1：重组质粒pFastBac HT-E0的鉴定
图2：重组E0蛋白纯化鉴定图
图3：胶体金组装示意图
背板1；金标垫2；样品垫3；硝酸纤维膜4；吸水垫5；质控线6；检测线7。
具体实施方式
以下为结合具体实施例来进一步描述本发明，这些实施例仅是范例性的，并不对本发明的范围构成任何限制。在不偏离本发明的精神和范围下可以对本发明的技术方案的细节和形式进行修改或替换，但这些修改和替换均应涵盖在本发明的保护范围之内，本发明实施例所用试剂，如未特殊说明，均购自生化试剂供应商，所用实验技术，如未特别说明，均为常规技术。
实施例1：牛病毒性腹泻病毒的E0蛋白克隆表达纯化及抗体制备
重组质粒构建
参照GenBank中登录的BVDV基因序列设计引物，其上游引物E0-F为SEQ ID No.3所列的序列，其下游引物E0-R为SEQ ID No.4所列的序列，用于克隆E0基因片段，在上、下游引物中引入了EcoRⅠ和Hind III 酶切位点，引物由上海生工生物工程技术服务有限公司合成。E0基因片段以pUC-E0质粒为模板，用PCR 方法扩增。反应条件：94 ℃预变性5min；94 ℃变性45 s，退火条件为55. 6 ℃ 45 s ，72 ℃延伸2 min，30 个循环后， 再72 ℃延伸7 min，4 ℃终止反应。纯化的PCR 产物与表达载体pFastBac HT-B分别进行双酶切后，用T4 DNA 连接酶4 ℃连接过夜，连接产物转化DH5α感受态细胞。阳性重组质粒命名为pFastBac HT-E0。
蛋白纯化鉴定
将阳性重组质粒转座DH10Bac感受态细胞，通过用KTG抗生素和蓝白斑筛选阳性克隆，提取Bacmid，转染SF9昆虫细胞，观察转染后的细胞病变并收集重组杆状病毒。将重组杆状病毒以MOI 0.1感染SF9昆虫细胞，4d后5000r/min离心10min收集上清。目的蛋白的纯化参照Ni-NTA纯化系统说明书进行，纯化后的蛋白用Bradford蛋白浓度测定试剂盒测定蛋白含量后分装，-80℃保存备用。纯化蛋白煮样后进行SDS-PAGE电泳，通过湿法转印至NC膜上。经5%脱脂奶粉封闭后一次加入一抗（BVDV阳性血清，1：100）和二抗（兔抗牛IgG HRP，1：3000），最后加入底物显色。
抗体制备
以纯化的E0蛋白为免疫抗原，以常规方法免疫Balb/C小鼠，多次免疫后测定血清效价，并加强免疫。随后取免疫小鼠的脾细胞与SP2/0细胞融合，经过筛选得到了能分泌抗E0蛋白特异性抗体的杂交瘤细胞株，命名为MAB106。
抗体鉴定
用亚型鉴定试剂盒对杂交瘤细胞株MAB106分泌的抗体进行鉴定，抗体亚型鉴定为IgG2b。
实施例2：MAB106胶体金标记及胶体金试纸条的组装
一、胶体金标记
按照常规手段，采用柠檬酸三钠还原法制备得到40nm的胶体金颗粒溶液，调节pH值至9.0备用。取一定量的胶体金颗粒溶液，置于磁力搅拌器上，然后将纯化后的单克隆抗体以PBS为稀释液按照1:2000的比例稀释后加入胶体金溶液中搅拌反应60分钟，然后将PEG20000溶液加入混合反应液中，使PEG的最终浓度约为1%，置于冷冻离心机（4℃）上慢速（速度为11000~13000rpm）离心10分钟后弃去上清液。将下层沉淀用PBS反复洗涤并继续离心弃去多余上清液。最后将沉淀物以PBS缓冲液重悬，使最终的蛋白浓度约为50μg/mL左右，保存于4℃备用。
二、胶体金试纸条组装
以PVC背板（1）为支撑物，在其上分别贴有硝酸纤维膜（4）、金标垫（2）、样品垫（3）、吸水垫（5）。其中金标垫（2）采用聚酯膜，经含有1% BSA和1%吐温-20的PBS处理后，将制备好的胶体金标记BVDV抗体按照40μL喷涂，完毕，37℃度烘干2h备用。
样品垫（3）采用聚酯纤维垫，采用自制含有表面活性剂的处理液浸泡后烘干备用。
硝酸纤维膜（4）上包被有两条线：分别为质控线（6）和检测线（7），其中检测线包被物为检测抗体，质控线包被物为羊抗鼠二抗（购自北京中杉金桥生物技术有限公司），分别将检测抗体和羊抗鼠二抗用含有1%BSA 的PBS稀释成浓度为0.8mg/mL和1.2mg/mL的溶液，然后用Biodot划膜仪喷涂于硝酸纤维膜上，完毕，37℃度烘干1h备用。
试纸条的组装按照图3模式进行。组装好的大板用切条机切成7-8mm的裸条备用。
实施例3：利用牛病毒性腹泻病毒特异性单克隆抗体检测牛血清中的BVDV抗原
（1）采样：用采血针（BD产品）采集牛尾静脉血于无菌采血管中，析出血清后备用。如果不能及时检测，样品可以冷藏于4℃内不超过24h。长期保存需分装后冻存于-20℃。
（2）样本的稀释：检测之前，将样本恢复至室温（19-25℃），如从冷藏环境中取出后至少在室温放置30min以上。利用pH7.2 0.1M PBS按照1：50的比例稀释样本，混匀后备用。
（3）检测：将准备好的样品用微量移液器吸取200μL至反应微孔中，插入准备好的试纸条，确保样品垫朝下插入溶液中，开始计时。
（4）判断：计时5-8min后，根据试纸条显色判定检测结果。试纸条显示两条线，表明检测样本中有BVDV抗原存在，样本为阳性；试纸条为一条线，即质控线显色，而检测限不显色，表明样本中不含有BVDV抗原，样本为阴性。
注意：当检测线不清楚，或者有弱影存在时，表明样本为可疑样本，需要重新测试或用其他方法进一步复核检测。
虽然上面的举例了一些特定实施例来说明和描述本发明，但并不意味着本发明仅局限于其中的各种细节。相反地，在等价于权利要求书的范畴和范围内可以不偏离本发明精神地在各种细节上做出各种修改。
SEQUENCE LISTING
<110> 北京纳百景弈生物科技有限公司
<120> 牛病毒病性腹泻病毒特异性单克隆抗体细胞株及其应用
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version 3.5
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<213> 制备该单克隆抗体所用基因片段的核苷酸
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ggagtcccat cccacctggc aacagacaca gaattgaaag gcatacaggg gatgatggac 180
gccagtgaga agaccaatta cacatgctgc agacttcagc gccatgaatg gaacaagcat 240
ggctggtgca actggtacaa catcgaacct tggatatgga tcatgaacaa aactcaagca 300
aacttaacag aaggccaacc actcagggaa tgtgcagtca cctgtagatt tgacaaggat 360
agtgatttaa acatagtgac ccaagccagg gacaggccta ccttgctaac tggttgcaag 420
aaggggaaaa atttttcctt ctcaggcatc ataacaacga gtccttgtaa tttcgaggtg 480
tctgcagaag acatcctgtt caaagatcca gattgctcta gcacgctcca tgacacagcc 540
atatacttgg tagacggggt aacaaataca ttggaaggag cccggcaagg taccgctaag 600
ctgaccacgt ggttggggaa acaattgggt atactaggta agaaactcga acacaaaagc 660
aagacatggt ttggagccta tgcataa 687
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<213> 牛病毒病性腹泻病毒特异性单克隆抗体免疫蛋白
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Claims (5)
1.一种牛病毒病性腹泻病毒特异性单克隆抗体细胞株，保藏于中国微生物菌种保藏管理委员会普通微生物中心,登记入册的编号为CGMCC No. 12033。
2.一种牛病毒病性腹泻病毒特异性单克隆抗体，其特征在于：所述单克隆抗体是由权利要求1所述的牛病毒病性腹泻病毒特异性单克隆抗体细胞株分泌产生的。
3.根据权利要求2所述的牛病毒病性腹泻病毒特异性单克隆抗体，其特征在于，制备该单克隆抗体所用基因片段的核苷酸序列如SEQ ID No.1所示，所用免疫蛋白的氨基酸序列如SEQ ID No.2所示。
4.权利要求2所述的牛病毒病性腹泻病毒特异性单克隆抗体在检测牛病毒病性腹泻病毒抗原检测装置中的应用。
5.根据权利要求1所述的牛病毒病性腹泻病毒特异性单克隆抗体细胞株或权利要求2所述的牛病毒病性腹泻病毒特异性单克隆抗体在制备牛病毒病性腹泻病毒抗原检测试剂盒中的应用。
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| CN112034169A (zh) * | 2020-08-27 | 2020-12-04 | 华威特（江苏）生物制药有限公司 | 用于检测牛病毒性腹泻病毒1型的直接免疫荧光试剂及其试剂盒 |
| CN115724957A (zh) * | 2022-10-21 | 2023-03-03 | 北京纳百生物科技有限公司 | 抗牛病毒性腹泻病毒ns3蛋白单克隆抗体的重链和轻链可变区 |
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