CN112034169A - 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 - Google Patents
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 Download PDFInfo
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- CN112034169A CN112034169A CN202010878307.7A CN202010878307A CN112034169A CN 112034169 A CN112034169 A CN 112034169A CN 202010878307 A CN202010878307 A CN 202010878307A CN 112034169 A CN112034169 A CN 112034169A
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- viral diarrhea
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- diarrhea virus
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Abstract
本发明涉及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，免疫荧光试剂是用异硫氰酸荧光素(FITC)标记的抗牛病毒性腹泻病毒1型的单克隆抗体；单克隆抗体，是利用牛病毒性腹泻病毒1型浓缩纯化的病毒粒子作为抗原，采用杂交瘤细胞单克隆抗体技术制备获得；检测牛病毒性腹泻病毒1型的免疫荧光试剂盒中的免疫试剂为检测牛病毒性腹泻病毒1型的免疫荧光试剂；本发明具有敏感性高特异性强、诊断速度快，可鉴别诊断1型、2型牛病毒性腹泻病毒等优点。
Description
技术领域
本发明属于生物技术领域，具体涉及检测牛病毒性腹泻病毒1型的免疫荧光试剂及其检测试剂盒，适用于牛病毒性腹泻病毒1型的诊断以及样品、血清或细胞中牛病毒性腹泻病毒1型的检测与鉴定。
背景技术
牛病毒性腹泻病毒（Bovine Viral Diarrhea Virus，BVDV）是单股正链RNA病毒，属于黄病毒科瘟病毒属，与猪瘟病毒和边界病毒在同一属，同源性较高，抗原性存在交叉。BVDV主要感染牛，也可感染猪、羊、鹿等其他偶蹄动物。BVDV感染怀孕母牛后，病毒可以穿过胎盘，造成胎儿流产、死胎和新生儿过早死亡。BVDV从基因型角度分为2个基因型，即牛病毒性腹泻病毒1型（BVDV1）、牛病毒性腹泻病毒2型（BVDV2），其中BVDV1（经典BVDV）的流行比较广泛，成世界性分布。
我国在1980年首次报道分离到了BVDV1，之后的一段时间内全国不同地方均报道了BVDV1的感染，表明其在我国牛群汇中已成普遍流行趋势。因BVDV1在牛群中存在着很高的感染率，牛的衍生产品牛血清、牛源细胞等都可能存在着BVDV1污染。牛血清和牛源细胞作为兽用生物制品行业重要的原辅材料，一旦原辅材料中出现了BVDV1，会对科研实验以及相关疫苗的生产造成不同程度的影响，尤其是牛用、猪用活疫苗污染BVDV1后可能会导致免疫动物感染BVDV1。
目前用于检测BVDV1型的方法主要有间接荧光抗体检测法、血清中和试验、ELISA试验以及PCR技术等，这些方法不是存在操作费时费力、灵敏度低缺点，就是具有假阳性率高、特异性弱，不能区分病毒活性的不足。为此，开发一种鉴别检测BVDV1的直接免疫荧光试剂盒，用于简化检测操作步骤，并进一步区分BVDV1、BVDV2以及猪瘟病毒就显得意义重大。
发明内容
本发明针对现有技术中存在的不足，提供了一种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，以解决现有技术中存在的问题。
为实现上述目的，本发明采用的技术方案如下：
一种检测牛病毒性腹泻病毒1型的直接免疫荧光试剂，是用异硫氰酸荧光素（FITC）标记的抗BVDV1的单克隆抗体。所述的单克隆抗体，是利用BVDV1浓缩纯化的病毒作为抗原，采用杂交瘤细胞单克隆抗体技术制备获得。经特异性检测，制备的单克隆抗体仅与BVDV1病毒反应。
单克隆抗体的亚类为IgG1。
单克隆抗体结合蛋白鉴定杂交瘤细胞分泌的单克隆抗体可与牛病毒性腹泻病毒1型的重组蛋白E2蛋白发生特异性反应；E2蛋白氨基酸序列为LKCRPDFSYAIAGSNKIGLLGAEDLTTTWQKYPVGMRLKDSMVEVWCKGGEIMFLKRCAREARYLATLHTRALPTSVVFLQLFSGQGLVESVGMEDNFEFGLCPCDARPVIKGKFNTTLLNGPAFQMVCPIGWTGSVECTLVNKDTLSTAIVRAYKRVRPFPHRQGCVTQKLVGEDLYDCLLGGNWTCITGDQVKYAGGQVKTCKWCGYNFQGSEGLPHYPIGKCKLENETGYRLVDDTPCNRNGVAIVPTGTLKCRIGDTVVQVIAMDTKLGPMPCRPYEITPSEGPVEKTACTFNYTKTLKNKYFEPRDSCFQQYMLKGEYQYWFDLDAIDHHKD。
一种检测牛病毒性腹泻病毒1型免疫荧光试剂盒，免疫荧光试剂为上述检测BVDV1的免疫荧光试剂。
本发明提供了检测牛病毒性腹泻病毒1型的免疫荧光试剂及其检测试剂盒，是应用本发明的检测BVDV1的免疫荧光试剂，建立的直接免疫荧光检测方法，可用于快速诊断牛病毒性腹泻病毒1型。直接免疫荧光检测时间短，60分钟左右即可报告结果；特异性强，仅与牛病毒性腹泻病毒1型反应；准确性强，与PCR、间接免疫荧光等方法的符合率可达95％以上。
由于采用了以上技术，本发明较现有技术相比，具有的有益效果如下：
（1）敏感性高：制备的单克隆抗体均有很高的效价，从而确保制备的免疫荧光抗体均有很好的敏感性。
（2）特异性强：制备的免疫荧光试剂与其他牛源病原体（BVDV2、IBR、PI3）无交叉反应，只能检测出牛病毒性腹泻病毒1型，具有很高的特异性。
（3）稳定性好：免疫荧光试剂可在2~8℃保存12个月，具有很好的稳定性。
（4）诊断速度快：60分钟左右即可报告结果，相比其他方法可节约1~2小时。
附图说明
图1为本发明实施例的试剂检测BVDV1阳性结果的显微镜图；
图2为本发明实施例的试剂检测BVDV2阴性结果的显微镜图；
图3为本发明实施例的试剂检测IBR阴性结果的显微镜图；
图4为本发明实施例的试剂检测BPIV3阴性结果的显微镜图；
图5为本发明实施例中细胞对照检测阴性结果的显微镜图。
具体实施方式
下面结合具体实施方式，进一步阐明本发明。
实施例1：牛病毒性腹泻病毒1型抗原的制备
将病毒液用过滤、离心去除细胞碎片；向病毒液中缓慢加入固体PEG和NaCl；2~8℃条件下，搅拌过夜；2~8℃条件下，以8000r/min离心50分钟，弃上清，用原体积1%的NTE缓冲液将沉淀物重悬；采用蔗糖密度梯度离心的方法纯化病毒。采用紫外分光光度计测定纯化后的病毒蛋白含量。
实施例2：抗牛病毒性腹泻病毒1型单克隆抗体的制备
1、动物免疫：取浓缩纯化后的病毒蛋白，用适量PBS稀释后与等体积弗氏完全佐剂乳化，腹腔接种BALB/c小鼠，50ug/只；首免后第14天和第28天分别与等体积弗氏不完全佐剂乳化后进行二免与三免；三免后第14天断尾采血分离血清，通过间接ELISA方法检测血清的抗体效价，当血清抗体效价大于1∶10000时，进行细胞融合；细胞融合前3天用不加佐剂的病毒液经尾静脉强化免疫1次，100ug/只。
2、细胞融合：将SP2/0细胞与免疫小鼠的脾细胞按1∶5的比例混合，以800r/min离心5分钟，弃上清，震荡细胞，使细胞松散混匀；然后于60秒内缓慢加预热的PEG-4000溶液；再缓慢加入无血清的1640培养基终止融合，静置2~3分钟后以700r/min离心5分钟，弃上清；用HAT培养基轻轻重悬，加入到已铺有饲养细胞的96孔细胞板中，100μl/孔；置37℃、含5%CO2培养箱中培养。
3、杂交瘤细胞株的筛选：采用间接ELISA方法和间接免疫荧光（IFA）方法检测其上清的特异性，其中间接ELISA方法检测时采用纯化的病毒蛋白作为检测抗原，间接免疫荧光方法检测时采用BVDV1、BVDV2、IBR和PI3病毒培养物为检测抗原。对ELISA与间接免疫荧光均检测为BVDV1阳性，且BVDV2、IBR和PI3检测为阴性的杂交瘤细胞进行克隆，直至所有细胞克隆孔均为阳性。经过多次克隆后，共获得1株杂交瘤细胞，选取命名为BV1株的杂交瘤细胞进行后期试验。
实施例3：单克隆抗体特性鉴定
1、单克隆抗体亚类鉴定按免疫球蛋白标准亚类鉴定试剂盒说明书进行，确定单克隆抗体的亚类为IgG1。
2、杂交瘤细胞株染色体分析采用秋水仙素法对杂交瘤细胞株进行染色体分析，结果显示BV1株杂交瘤细胞的染色体数目为102条。
3、单克隆抗体结合蛋白鉴定杂交瘤细胞分泌的单克隆抗体可与牛病毒性腹泻病毒1型的重组蛋白E2蛋白发生特异性反应。E2蛋白氨基酸序列为LKCRPDFSYAIAGSNKIGLLGAEDLTTTWQKYPVGMRLKDSMVEVWCKGGEIMFLKRCAREARYLATLHTRALPTSVVFLQLFSGQGLVESVGMEDNFEFGLCPCDARPVIKGKFNTTLLNGPAFQMVCPIGWTGSVECTLVNKDTLSTAIVRAYKRVRPFPHRQGCVTQKLVGEDLYDCLLGGNWTCITGDQVKYAGGQVKTCKWCGYNFQGSEGLPHYPIGKCKLENETGYRLVDDTPCNRNGVAIVPTGTLKCRIGDTVVQVIAMDTKLGPMPCRPYEITPSEGPVEKTACTFNYTKTLKNKYFEPRDSCFQQYMLKGEYQYWFDLDAIDHHKD。
4、抗体特异性鉴定将BV1株杂交瘤细胞分泌的单克隆抗体分别于牛病毒性腹泻病毒2型（BVDV2）、牛传染性鼻气管炎病毒（IBRV）和牛副流感病毒3型（BPIV3）进行间接免疫荧光试验。结果显示，BV1株杂交瘤细胞分泌的单克隆抗体只与BVDV1发生反应，与其他病毒均无交叉反应性，鉴定结果见表1。
表1 BV1株杂交瘤细胞分泌的单克隆抗体特异性鉴定结果
| 样品名称 | BVDV1 | BVDV2 | IBRV | BPIV3 | 细胞对照 |
| 结果 | + | - | - | - | - |
注：“-”未见特异性荧光，“+”出现特异性荧光。
5、单克隆抗体制备选择6~10周龄SPF级BALB/c小鼠，腹腔接种弗氏不完全佐剂，0.5ml/只；7天后腹腔注射杂交瘤细胞，0.5ml/只；7~10天后，待小鼠腹腔明显隆起时无菌采集腹水，2~8℃静置过夜，8000r/min离心，弃上层油脂和下层细胞碎片等杂质，留取中间淡黄色或淡红色液体，加入防腐剂Proclin300至终浓度为0.02％，于-70℃以下保存。
6、单克隆抗体纯化取1份腹水加入2份醋酸缓冲液（0.06mol/L），调pH值至4.8；按33μl/ml将正辛酸逐滴加入腹水中，边加边搅拌，在室温下混匀30分钟；2~8℃静置至少2小时，10000r/min离心35分钟，取上清。按上清体积的1/10加入PBS，调pH值至7.2；在冰水浴中按0.277～0.310g/ml将硫酸铵慢慢加入腹水中，边加边搅拌，至45%～50%饱和度，冰水浴中混匀30分钟；2~8℃静置至少2小时，10000r/min离心60分钟，弃上清。用适量PBS溶解沉淀，在2~8℃条件下用约1000倍体积的PBS透析24~48小时，期间更换PB至少3次，10000r/min离心10分钟，取上清采用紫外分光光度计测定蛋白浓度后，分装于-70℃以下保存。
实施例4：免疫荧光试剂的制备
1、单克隆抗体稀释将纯化后BVDV1单克隆抗体用碳酸盐缓冲液（0.1mol/L，pH值9.0）稀释至蛋白浓度为5mg/ml。
2、荧光素溶液制备称取异硫氰酸荧光素（FITC），避光条件下，用碳酸盐缓冲液（0.1mol/L，pH值9.0）配制成1mg/ml。
3、搅拌法标记按1ml 单克隆抗体溶液（5mg/ml）加250ul FITC溶液（1mg/ml）的比例，向纯化的单克隆抗体溶液中逐滴加入FITC溶液（1mg/ml），边加边搅拌，在室温条件下，避光轻柔混匀2小时。
4、游离荧光素的去除使用葡聚糖G-25M柱子去除游离荧光素，分管收集洗脱液。用紫外分光光度计在280nm波长下检测每管洗脱液的吸收值，可见有2个洗脱峰，偶联物出现在第一个洗脱峰；汇集主要的偶联物，收集A280≥0.4的洗脱液，2~8℃避光保存。
5、荧光试剂制备按0.01g/ml将BSA溶解于收集的洗脱液中，加入防腐剂Proclin300至终浓度为0.02％，混合均匀，过滤除菌，无菌分装于灭菌避光管中，2~8℃保存。即为牛病毒性腹泻病毒1型免疫荧光试剂。
实施例5：直接免疫荧光检测方法的建立
1、牛病毒性腹泻病毒1型感染细胞板的制备：将牛病毒性腹泻病毒1型接种已长成单层的96孔MDBK细胞培养板，同时设正常MDBK细胞作为阴性对照；于37℃、5％CO2条件下培养24小时。弃去培养液，用PBST（含0.5‰ Tween-20的PBS，0.01mol/L，pH值7.2）洗涤2次，200μl/孔，室温静置5分钟后弃液。置于干净的吸水纸上吸干，加入4%甲醛固定液，100μl /孔，25℃作用10分钟后，用PBST洗涤2次。-20℃冻存备用。
2、直接免疫荧光操作方法：取制备的牛病毒性腹泻病毒1型感染细胞板，加入0.5%Triton X-100 0.2ml，25℃作用10分钟；弃液，用PBST洗涤2次；加入用含1% BSA的PBS稀释至工作浓度的荧光抗体试剂，100μl/孔，置37℃湿盒中孵育45-60分钟；弃荧光抗体，用PBST洗涤2次，置荧光显微镜下观察，以出现特异性绿色荧光判为阳性，细胞对照孔应未出现特异性绿色荧光。
实施例6：免疫荧光试剂特异性鉴定
1、荧光试剂工作浓度的确定：取制备的牛病毒性腹泻病毒1型感染细胞板，每孔分别加入用含1% BSA的PBS进行10倍系列稀释的免疫荧光试剂，进行直接免疫荧光试验，以出现清晰、明亮、特异性荧光的稀释倍数为试剂的最大稀释倍数。为保证荧光试剂的敏感性和有效性，将最大稀释倍数的上一个稀释度确定为工作浓度。
2、荧光试剂的特异性检测：将荧光抗体进行适当稀释后，分别与制备的牛病毒性腹泻病毒1型（BVDV1）、牛病毒性腹泻病毒2型（BVDV2）、牛副流感病毒3型（BPIV3）、牛传染性鼻气管炎病毒（IBRV）感染细胞以及正常细胞对照，进行直接免疫荧光试验。结果显示，在荧光显微镜下仅BVDV1感染细胞孔出席年特异性绿色荧光，呈阳性反应；BVDV2和IBRV感染细胞孔未出现特异性绿色荧光，呈阴性反应；细胞对照孔应未出现特异性绿色荧光，结果见表2。表明制备的免疫荧光试剂特异性好，可用于BVDV1的检测与鉴定。
表2 免疫荧光试剂特异性鉴定结果
| 样品名称 | BVDV1 | BVDV2 | IBRV | BPIV3 | 细胞对照 |
| 结果 | + | - | - | - | - |
3、免疫荧光试剂的重复性和稳定性检测：将3批BVDV1免疫荧光试剂置2~8℃保存3、6、9、12和15个月后，随机抽样对BVDV1感染细胞进行直接免疫荧光试验，测定试剂的保存期和批内、批间的稳定性。结果显示，免疫荧光试剂在2~8℃保存15个月后质量稳定，批内和批间的重复性良好。为保证试剂的质量，将保存期暂定为2~8℃保存12个月。
免疫荧光试剂盒的临床应用使用制备2017001批、2017002批、2017003批牛病毒性腹泻病毒1型荧光抗体试剂盒分别对1106份临床样品进行检测，阳性检测率分别为7.32%、7.23%、7.23%。
上述实施例仅为本发明的优选技术方案，而不应视为对于本发明的限制，本发明的保护范围应以权利要求记载的技术方案，包括权利要求记载的技术方案中技术特征的等同替换方案为保护范围，即在此范围内的等同替换改进，也在本发明的保护范围之内。
序列表
<110> 华威特（江苏）生物制药有限公司
<120> 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
<160> 1
<170> SIPOSequenceListing 1.0
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<212> PRT
<213> 人工序列(Artificial Sequence)
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Asp
Claims (6)
1.一种检测牛病毒性腹泻病毒1型的直接免疫荧光试剂，其特征在于：所述免疫荧光试剂是用荧光染料标记的抗牛病毒性腹泻病毒1型的单克隆抗体。
2.根据权利要求1所述的一种检测牛病毒性腹泻病毒1型的直接免疫荧光试剂，其特征在于：所述荧光染料为异硫氰酸荧光素（FITC）。
3.根据权利要求1所述的一种检测牛病毒性腹泻病毒1型的直接免疫荧光试剂，其特征在于：所述单克隆抗体，是利用BVDV1浓缩纯化的病毒作为抗原，采用杂交瘤细胞单克隆抗体制备获得。
4.根据权利要求1所述的一种检测牛病毒性腹泻病毒1型的直接免疫荧光试剂，其特征在于：单克隆抗体的亚类为IgG1。
5.根据权利要求1所述的一种检测牛病毒性腹泻病毒1型的直接免疫荧光试剂，其特征在于：单克隆抗体结合蛋白鉴定杂交瘤细胞分泌的单克隆抗体与牛病毒性腹泻病毒1型的重组蛋白E2蛋白发生特异性反应；E2蛋白氨基酸序列为LKCRPDFSYAIAGSNKIGLLGAEDLTTTWQKYPVGMRLKDSMVEVWCKGGEIMFLKRCAREARYLATLHTRALPTSVVFLQLFSGQGLVESVGMEDNFEFGLCPCDARPVIKGKFNTTLLNGPAFQMVCPIGWTGSVECTLVNKDTLSTAIVRAYKRVRPFPHRQGCVTQKLVGEDLYDCLLGGNWTCITGDQVKYAGGQVKTCKWCGYNFQGSEGLPHYPIGKCKLENETGYRLVDDTPCNRNGVAIVPTGTLKCRIGDTVVQVIAMDTKLGPMPCRPYEITPSEGPVEKTACTFNYTKTLKNKYFEPRDSCFQQYMLKGEYQYWFDLDAIDHHKD。
6.一种检测牛病毒性腹泻病毒1型免疫荧光试剂盒，其特征在于，免疫试剂为权利要求1所述的检测牛病毒性腹泻病毒1型的免疫荧光试剂。
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