CN107656066A - 一种鸭坦布苏病毒e蛋白截短蛋白及应用 - Google Patents
一种鸭坦布苏病毒e蛋白截短蛋白及应用 Download PDFInfo
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- CN107656066A CN107656066A CN201710798615.7A CN201710798615A CN107656066A CN 107656066 A CN107656066 A CN 107656066A CN 201710798615 A CN201710798615 A CN 201710798615A CN 107656066 A CN107656066 A CN 107656066A
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Abstract
本发明属于动物病毒学与动物传染病学检测技术领域,具体公开了一种鸭坦布苏病毒E蛋白截短蛋白及应用。利用SEQ ID NO.2所示的氨基酸序列的蛋白作为包被抗原,保藏编号为CCTCC NO:C2017169的杂交瘤细胞株制备的鸭坦布苏病毒血清检测试剂盒,对被野毒感染过的鸭群血清及蛋黄抗体进行检测,具有很好的特异性;灵敏度高,鸭坦布苏病毒阳性血清在稀释到1:12800时仍能检测阳性,与传统的琼脂扩散试验相比,符合率高,操作简便,检测时间更短,适合大量样本同时检测。
Description
技术领域
本发明属于动物病毒学与动物传染病学检测技术领域。具体涉及一种鸭坦布苏病毒E蛋白截短蛋白及应用。
背景技术
鸭坦布苏病毒DTMUV(Duck Tembusu Virus)是具有囊膜的单股正链RNA病毒,属于黄病毒科黄病毒属蚊媒病毒类恩塔亚病毒群,外观为球形。临床上引起以鸭死亡和蛋鸭产蛋下降为主要特征的传染病。根据已有的研究报道,该病毒可感染鸡、鸭、鹅、鸽等多种动物发病。2011年中国畜牧兽医学会第一届水禽疫病防控研讨会将该病毒引发的疾病统一命名为禽坦布苏病毒病(Avian Tembusu Virus Disease,ATMUVD),引起鸭致病的称之为鸭坦布苏病毒病。
2010年4月鸭坦布苏病毒病在我国华东地区首次爆发,并迅速传播至浙江、安徽、福建、江苏、广东、江西等主要鸭养殖地,主要的感染宿主有种(蛋)鸭、蛋鸡、鹅、肉鸭、麻雀,发病品种有鸡、鸭、鹅、鸟,发病鸭中以麻鸭最多,其次为樱桃谷鸭、番鸭等。鸭坦布苏病毒病传播途径广,传播速度快,宿主范围广,一年四季均可发病,其中以春秋季节多发,群内发病率为100%,不同地区不同的养殖场病死率高低不一,一般在5%-30%,可与其它病原混合感染,对养禽业形成较大的威胁并造成较大的损失。
DTMUV为约由10990nt组成的不分节段的单股正链RNA病毒,基因组由5’端和3’端非编码区及一个开放阅读框(ORF)组成。ORF编码3个结构蛋白(C、prM、E)及7个非结构蛋白。E蛋白分子量大小约为54KD,由501个氨基酸组成,富含甘氨酸和丙氨酸,有一个糖基化位点。
E蛋白是DTMUV最主要的结构蛋白,具有多个抗原决定簇,是引起宿主产生免疫抗体的主要蛋白。与病毒的吸附、穿入、组织嗜性、致病性、血凝反应、血清特异性和诱导宿主的免疫应答等作用密切相关。将E基因编码的氨基酸序列利用蛋白质序列软件分析,发现包含一个胞内区,两个跨膜区,较为简短的胞外区,作为囊膜蛋白,E蛋白的疏水性导致其全长表达难度增加。申请人根据一些已报道的文献,构建了几种不同长度的质粒,原核表达了相应的截短蛋白,发现有些与鸭坦布苏病毒鸭阳性血清无特异性反应,有些表达量不稳定,目前尚无成熟的鸭坦布苏病毒E蛋白表达后用于检测血清的相关报道。
发明内容
针对上述问题,本发明提供了一种鸭坦布苏病毒E蛋白截短蛋白在制备鸭坦布苏病毒血清检测试剂盒中的应用,所述的鸭坦布苏病毒E蛋白截短蛋白的核苷酸序列为SEQID NO.1所示,氨基酸序列为SEQ ID NO.2所示,该蛋白与鸭坦布苏病毒血清呈明显的阳性反应,适用于血清的ELISA检测。
本发明的另一个目的在于提供了鸭IgG的单克隆抗体,所述的抗体由杂交瘤细胞株4A10分泌得到。
本发明还有一个目的在于提供了一株杂交瘤细胞株,该杂交瘤细胞株已于2017年9月1日送至中国典型培养物保藏中心保藏,分类命名:杂交瘤细胞株4A10,保藏编号:CCTCCNO:C2017169地址:中国武汉武汉大学。
本发明最后一个目的在于提供了一种鸭坦布苏病毒血清检测试剂盒,所述试剂盒的包被抗原为SEQ ID NO.2所示的氨基酸序列,酶标二抗为酶标鸭IgG单抗,所述的单抗由保藏编号为CCTCC NO:C2017169的杂交瘤细胞分泌得到。
为达到上述目的,本发明通过以下技术方案实现:
鸭坦布苏病毒E蛋白截短蛋白在制备鸭坦布苏病毒血清检测试剂盒中的应用,包括利用本发明提供的SEQ ID NO.2所示的蛋白作为抗原包被板;
以上所述的方案中,优选的,酶标二抗为酶标鸭IgG单抗。
以上所述的方案中,优选的,所述的单抗由杂交瘤细胞株4A10分泌得到。该杂交瘤细胞株已于2017年9月1日送至中国典型培养物保藏中心保藏,分类命名:杂交瘤细胞株4A10,保藏编号:CCTCC NO:C2017169地址:中国武汉武汉大学。
杂交瘤细胞株4A10的生理生化特性:杂交瘤细胞4A10的原代细胞、传代细胞及液氮中保存的能稳定分泌单克隆抗体的定株4A10细胞在RPMI-1640完全培养基中,在含5%CO2,37℃条件下培养48小时,显微镜下观察,细胞生长良好,呈葡萄串状、集落浑圆透亮,符合杂交瘤细胞的培养特性。
本发明首先利用RT-PCR,扩增出E基因,连接至Pgex-kg载体,转化到Rosetta菌株内,通过IPTG诱导表达,大量制备并通过在不同浓度的脲溶液中变性复性纯化从而得到鸭坦布苏病毒E蛋白。
其次采集健康鸭全血,分离血清,利用辛酸硫酸铵法提纯,免疫6-8周龄Balb/c小鼠,通过杂交瘤细胞融合技术,制备并筛选得到一株稳定分泌鼠抗鸭的杂交瘤细胞株4A10,大量制备4A10杂交瘤细胞株小鼠腹水,辛酸硫酸铵法纯化后进行HRP标记。将制备的E蛋白作为抗原,HRP标记物作为酶标二抗,建立了鸭坦布苏病毒E蛋白ELISA抗体检测方法,该方法特异性强,敏感性高,易于操作,节省时间。
与现有技术相比,本发明具有以下优点:
1.本发明的鸭坦布苏病毒E蛋白ELISA抗体检测方法适用于对经过鸭坦布苏病毒野毒感染的,鸭坦布苏病毒活苗(弱毒苗),灭活苗免疫的鸭群血清抗体水平进行检测,而对鸭类其他病毒感染或其他鸭类疫苗免疫过的鸭血清则不反应,具有很好的特异性。
2.本发明的鸭坦布苏病毒E蛋白ELISA抗体检测方法与传统的琼脂扩散试验抗体检测方法符合率高。
3.本发明将所需的各种试剂组装成试剂盒,操作简单,用时短,试剂盒稳定性好、保存期长。
4.本发明同时处理样本量大,适用于临床大规模适用,并且可以用于科研。
5.本发明表达的E基因截短蛋白由于碱基密码子含量较多,故选用BL21(DE3)的优化表达菌株Rosetta,通过摸索不同诱导浓度的IPTG,最终优化条件,获得稳定表达E蛋白的最佳条件,在该条件下,E蛋白表达量大,易于纯化,不仅可与载体的标签抗体发生特异性反应,也可很好地识别经过鸭坦布苏疫苗免疫的鸭阳性血清,说明该截短蛋白已经含有了能很好地识别血清抗体的抗原表位,可以作为检测抗体用抗原。
6.利用本发明提供的抗原和二抗制备的试剂盒,灵敏度高,鸭坦布苏病毒阳性血清在稀释到1:12800时仍能检测阳性;特异性好,本发明的试剂盒仅能特异性的识别DTMUV鸭阳性血清,而与其他4种禽类病毒的阳性血清没有交叉反应;符合率高,与传统的琼脂扩散进行比较,准确率高达87.5%,适用于鸭坦布苏病毒血清抗体的快速检测。
附图说明
图1鸭坦布苏病毒E基因截短基因扩增的DNA电泳图。
图2鸭坦布苏病毒E蛋白的截短蛋白的SDS-PAGE电泳图;
其中泳道1为Pgex-kg空载对照,2为37℃诱导表达的上清,3为37℃诱导表达的沉淀,4为大量诱导表达纯化浓缩产物。
泳道3箭头所指条带为在沉淀中表达的E蛋白,大小约为76KD,泳道4箭头所指条带为纯化浓缩后的E蛋白。
图3A为鸭坦布苏病毒E蛋白截短蛋白与GST标签抗体Western-blot示意图;
其中泳道1为诱导表达的Pgex-kg空载,2为E蛋白;
泳道1箭头所指为Pgex-kg空载表达的标签蛋白与标签抗体杂交条带,大小约为26KD,泳道2为重组质粒E-Pgex-kg经IPTG诱导表达的重组蛋白与标签抗体杂交条带,大小约为76KD。
图3B为鸭坦布苏病毒E蛋白截短蛋白与鸭坦布苏病毒鸭阳性血清Western-blot示意图;
其中泳道1为诱导表达的Pgex-kg空载,2为E蛋白。
泳道2箭头所指为为重组质粒E-Pgex-kg经IPTG诱导表达的重组蛋白与鸭坦布苏病毒鸭阳性血清杂交条带,大小约为76KD。
图4A为从健康鸭血清提取的IgG SDS-PAGE电泳图;
其中泳道1为鸭血清IgG,180KD所示箭头为鸭IgG未解链完整条带,55KD所示箭头为解链后的鸭IgG部分重链条带。
图4B为从健康鸭血清提取的IgG与鼠抗鸭IgG抗体Western-blot图;
180KD所示箭头为鸭IgG未解链完整条带与鼠抗鸭IgG特异性反应条带,,55KD所示箭头为解链后的鸭IgG部分重链条带与鼠抗鸭IgG特异性反应条带。
具体实施方式
本发明所用试剂及其组分如下:
1.碳酸盐包被缓冲液(pH 9.6):Na2CO3 1.59g,NaHCO3 2.93g,用去离子水定容至1000mL;
2.BSA封闭液(pH7.4):NaCl 8g,KCl 0.2g,Na2HPO4·12H2O 2.9g,KH2PO4 0.2g,BSA 5g,用去离子水定容至1000mL(pH=7.4);
3.样品稀释液:NaCl 8g,KCl 0.2g,Na2HPO4·12H2O 2.9g,KH2PO4 0.2g,Tween-200.5mL,用去离子水定容至1000mL;
4.显色液A:Na2HPO4·12H2O 14.60g,柠檬酸9.33g,30%的双氧水2mL,加去离子水溶解并定容至1000mL,调pH至5.0~5.4,分装,10mL/瓶。
5.显色液B:四甲基联苯胺(TMB)20.00mg、无水乙醇10.00mL,加去离子水溶解定容至1000mL,分装成10mL/瓶。
6.终止液1:2.5mL氢氟酸(HF)加到900mL去离子水中,定容至1000mL,分装,10mL/瓶。
7.20倍浓缩洗涤液:NaCl 160g,KCl 4g,Na2HPO4·12H2O 58g,KH2PO4 4g,Tween-2010mL,用去离子水定容至1000mL(pH=7.4)。
8.鸭坦白苏病毒E蛋白ELISA抗体检测试剂盒中阴、阳性对照血清:阳性对照为购买瑞普(保定)生物股份有限公司鸭坦布苏病毒灭活苗免疫健康开产蛋鸭,采集全血,分离血清,经效价测定,筛选而得;阴性血清为采集健康蛋鸭全血,分离血清,测定其效价,筛选而得。
下面结合具体实施例来进一步阐述本发明。本发明所述技术方案,如无特别说明,均为本领域的常规方案。所述试剂或材料,如无特别说明,均为购买的商品化试剂。
实施例1:
DTMUV DF2株E截短蛋白的获得:
(1)DTMUVDF2株鸡胚毒,取200μL,加入Trizol试剂800μL,混匀,室温静置5min,加入200μL三氯甲烷,剧烈振摇15s,冰浴10min,4℃,12000rpm,离心15min,取600μL上清至洁净EP管内,加入等体积异丙醇溶液,轻轻混匀,室温静置10min,4℃,12000rpm,离心10min,弃上清,在EP管内加入1mL无水乙醇,轻轻混匀,4℃,7500rpm,离心5min,弃上清,室温晾干,加入13μL DEPC水溶解,-20℃存放。
(1)反转录:
按如下体系加入各组份:AMV反转录酶0.5μL,5倍AMV反转录buffer 4μL,RRI 1μL,随机引物1μL,dNTPS 2μL,RNA模板11.5μL,共20μL。反转录程序:42℃1h,72℃15min,-20℃存放。
(2)PCR扩增E基因:
上游引物P1:ATGGATCCTTCAGCTGTCTGGGGATGCAGA;
下游引物P2:CACTCGAGTTATTTAAATGCTGAGCCGAAAACCT。
按下述体系:cDNA 10μL,10mM dNTPs 4μL,P1 5μL,P2 5μL,TAQ酶1μL,10倍TAQbuffer 5μL,DEPC H2O 20μL,共50μL;
扩增程序如下:
95℃5min,95℃30s,55℃40s,72℃90s,72℃10min,获得截短的E基因(以下简称E基因),序列为SEQ ID NO.1所示,-20℃保存。
(3)酶切体系:
BamHI 1.5μL,XhoI 1.5μL,10倍K 5μL,E基因PCR回收产物13μL,DEPC H2O 29μL,共50μL。37℃3h。酶切完毕,用MG公司胶回收试剂盒分别进行回收。
(4)连接、转化及质粒的提取
连接体系:T4DNA Ligase 1μL,10倍T4buffer 1μL,Pgex-kg 4μL,E基因4μL,共10μL,4℃15h。在连接产物内加入50μL DH5a感受态细胞,冰浴30min,42℃热击90s,冰浴2mins,加入800μL LB液体,37℃,180rpm,45min,取100μL涂布含有1‰氨苄的LB琼脂平板,37℃倒置培养过夜。挑取平板上单克隆菌落,置于含1‰氨苄的LB培养液中37℃,220rpm,培养过夜,参照质粒提取试剂盒提取质粒,送测序鉴定。
(5)转化表达菌株,小量表达鉴定
将测序正确的质粒按上述转化方法,转入Rosetta表达菌株,涂布含1‰氨苄及1‰氯霉素的LB琼脂平板,挑取单克隆菌落,置于含1‰氨苄及1‰氯霉素的LB液体培养基中37℃,220rpm,培养至OD600为0.6-0.8,加入终浓度为1mM/L的IPTG诱导剂,,37℃,220rpm,诱导5h,收集菌体,超声波破碎,用SDS-PAGE及Western-blot进行鉴定。
(6)大量制备、纯化及鉴定
将小量表达鉴定正确的菌种按1‰接种20mL含1‰氨苄及1‰氯霉素的LB液体培养基中37℃,220rpm过夜进行复苏。将复苏的种子液按1%比例接种含1‰氨苄及1‰氯霉素的2L LB液体培养基中37℃,220rpm培养至OD600为0.6-0.8,加入终浓度为1mM/L的IPTG诱导剂,,37℃,220rpm,诱导5h,收集菌体,加入pH8.5的50mM Tris-cl溶液,重悬后,4℃,1200磅压力破碎三次。将破碎后的溶液4℃,12000rpm,离心10mins,分离上清沉淀。在沉淀中加入200mL 2Mol/L的尿素溶液,重悬均匀后,再次离心,弃上清,在沉淀中加入8M尿素溶液,重悬均匀后,再次离心,弃沉淀。将上清装入洁净透析袋中,置于2L 4M尿素溶液中4℃透析12h,2L 2M尿素溶液中透析12h,2L 1M尿素溶液中透析12h,4L50Mm Tris-cl溶液中透析24h,取出透析袋,置于蔗糖晶体中浓缩至约一半体积,收集,4℃,12000rpm,离心10min,弃去沉淀,按终浓度10%加入无菌甘油,磁力搅拌均匀后,分装,每管1.5mL,-80℃保存,同时用A280测定E蛋白的最终浓度,浓度为2.67mg/ml,即为E蛋白截短蛋白,序列为SEQ ID NO.2所示,用于实施例4。
(7)将纯化的蛋白取80μL加入20μL 5倍Loading Buffer,沸水中加热10min,进行SDS-PAGE及Western-bolt验证(图3A和图3B)。
由图3A和图3B可见,该重组蛋白既可以与标签抗体发生特异性反应,同时也能与鸭坦布苏病毒全毒灭活苗免疫鸭群后分离的阳性血清发生特异性反应,反应条带清晰,大小正确,可以作为检测鸭坦布苏病毒血清抗体的抗原。
实施例2:
杂交瘤细胞株4A10的获得:
(1)取三只健康鸭,颈动脉釆血,置于37℃30min,4℃过夜后分离,收集血清。用辛酸-硫酸铵法提取鸭血清IgG,具体操作如下:将收集的鸭血清4℃4000rpm离心3min,取上清,加入4倍体积的60mM/L的pH4.5的醋酸钠溶液,边加边搅拌,按每1mL上述血清加正辛酸25μL,室温静置30min,4℃,12000rpm,离心10min,取上清,用滤纸过滤,滤后调节pH至7.4。边搅拌边加入终体积小于等于45%饱和硫酸铵溶液,室温搅拌30min,4℃4000rpm离心10min,去上清,沉淀用适量10mM Tris-cl重悬,装入透析袋,10mM Tris-cl中透析24h,收集,分装,-20℃保存。
将收集的透析液,取80μL,加入20μL 5倍Loading Buffer,沸水中加热10min,SDS-PAGE检测;按同样的方法制备健康鸡IgG,以在筛选单克隆抗体时作为辅筛用。
(2)鼠抗鸭IgG的制备与检验小鼠的免疫及免疫效果检测:
将鸭IgG加入等体积弗氏完全佐剂充分乳化,按100μg/只,体积不超过0.2mL/只,颈背部皮下多点注射免疫6-8周龄Balb/c健康雌性小鼠,二周后等量等体积二免,四周后三免,(二免,三免均采用弗氏不完全佐剂)。三免后十天,尾部静脉釆血,间接ELISA方法测定免疫效价,效价达到1:6400后,按100ug/只,体积不超过0.2mL/只,腹腔加强免疫鸭IgG,三天后与SP2/0骨髓瘤细胞进行融合试验。
(3)免疫脾细胞与SP2/0细胞的融合:
取一只正常饲养的健康小鼠,按常规方法,取脾,研磨,制备饲养脾细胞备用;取一瓶T75瓶铺满单层的SP2/0小鼠骨髓瘤细胞,吹落,离心,去上清,备用;取免疫小鼠脾,制备脾细胞,将免疫脾细胞与小鼠骨髓瘤细胞置于同一管内,37℃水浴条件下,取1mL PEG2450,1min内缓慢滴加至混合细胞中,加完轻轻搅拌细胞30s,静置1min,加入40mL
RPMI培养基终止反应,离心去上清,加入40mL含饲养细胞的HAT培养基,混匀,铺四块96孔板,37℃5%CO2培养箱内静置培养。
(4)用间接ELISA方法对融合细胞进行筛选,具体操作如下:
将制备的鸭和鸡IgG溶液按500ng/孔进行分别包被,封闭。取融合细胞的上清各70μL,一一对应加入到包被的酶标板中用保鲜膜密封,37℃作用1h,弃去孔内液体,洗涤三次,分别加入1:5000稀释的HRP标记的羊抗鼠IgG,用保鲜膜密封后37℃作用1h,弃去孔内液体,洗涤三次,每孔内分别加入底物液A,B各50μL,室温避光静置10mins,加入终止液50μL,在酶标仪上读取OD630数值。选取跟鸭IgG有反应,而跟鸡IgG无反应,且OD630读值最大的四个孔,作为特异性杂交瘤细胞,利用有限稀释法,进行三到四次亚克隆,选取读数高,且亚克隆时阳性率为100%的细胞株进行定株并进行保藏,所述细胞株已于2017年9月1日送至中国典型培养物保藏中心保藏,分类命名:杂交瘤细胞株4A10,保藏编号:CCTCC NO:C2017169,地址:中国武汉武汉大学,其分泌的单克隆抗体命名为4A10单抗。
实施例3:
酶标二抗的获得
(1)鼠抗鸭IgG小鼠腹水的制备及纯化:
将杂交瘤细胞株4A10从液氮罐取出,复苏,扩大培养。提前三天至一周在8-10周龄BALB/C雌性小鼠腹腔内按每只0.5mL注射弗氏不完全佐剂。将扩大培养的杂交瘤细胞用细胞计数板计数,按5*105个/只腹腔注射小鼠,7-10天后,每天收集小鼠腹水,直至小鼠死亡。将收集的腹水12000rpm,离心5mins,弃沉淀,在上清中加入终浓度为0.01%的硫柳汞,分装后-80℃保存。按辛酸-硫酸铵法进行纯化,得到4A10单抗,即鼠抗鸭IgG。
(2)HRP标记鼠抗鸭IgG:
取5mg辣根过氧化物酶(HRP)溶于0.5mL注射用水中,加入0.5mL0.06mol/L的NaIO4溶液,2~8℃作用30min后,加入0.16mol/L的乙二醇0.5mL,室温避光作用30min;加入5mg纯化的4A10单抗,装透析袋(MW:8000~14000),用碳酸盐缓冲液2~8℃透析14h;次日,吸出透析袋内的样品于EP管中,加入0.2mL 5mg/mL的NaBH4溶液置于2~8℃作用2h后,加入等体积的饱和硫酸铵溶液,置于2~8℃沉淀30分钟,10000r/min离心10min,弃上清,沉淀用适量磷酸盐缓冲液重悬后,在该磷酸盐缓冲液中2~8℃条件下透析14h;次日,10000r/min离心5min,取上清用磷酸盐缓冲液定容至5mL,A280测定浓度为12.2mg/ml,分装,100μL/管,即为HRP标记的4A10单抗,HRP-4A10,-70℃保存。
实施例4:
鸭坦布苏病毒E蛋白截短蛋白在制备鸭坦布苏病毒血清检测试剂盒中的应用,本发明利用鸭坦布苏病毒E蛋白截短蛋白和HRP标记的鼠抗鸭IgG建立的间接ELISA方法,包被蛋白最佳工作浓度为2μg/m L,酶标单抗的最佳稀释倍数为1:16000。
具体使用步骤:
(1)包被:2μg/mL实施例1制备的E蛋白截短蛋白,为100μL/孔,4℃过夜。
(2)封闭:弃孔中液体,拍干,加入封闭液,120μL/孔,4℃过夜。
(3)加样:抗原包被板用洗涤液洗3遍,在纱布上拍干。将稀释好的血清样品按照相同的布局转移100μL到抗原包被板上,37℃30min。
(4)洗涤:取出酶标板,将其甩干,用洗涤液洗3次,在吸水纸上拍干,加入1:16000倍稀释的HRP-4A10,100μL/孔,37℃孵育30min。
(5)洗涤:取出酶标板,将其甩干,用洗涤液洗5次,在吸水纸上拍干。
(6)显色:加入显色液A、B各50μL/孔,室温显色10min,用终止液1终止反应,50μL/孔。用酶标仪测定OD630。
(7)结果判定:通过对40份已知背景的鸭坦布苏病毒阴性血清样品进行检测,得到结果,求其平均值0.13;标准偏差SD=0.01;根据公式阴阳性临界值=阴性样本OD630nm平均值+3SD(标准偏差),得到阴阳性临界值0.16。即样品的OD630nm≤0.16则判为阴性,OD630nm>0.16则判为阳性。ELISA试验成立的条件是阳性对照值大于1.0,阴性对照值小于0.16。
上述间接ELISA方法的特异性试验和敏感性试验
(1)特异性试验
将禽流感,鸭呼肠孤病毒,鸭病毒性肝炎,鸭疫里氏杆菌的阳性鸭血清按照1:500稀释,同时将鸭DTMUV阴阳性血清作为对照,按照上述ELISA方法进行检测。
表1五种不同病毒阳性血清的测定
AIV表示禽流感病毒、DRV表示鸭呼肠孤病毒、PA表示鸭巴氏杆菌、DHV表示鸭病毒性肝炎病毒、DTMUV表示鸭坦布苏病毒。
由表1可知,本发明建立的间接ELISA方法仅能特异性的识别DTMUV鸭阳性血清,而与其他4种禽类病毒的阳性血清没有交叉反应。
(2)敏感性试验
将鸭DTMUV阳性血清从1:100依次倍比稀释到1:12800,每个梯度设置两个重复,按照建立的ELISA方法进行检测
表2阳性血清和阴性血清稀释不同倍数的OD630测定
随着稀释倍数增加,阳性血清和阴性血清OD630值均持续下降,参照临界值的判定标准,阳性血清在稀释到1:12800时,仍能检测出阳性。
(3)符合率试验
用本发明的鸭坦布苏病毒E蛋白ELISA抗体检测试剂盒和传统的琼脂扩散实验共同检测临床样本200份,结果见表3,二者符合率为87.5%。
表3符合率试验
序列表
<110> 华中农业大学
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Claims (2)
1.鸭坦布苏病毒E蛋白截短蛋白在制备鸭坦布苏病毒血清检测试剂盒中的应用,所述的鸭坦布苏病毒E蛋白截短蛋白的核苷酸序列为SEQ ID NO.1所示,氨基酸序列为SEQ IDNO.2所示。
2.根据权利要求1所述的应用,所述的试剂盒的包被抗原为SEQ ID NO.2所示的氨基酸序列,二抗为保藏编号为CCTCC NO:C2017169的杂交瘤细胞分泌得到的单抗。
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CN108484737A (zh) * | 2018-02-09 | 2018-09-04 | 四川农业大学 | 坦布苏病毒非结构蛋白ns4b截短蛋白的表达方法及其产品和应用 |
CN108484736A (zh) * | 2018-02-09 | 2018-09-04 | 四川农业大学 | 坦布苏病毒非结构蛋白ns2a截短蛋白的表达方法及其产品和应用 |
CN110386965A (zh) * | 2018-04-18 | 2019-10-29 | 江苏省农业科学院 | 一种坦布苏病毒e蛋白b细胞表位及其编码基因和应用 |
CN110237244A (zh) * | 2019-06-19 | 2019-09-17 | 苏州世诺生物技术有限公司 | 鸭坦布苏病毒基因工程亚单位疫苗及其制备方法和应用 |
CN114380921A (zh) * | 2022-01-19 | 2022-04-22 | 中国农业科学院北京畜牧兽医研究所 | 基于人铁蛋白的鸭坦布苏病毒e蛋白的纳米疫苗、抗原及其应用 |
CN114380921B (zh) * | 2022-01-19 | 2023-05-30 | 中国农业科学院北京畜牧兽医研究所 | 基于人铁蛋白的鸭坦布苏病毒e蛋白的纳米疫苗、抗原及其应用 |
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