CN112485424A - 一种检测新型鸭呼肠孤病毒感染抗体的间接elisa试剂盒 - Google Patents
一种检测新型鸭呼肠孤病毒感染抗体的间接elisa试剂盒 Download PDFInfo
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- CN112485424A CN112485424A CN202011278310.1A CN202011278310A CN112485424A CN 112485424 A CN112485424 A CN 112485424A CN 202011278310 A CN202011278310 A CN 202011278310A CN 112485424 A CN112485424 A CN 112485424A
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Abstract
本发明提供一种检测新型鸭呼肠孤病毒感染抗体的间接ELISA试剂盒,属于生物技术领域的免疫学检测方法。所述新型鸭呼肠孤病毒非结构蛋白p18的序列如SEQ ID No:2所示。本发明检测新型鸭呼肠孤病毒感染抗体的间接ELISA试剂盒,能够区分抗体是源于病毒感染还是灭活疫苗免疫,从而可以移除新型鸭呼肠孤病毒感染禽群,进而有效控制新型呼肠孤病毒的传播。
Description
技术领域
本发明属于生物技术领域的免疫学检测方法,具体涉及一种检测新型鸭呼肠孤病毒感染抗体的间接ELISA试剂盒。
背景技术
鸭呼肠孤病毒病是由鸭呼肠孤病毒(Duck reovirus,DRV)引起的番鸭、半番鸭、肉鸭(含北京鸭、樱桃谷肉鸭)、麻鸭和鹅等水禽的一种急性传染病,可以分为番鸭呼肠孤病毒(Muscovy duck reovirus,MDRV)和新型鸭呼肠孤病毒(Novel duck reovirus,NDRV)两种基因型。NDRV可以感染番鸭、半番鸭、肉鸭和鹅等多种水禽,引起番鸭“出血性坏死性肝炎”(也称为番鸭”新肝病”)和肉鸭“脾坏死症”。NDRV自然感染引起的死亡率较低,发病鸭耐过后成为僵鸭;并发感染时的发病率和死亡率升高,还可以引起免疫抑制和生长发育障碍,现已成为危害水禽养殖的重要传染病之一。目前,对该病没有商品化的疫苗,灭活疫苗正在临床试验阶段。
ELISA方法具有方便、快速、准确等特点,采用该方法开发的试剂盒已被广泛应用于多种动物疫病的快速诊断和检测中。但是,目前已报道检测NDRV血清抗体的ELISA方法并不能区分新型鸭呼肠孤病毒抗体是来自灭活疫苗免疫还是病毒感染,不利于养殖场对该病毒的净化。
发明内容
本发明的目的是提供一种检测新型鸭呼肠孤病毒感染抗体的间接ELISA试剂盒,能够区分抗体是源于病毒感染还是灭活疫苗免疫,从而可以移除新型鸭呼肠孤病毒感染禽群,进而有效控制新型呼肠孤病毒的传播。
本发明的目的采用如下技术方案实现:
一种检测新型鸭呼肠孤病毒感染抗体的间接ELISA试剂盒,所述ELISA试剂盒包括包被有新型鸭呼肠孤病毒非结构蛋白p18的酶标板,所述新型鸭呼肠孤病毒非结构蛋白p18的序列如SEQ ID No:2所示。
在本发明中,所述非结构蛋白p18采用包括如下步骤的方法制备:使所述新型鸭呼肠孤病毒非结构蛋白p18的编码基因在生物中进行表达得到所述蛋白质;所述生物为微生物或植物。
在本发明中,使所述新型鸭呼肠孤病毒非结构蛋白p18的编码基因在生物中进行表达包括将所述新型鸭呼肠孤病毒非结构蛋白p18的编码基因导入受体微生物,得到表达所述重组新型鸭呼肠孤病毒非结构蛋白p18的微生物,培养所述重组微生物,表达得到所述新型鸭呼肠孤病毒非结构蛋白p18。
在本发明中,所述非结构蛋白p18采用包括如下步骤的方法制备:将p18编码基因插入pET-32a(+)中,然后导入大肠杆菌BL21(DE3)中,诱导表达重组菌后得到的所述蛋白质。
在本发明中,所述新型鸭呼肠孤病毒非结构蛋白p18的编码基因的序列如SEQ IDNo:1所示。
在本发明中,诱导表达条件是用1.0mmol/L的IPTG在37℃诱导6h。
在本发明中,所述试剂盒还包括洗涤液、稀释液、HRP标记的羊抗鸭IgG(H+L)抗体、新型鸭呼肠孤病毒感染阳性对照血清和阴性对照血清、显色液和终止液;所述HRP标记的羊抗鸭IgG(H+L)抗体购自美国KPL公司,编号为04-25-06。
在本发明中,所述酶标板采用含有BSA的PBS缓冲液封闭。
本发明的有益效果是:本发明适用于新型鸭呼肠孤病毒感染血清抗体的快速检测,能够区分抗体是源于病毒感染还是灭活疫苗免疫,从而移除新型呼肠孤病毒感染禽群,进而有效控制新型呼肠孤病毒的快速传播。本发明试剂盒具有检测灵敏、准确,操作简便、重复性好以及特异性强的优点,成本较低,适合大规模的样品检测,同时检测较为快速,可在5小时左右完成。
附图说明
图1为重组菌株表达蛋白的SDS-PAGE电泳图谱,M为PageRuler UnstainedProtein LadderMarker,1为纯化的p18重组蛋白。
图2为重组蛋白免疫印迹图,M为Page Ruler Prestained Protein LadderMarker;1为纯化的p18重组蛋白的免疫印迹图。
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细描述,给出的实施例仅为了阐明本发明,而不是为了限制本发明的范围。下述实施例中的实验方法,如无特殊说明,均为常规方法。下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
1、下述实施例中的pET-32a(+)为Novagen公司产品(Cat No.69909.3)。
实施例1、新型鸭呼肠孤病毒p18重组蛋白的制备
(1)p18蛋白基因的克隆
新型鸭呼肠孤病毒的非结构蛋白p18蛋白序列如SEQID NO:2。以新型鸭呼肠孤病毒NDRV SY株(GenBankMK955818to MK955827.)的基因组DNA为模板,以P18-F、P18-R为引物,利用高保真酶PCR扩增非结构蛋白p18蛋白的基因,并在p18蛋白基因两端分别引入限制性酶切位点Bam HI和Hind III。
P18–F的序列如下:5’-CCA GGA TCC ATG TCA CTC CCG CCA ACC-3’(斜体部分是BamH I酶切位点);P18–R的序列如下:5’-CCA AAG CTT G GCA GTT GTT GAT TGT AGA T-3’(斜体部分是Hind III酶切位点)。
将扩增得到的目的基因(SEQ ID NO:1)克隆到载体pEASY-Blunt(北京全式金生物技术有限公司)中,由南京擎科生物技术有限公司测序,获得成功插入了目的基因的重组质粒,命名为pEASY-Blunt-p18质粒。
(2)重组菌构建
将pET-32a(+)质粒和pEASY-Blunt-p18质粒分别用限制性内切酶Bam HI和HindIII(宝生物工程(大连)有限公司)双酶切,回收酶切后的pET-32a(+)片段和p18基因,用T4DNA连接酶连接回收的片段,得到质粒pET-32a(+)-p18,并转化BL21(DE3)大肠杆菌感受态细胞,采用Bam HI和Hind双酶切筛选出阳性重组菌,命名为pET-32a(+)-p18-BL21(DE3)。
(3)p18重组蛋白的表达、纯化
按体积比为1:100,将重组菌pET-32a(+)-p18-BL21(DE3)菌液接种至500mL、含100μg/mL氨苄青霉素的LB培养基,在37℃培养至OD600=0.8,加入终浓度为1.0mmol/L的IPTG,37℃诱导表达6h。将发酵液8000rpm离心15min,取菌体沉淀用0.01M的PBS溶液洗涤2次,再次离心,取菌体沉淀用超声波仪裂解,得到菌体裂解液。将菌体裂解液在4℃、8 000rpm离心15min,收集沉淀,将该沉淀采用Ni-NTA琼脂糖柱(GE Healthcare),按照说明书中方法纯化p18重组蛋白。取纯化后的p18重组蛋白,10μg/孔,采用12%聚丙烯酰胺凝胶电泳分离,用蛋白转印仪转印硝酸纤维素膜,5%的脱脂奶粉溶液37℃封闭3h,0.1%TBST洗涤3次,加入1:2000稀释的新型鸭呼肠孤病毒感染阳性对照血清(实施例2中方法制备),在37℃孵育1.5h,0.1%TBST洗涤3次,加入1:500稀释的HRP标记的羊抗鸭IgG(H+L)抗体(北京索莱宝科技有限公司抗体),0.1%TBST洗涤5次,DAB显色试剂(博士德生物)显色。从图1可以看到纯化的p18重组蛋白大小为38KDa,与预期目的蛋白大小相等。从图2可以看到P18重组蛋白与新型鸭呼肠孤病毒感染阳性对照血清发生特异性反应,表明P18重组蛋白具有反应原性。
实施例2、阴阳性对照血清制备
1、新型鸭呼肠孤病毒感染阳性对照血清:取3~5日龄后的健康非免鸭60只,采用新型鸭呼肠孤病毒NDRV-SY株(GenBank登录号:MK955818-MK955827)的鸭胚组织毒按照每只鸭1000ELD50接种,每周采血分离血清,共采集3次,将每只鸭血清合并混匀后,经间接免疫荧光试验检测为NDRV抗体阳性的,作为新型鸭呼肠孤病毒感染阳性对照血清。新型鸭呼肠孤病毒野毒感染后的阳性血清和新型鸭呼肠孤病毒感染阳性对照血清均含有NDRV抗体(P18抗体)。
间接免疫荧光试验检测的具体方法如下:将100PFU病毒量的NDRV-SY株接种于24孔细胞培养板的每孔中,培养24h后,吸去生长液,PBS洗3次,用4℃、体积比为3:1的丙酮甲醇混合溶液,在-20℃固定20min,PBS洗3次,每个孔中加入0.5mL经PBS按照体积比为1∶500稀释的免疫鸭血清,并设置健康非免鸭(食欲、饮水、精神状态、呼吸、生长、被毛、粪便情况良好,未免疫新型鸭呼肠孤病毒病疫苗的鸭)血清对照孔作为阴性对照,放置于37℃恒温培养箱中反应1.5h,用PBS清洗3次,然后每孔加入0.5mL经PBS按照体积比为1:200稀释的FITC标记小鼠抗鸭IgG荧光抗体(北京百奥莱博科技有限公司),放置于37℃恒温生化培养箱中反应1h,PBS洗3次,拍干,在倒置荧光显微镜下观察,健康非免鸭血清对照孔未出现绿色荧光病灶,说明是NDRV阴性,免疫鸭血清孔出现亮绿色荧光病灶,说明免疫鸭血清中含有NDRV抗体,是NDRV抗体阳性。
2、阴性对照血清:将健康非免鸭(食欲、饮水、精神状态、呼吸、生长、被毛、粪便情况良好,未免疫新型鸭呼肠孤病毒病疫苗的鸭)采血分离血清,用上述间接免疫荧光试验检测为NDRV抗体阴性后,分装后-20℃保存,作为阴性对照血清。
3、新型鸭呼肠孤病毒灭活疫苗免疫血清样本:将新型鸭呼肠孤病毒NDRV-SY株采用细胞培养后所得病毒液灭活。将灭活病毒液与白油按照体积比为1:3混合,乳化,得到灭活疫苗。将灭活疫苗免疫3~5日龄健康非免鸭60只,接种剂量为每只7.88×106TCID50,免疫后14天、21天,无菌采集鸭血分离血清,将每只鸭血清合并混匀后,经上述间接免疫荧光试验检测为阳性后,作为新型鸭呼肠孤病毒灭活疫苗免疫血清样本。
4、3~5日龄健康非免鸭(食欲、饮水、精神状态、呼吸、生长、被毛、粪便情况良好,未免疫新型呼肠孤病毒病疫苗的鸭):种蛋购于江苏省农业科学院黄梅养殖基地,实验室自行孵化。
实施例3、新型鸭呼肠孤病毒感染抗体间接ELISA试剂盒及检测方法
1.新型鸭呼肠孤病毒感染抗体间接ELISA试剂盒的组成
新型鸭呼肠孤病毒感染抗体间接ELISA试剂盒包括:洗涤液、稀释液、以p18重组蛋白包被的酶标板、酶标二抗、新型鸭呼肠孤病毒感染阳性对照血清和阴性对照血清、显色液和终止液。
(1)洗涤液
洗涤液(PBST缓冲液)是含有0.05%(体积百分浓度)Tween-20的PBS缓冲液。按照如下方法配制:在浓度为0.01M、pH值为7.4的PBS缓冲液中加入终浓度(体积百分浓度)为0.05%的Tween-20和终浓度为5g/L的叠氮化钠。
浓度为0.01M、pH值为7.4的PBS缓冲液配制方法如下:取8.5g NaCl、0.2g KCl、2.9g Na2HPO4·12H2O和0.59g NaH2PO4·2H2O溶于去离子水,然后定容至1L。
(2)稀释液
稀释液是含0.01%(体积百分浓度)Proclin300和10g/L牛血清白蛋白(BSA)的PBST缓冲液。配制方法:在PBST缓冲液中添加终浓度为10g/L的牛血清白蛋白和终浓度(体积百分浓度)为0.01%的Proclin300,0.22μm滤器过滤除菌,4℃保存,其中PBST缓冲液就是标题(1)中的洗涤液。
Proclin300是防腐剂。
(3)以p18重组蛋白包被的酶标板
用包被缓冲液将纯化后的p18重组蛋白(实施例1制备)稀释至1.22μg/mL,在96孔板的每孔中加入100μL,4℃包被过夜。用洗涤液洗涤3次,拍干。每孔加入100μL封闭液,在37℃封闭2h,再用洗涤液洗涤3遍,拍干,得到以p18重组蛋白(实施例1制备)包被的酶标板。
其中包被缓冲液是pH9.6、0.05mol/L的碳酸钠-碳酸氢钠缓冲液,配制方法如下:取1.59g的Na2CO3和2.93g的NaHCO3,溶于去离子水,然后采用去离子水定容至1L。洗涤液同本实施例标题(1)。封闭液是含有10g/L BSA(牛血清白蛋白)的PBS缓冲液,按照如下方法配制:在浓度为0.01M、pH值为7.4的PBS缓冲液中添加终浓度(质量百分浓度)为1%的BSA所得。
(4)酶标二抗
酶标二抗为HRP标记的羊抗鸭IgG(H+L)抗体,购自美国KPL公司,产品编号为04-25-06。
(5)新型鸭呼肠孤病毒感染阳性对照血清和阴性对照血清
采用实施例2中方法制备得到新型鸭呼肠孤病毒感染阳性对照血清和阴性对照血清。
(6)显色液
显色液为单组分TMB显色液I,购自湖州英创生物科技有限公司,产品编号为TMB-S-001。
(7)终止液
终止液为2mol/L的硫酸水溶液。
上述新型鸭呼肠孤病毒感染抗体间接ELISA试剂盒记为本发明试剂盒。
2.本发明试剂盒的使用方法
采用本发明试剂盒检测新型鸭呼肠孤病毒感染抗体的具体步骤如下:
(1)加样:用稀释液将待检血清样品、新型鸭呼肠孤病毒感染阳性对照血清和阴性对照血清分别稀释800倍,然后分别加入以p18重组蛋白包被的酶标板中,每孔100μL,37℃反应1h,倾去孔内液体,然后用洗涤液洗涤5次。
(2)加酶标二抗:加入用稀释液按照稀释度为1:500稀释的酶标二抗,100μL/孔,37℃作用45min。
(3)显色:加入显色液,100μL/孔,反应7min。
(4)终止:加入终止液终止反应,50μL/孔。
(5)测定:用酶标仪读取各孔在450nm波长下的吸光值OD450。
本发明试剂盒的判定标准确定:采用本发明试剂盒对按照实施例2中方法制备的100份阴性对照血清进行检测,计算OD450的平均值和标准偏差(SD)。结果表明,该100份新型鸭呼肠孤病毒抗体阴性的鸭血清OD450平均值为0.35,SD为0.05,因此阳性临界值为0.5,阴性临界值为0.45。因此,本发明试剂盒的判断标准如下:当待检血清的OD450>0.5时,检测结果判为阳性,即该待检血清样品含有新型鸭呼肠孤病毒感染抗体;当OD450<0.45时,检测结果判为阴性,即该待检血清样品不含有新型鸭呼肠孤病毒感染抗体,该待检血清来源于没有感染新型鸭呼肠孤病毒的鸭只或来源于免疫了灭活疫苗的鸭只;当0.45≤OD≤0.5判为可疑,可以复检。
利用本发明试剂盒对实施例2中制备的50份阴性对照血清及20份新型鸭呼肠孤病毒感染阳性对照血清进行检测,检测结果显示:本发明试剂盒检测阴性对照血清及新型鸭呼肠孤病毒感染阳性对照血清与间接免疫荧光试验的符合率分别为96%和100%。
实施例4、本发明试剂盒与PCR检测方法符合性试验
采用本发明试剂盒,对临床采集的50份疑似新型鸭呼肠孤病毒感染鸭的血清样品进行检测,并与实时荧光定量RT-PCR方法(袁远华,吴志新,王俊峰等,新型鸭呼肠孤病毒SYBR Green I实时荧光定量PCR检测方法的建立,中国预防兽医学报[J],2013,35(9):738-741)进行比较。结果显示,本发明试剂盒检测50份待检样品时有32份为阳性,实时荧光定量RT-PCR检测时有32份为阳性,而且本发明试剂盒检测出的阳性样品用实时荧光定量RT-PCR检测时均为阳性,符合率为100%。
实施例5、新型鸭呼肠孤病毒灭活疫苗免疫血清样本、新型鸭呼肠孤病毒感染阳性对照血清的检测
利用本发明试剂盒对采用实施例2中方法制备的40份新型鸭呼肠孤病毒灭活疫苗免疫血清样本和40份新型鸭呼肠孤病毒感染阳性对照血清样本进行检测。结果如表1所示:新型鸭呼肠孤病毒灭活疫苗免疫血清样本检测的OD450值均<0.45,新型鸭呼肠孤病毒感染阳性对照血清检测的OD450值>0.5,说明本发明试剂盒能够检测新型呼肠孤病毒感染抗体。
表1各血清样本采用本发明试剂盒的检测结果
SEQUENCE LISTING
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Claims (8)
1.一种检测新型鸭呼肠孤病毒感染抗体的间接ELISA试剂盒,其特征在于所述ELISA试剂盒包括包被有新型鸭呼肠孤病毒非结构蛋白p18的酶标板,所述新型鸭呼肠孤病毒非结构蛋白p18的序列如SEQ ID No:2所示。
2.如权利要求1所述检测新型鸭呼肠孤病毒感染抗体的间接ELISA试剂盒,其特征在于,所述非结构蛋白p18采用包括如下步骤的方法制备:使所述新型鸭呼肠孤病毒非结构蛋白p18的编码基因在生物中进行表达得到所述蛋白质;所述生物为微生物或植物。
3.根据权利要求2所述检测新型鸭呼肠孤病毒感染抗体的间接ELISA试剂盒,其特征在于:使所述新型鸭呼肠孤病毒非结构蛋白p18的编码基因在生物中进行表达包括将所述新型鸭呼肠孤病毒非结构蛋白p18的编码基因导入受体微生物,得到表达所述重组新型鸭呼肠孤病毒非结构蛋白p18的微生物,培养所述重组微生物,表达得到所述新型鸭呼肠孤病毒非结构蛋白p18。
4.根据权利要求3所述试剂盒,其特征在于:所述非结构蛋白p18采用包括如下步骤的方法制备:将p18编码基因插入pET-32a(+)中,然后导入大肠杆菌BL21(DE3)中,诱导表达重组菌后得到的所述蛋白质。
5.根据权利要求1-4中任一所述的试剂盒,其特征在于:所述新型鸭呼肠孤病毒非结构蛋白p18的编码基因的序列如SEQ ID No:1所示。
6.根据权利要求5所述试剂盒,其特征在于:诱导表达条件是用1.0 mmol/L的IPTG在37℃诱导6 h。
7.根据权利要求1-6之一所述试剂盒,其特征在于所述试剂盒还包括洗涤液、稀释液、HRP标记的羊抗鸭IgG(H+L)抗体、新型鸭呼肠孤病毒感染阳性对照血清和阴性对照血清、显色液和终止液;所述HRP标记的羊抗鸭IgG(H+L)抗体购自美国KPL公司,编号为04-25-06。
8.根据权利要求7所述试剂盒,其特征在于所述酶标板采用含有BSA的PBS缓冲液封闭。
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