CN113265006B - 一种用于捕获prrsv核衣壳蛋白抗体的融合蛋白3an及其应用 - Google Patents
一种用于捕获prrsv核衣壳蛋白抗体的融合蛋白3an及其应用 Download PDFInfo
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- CN113265006B CN113265006B CN202110598098.5A CN202110598098A CN113265006B CN 113265006 B CN113265006 B CN 113265006B CN 202110598098 A CN202110598098 A CN 202110598098A CN 113265006 B CN113265006 B CN 113265006B
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Abstract
本发明提供了一种用于捕获PRRSV核衣壳蛋白抗体的融合蛋白3AN及其应用，属于病毒检测技术领域。本发明提供了一种用于捕获猪繁殖与呼吸综合征病毒核衣壳蛋白抗体的融合蛋白3AN，氨基酸序列如SEQ ID NO：3所示。试验表明以FMDV的3A表位单抗3A24作为包被抗体，捕获3AN融合蛋白后形成包被酶标板，以C8单抗作为检测抗体，采用竞争ELISA反应模式检测猪血清PRRSV抗体，制备的PRRSV抗体检测试剂盒，具有很好的敏感性和特异性，为PRRSV血清流行病学调查提供了一种新工具。
Description
技术领域
本发明属于病毒检测技术领域，具体为一种用于捕获PRRSV核衣壳蛋白抗体的融合蛋白3AN及其应用。
背景技术
猪繁殖与呼吸综合征(Porcine reproductive and respiratory syndrome,PRRS)是由猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndromevirus,PRRSV)引起的一种高度接触性传染病，临床症状主要是以母猪的繁殖障碍，仔猪呼吸道症状以及哺乳仔猪的高死亡率为特征。该病对全球养猪业造成了极大的经济损失，是目前影响养猪业健康发展的重要的疫病之一。PRRSV是一种有囊膜的单股正链RNA病毒，极易发生变异，流行毒株众多，而商品化弱毒疫苗的使用，也会导致流行毒与疫苗毒重组现象的发生，造成PRRS防控难度加大。
目前，我国PRRS的防控主要依靠弱毒疫苗免疫和严格的生物安全措施。在感染早期对该病快速准确的检测，有利于最大限度的控制PRRS的流行。ELISA检测技术是检测PRRSV抗体的常用方法，检测的靶标多为N蛋白抗体，通过检测抗体可以对群体感染或免疫状况作出判断。N蛋白是PRRSV的核衣壳蛋白，具有高度的保守性和极强的免疫原性，感染后7天即可在血清中检测到N蛋白抗体，因此，N蛋白抗体是PRRS感染状况监测评估的主要指标。竞争ELISA法是检测PRRSV的N蛋白抗体的有效方法，然而目前缺乏能够有效捕获N蛋白抗体的抗原，这对采用竞争ELISA法检测PRRSV的N蛋白抗体形成阻碍。
发明内容
有鉴于此，本发明的目的在于提供一种用于捕获PRRSV核衣壳蛋白抗体的融合蛋白3AN，同时对口蹄疫病毒3A蛋白的单克隆抗体3A24和猪繁殖与呼吸综合征病毒的N蛋白抗体具有较高的结合特异性。
本发明的目的还在于提供一种竞争ELISA检测猪繁殖与呼吸综合征病毒N蛋白抗体的抗原抗体组合物及其应用，用于PRRS感染或免疫状况的监测。
本发明提供了一种用于捕获猪繁殖与呼吸综合征病毒核衣壳蛋白抗体的融合蛋白3AN，所述融合蛋白3AN的氨基酸序列如SEQ ID NO：3所示。
本发明提供了一组基于竞争法检测猪繁殖与呼吸综合征病毒核衣壳蛋白抗体的抗原抗体组合物，包括所述融合蛋白3AN和PRRSV N蛋白抗体C8。
优选的，所述PRRSVN蛋白抗体C8包括氨基酸序列如SEQ ID NO：1所示的重链可变区基因HV1和氨基酸序列如SEQ ID NO：12所示的轻链可变区基因LV1。
优选的，所述抗原抗体组合物还包括口蹄疫病毒3A蛋白的单克隆抗体3A24。
本发明提供了所述融合蛋白3AN或所述抗原抗体组合物在制备免疫检测猪繁殖与呼吸综合征病毒感染或免疫动物后N蛋白抗体的试剂或试剂盒中的应用。
优选的，所述免疫检测包括胶体金免疫检测、免疫沉淀检测、荧光免疫检测和/或酶联免疫检测。
优选的，所述试剂盒包括抗原反应板；所述抗原反应板是预先包被单克隆抗体3A24再结合3A单抗包被所述融合蛋白3AN制备得到。
优选的，所述试剂盒包括100×浓缩生物素标记抗体；
所述100×浓缩生物素标记抗体为生物素标记的所述猪源单个B细胞抗体C8。
优选的，所述猪源单个B细胞抗体C8的标记浓度为0.06～0.25μg/mL；
所述100×浓缩生物素标记抗体的工作浓度为1：10000～30000。
优选的，所述试剂盒还包括以下组分：100×浓缩酶标亲和素、25倍浓缩洗涤液、血清稀释液、底物溶液、终止液、阳性对照血清和阴性对照血清。
本发明提供的用于捕获猪繁殖与呼吸综合征病毒核衣壳蛋白抗体的融合蛋白3AN，氨基酸序列如SEQ ID NO：3所示。所述融合蛋白3AN不仅对PRRSV的N蛋白抗体具有较高的特异性结合能力，而且对口蹄疫病毒的3A蛋白单克隆抗体3A24也具有较强的特异性结合能力，能够用于竞争法检测PRRSV的N蛋白抗体的产品中。
本发明提供的抗原抗体组合物，包括所述融合蛋白3AN和所述的猪繁殖与呼吸综合征病毒核衣壳蛋白抗体C8。所述抗体C8是利用单个B细胞抗体技术制备的一株猪源PRRSVN蛋白单抗，能够识别PRRSVN蛋白C端的抗原表位，因此，能够实现PRRSV的特异性识别和检测。3AN和C8具有较强的特异性结合能力，可用于基于竞争法检测PRRSVN蛋白抗体的产品或方法中。
进一步的，本发明提供的抗原抗体组合物还包括单抗3A24。用本实验室研制的3A单抗3A24捕获3AN蛋白制备抗原反应板，生物素标记的猪源单个B细胞C8单抗作为检测抗体，采用竞争ELISA检测模式，能够区分PRRSV抗体阴性血清与阳性血清，因此，该抗体组合物可用于猪繁殖与呼吸综合征病毒的抗体检测，且具有较好的敏感性与特异性。
本发明提供了所述抗原抗体组合物在制备免疫检测猪繁殖与呼吸综合征病毒感染或免疫动物后N蛋白抗体的试剂或试剂盒中的应用。采用制备的试剂盒对204份来自感染后21天猪检测敏感性为98.5％，对65份健康猪血清的检测特异性为98.5％。
附图说明
图1为Marc-145接种VR-2332毒株后间接免疫荧光实验检测9株抗体与PRRSV反应性的结果；
图2为293T细胞中转染GSWW/2015(A)和VR2332(B)毒株N蛋白表达质粒，Western-blot验证9株抗体识别N蛋白的结果；
图3为Western-blot验证C8单抗与GST融合N蛋白截短体的反应结果；
图4为SDS-PAGE及考马斯亮蓝染色检测3AN蛋白的结果；
图5为3A24抗体不同浓度下阳性标准血清阻断率；
图6为不同C8抗体浓度及3AN融合蛋白抗原浓度下阳性标准血清阻断率；
图7为不同稀释度标准阳性血清阻断率；
图8为37℃反应不同时间三份阳性血清平均阻断率；
图9为65份猪的阴性血清和204份阳性血清检测阻断率；
图10为根据65份猪的阴性血清和204份阳性血清检测阻断率绘制的ROC曲线；
图11为以3A24为包被抗体、C8为检测抗体制备的试剂盒稳定性检测结果；
图12为以C8为包被抗体，D11为检测抗体制备的试剂盒稳定性检测结果。
具体实施方式
本发明提供了一种用于捕获猪繁殖与呼吸综合征病毒核衣壳蛋白抗体的融合蛋白3AN，所述融合蛋白3AN的氨基酸序列如SEQ ID NO：3所示。
本发明将猪繁殖与呼吸综合征病毒N蛋白序列与口蹄疫病毒3A24单抗识别表位序列融合表达得到融合蛋白3AN，中间加入“GPGPGP”联接肽序列，N端与C端均加有6×组氨酸标签，方便进行融合蛋白的纯化。融合蛋白3AN的氨基酸序列为“GHHHHHHVDDAVNEYIEKANITTDDKTLDEAEKNPLETSGAATVGKTLPGHKAGPGPGPMPNNNGKQQKKKKGNGQPVNQLCQMLGKIIAQQNQSRGKGPGKKNRKKNPEKPHFPLATEDDVRHHFTPSERQLCLSSIQTAFNQGAGTCALSDSGRISYTVEFSLPTQHTVRLIRATASPSAHHHHHH”(SEQ ID NO:3)。本发明对所述融合蛋白3AN的来源没有特殊限制，采用本领域所熟知的融合蛋白的制备方法即可。
本发明提供了一组基于竞争法检测猪繁殖与呼吸综合征病毒核衣壳蛋白抗体的抗原抗体组合物，包括所述融合蛋白3AN和PRRSV N蛋白抗体C8。
在本发明中，所述PRRSVN蛋白抗体C8的氨基酸序列包括重链可变区氨基酸序列HV1(SEQ ID NO：1:GLVQPGGSLRLSCVASGFTFSSYIVTWVRQSPGKGLEWLAGTGVGEYALYYRNSVRGRFTLSRDNSQNTAYLQMNSLRVEETGRYFCRRGAAESVDLWGPGVEVVVSS)和轻链可变区氨基酸序列LV1(SEQ ID NO：2：QEPAMSVSLGGTVTLTCAFSSGSVTRSHWPSWFQLTPGQPPRTLIVSTDSRPTGVPSRFSGAISGYKAALTITGAQPEDEADYVCGVYFTFTKRPFGGGTHLTVLG)。
在本发明中，所述单克隆抗体C8的制备方法，优选包括以下步骤：将所述单克隆抗体C8的重链可变区和轻链可变区的编码序列分别与猪IgG重链和轻链的恒定区序列连接后分别插入pcDNA3.4真核表达载体中，重链重组表达质粒和轻链重组表达质粒按摩尔数比2:3混合转染CHO悬浮培养细胞，表达获得完整抗体，抗体经亲合层析纯化得到。本发明对重组表达质粒转染方法、表达方法及亲和层析纯化方法不做具体限定，采用本领域所熟知的技术方案即可。
在本发明中，所述抗原抗体组合物优选还包括口蹄疫病毒3A蛋白的单克隆抗体3A24。3A24单抗识别的表位序列为VDDAVNEYIEKANITTDDKTLDEAEKNPLETSGAATVGKTLPGHKA(SEQ ID NO:4)。3A24的制备方法参见现有技术(Fu Y,Li P,Cao Y,et al.Development ofa blocking ELISAusing a monoclonal antibody to a dominant epitope in non-structural protein 3A of foot-and-mouth disease virus,as a matching test fora negative-marker vaccine[J].Plos One,2017,12(1):e0170560.)。在所述抗原抗体组合物中，所述单克隆抗体3A24和抗体C8的配比没有特殊限制，根据制备试剂的实际使用浓度确定两种抗体的配合使用浓度。
本发明提供了所述融合蛋白3AN或所述抗原抗体组合物在制备免疫检测猪繁殖与呼吸综合征病毒感染或免疫动物后N蛋白抗体的试剂或试剂盒中的应用。
在本发明中，所述免疫检测优选包括胶体金免疫检测、免疫沉淀检测、荧光免疫检测和/或酶联免疫检测。本发明以制备酶联免疫试剂盒为例加以说明试剂盒的具体成分及其制备方法和使用方法。
在本发明中，所述试剂盒优选包括抗原反应板；所述抗原反应板优选是预先包被单克隆抗体3A24再结合3A单抗包被所述融合蛋白3AN制备得到。所述抗原反应板优选采用抗原间接包被方法制备得到。采用抗原间接包被法包被反应板的方法优选包括先在反应板上包被所述单克隆抗体组合物中的单克隆抗体3A24，再利用所述单克隆抗体3A24捕获融合蛋白3AN抗原，加入固相抗原稳定液(含质量浓度5％～10％蔗糖+1％BSA的PBS液)后，孵育，吸弃上清，吹干酶标板后，密封。所述单克隆抗体3A24的包被浓度优选为0.12～3.9μg/mL，最优选为0.49μg/mL；所述3AN融合蛋白抗原的工作浓度优选为0.1μg/mL。
在本发明中，所述试剂盒优选包括100×浓缩生物素标记抗体。所述100×浓缩生物素标记抗体优选为生物素标记的所述PRRSVN蛋白抗体C8。所述100×浓缩生物素标记抗体的工作浓度优选为1：10000～30000，更优选为1:2000。所述PRRSV N蛋白抗体C8的标记浓度优选为0.06～0.25μg/mL，更优选为0.13μg/mL。本发明对所述生物素标记单抗的制备方法没有特殊限制，采用本领域所熟知的标记方法即可。
在本发明中，所述试剂盒包括100×浓缩酶标亲和素。所述100×浓缩酶标亲和素优选为辣根过氧化物酶(HRP)标记的亲和素。HRP标记的亲和素的来源没有特殊限制采用本领域所熟知的HRP标记的亲和素即可。
在本发明中，所述试剂盒优选还包括以下组分：25倍浓缩洗涤液、血清稀释液、底物溶液、终止液、阳性对照血清和阴性对照血清。
在本发明中，所述试剂盒包括25×浓缩洗涤液。所述25×浓缩洗涤液的制备方法优选如下：每500mL超纯水中加NaCl 200g、KCl 5g、Na2HPO4·12H2O 72.5g、KH2PO45g和12.5mL吐温20，补超纯水定容至1000mL，pH值调整为7.4，经高压灭菌，室温存放备用。
在本发明中，所述试剂盒包括血清稀释液。所述血清稀释液的制备方法如下：配制含2.0％山羊血清、1.0％牛血清白蛋白(纯度大于98％)和0.02％硫柳汞防腐剂的磷酸盐缓冲液，过滤除菌后，2～8℃保存备用。
在本发明中，所述试剂盒包括底物溶液。所述底物溶液的种类根据酶标记亲和素中酶的种类确定。当HRP标记亲和素时，所述底物溶液为3,3’,5,5’-四甲基联苯胺(TMB)底物。
在本发明中，所述试剂盒包括终止液，所述终止液优选为0.3mol/L H2SO4溶液。
在本发明中，所述试剂盒包括阳性对照血清和阴性对照血清。所述阳性对照血清为采自PRRSV攻毒后120天康复猪，经检测，N蛋白抗体为阳性，经无菌处理后加保存剂制备。所述阴性对照血清为采自非免疫健康猪，经检测PRRSVN蛋白抗体为阴性，经无菌处理后加保存剂保存。
在本发明中，所述试剂盒的使用方法优选包括以下步骤：
A1.于抗原包被板中，每孔加入30μL血清稀释液，然后依次加入待测血清样本、阴性对照、阳性对照血清样本与PBS对照，每孔20μL，待测样本加1孔，阴性、阳性对照与PBS空白对照各加两孔；然后，用血清稀释液1∶100稀释100倍工作浓度的生物素标记C8单抗(2.5μg/ml)，每孔50μL，轻振混匀，37℃作用1h；所述血清的最终稀释度优选为1∶5；
A2.用1×PBST洗涤液洗板5次，最后一次拍干；
A3.将HRP标记亲和素按1∶30000稀释至血清稀释液中，加入酶标板，每孔100μL，37℃作用15min；然后同A2洗涤；
A4.每孔加入100μL的底物溶液，37℃作用15min；
A5.加终止液，每孔100μL，轻振混匀，于10min内用酶标仪读450nm吸光值(OD450nm)；
A6.阻断率计算：阻断率(PI)＝(1-样品OD450nm值/空白对照OD 450nm值)×100％；
A7.试验成立的条件：空白对照OD450nm值-阳性对照OD450nm值≥1.0，且阳性对照阻断率应大于50％；阴性对照阻断率应小于25％。
A8.判定标准：是否存在PRRSV抗体，通过计算每个血清样品的PI值判定：PI值大于或等于40％，判为PRRSV抗体阳性；PI值小于40％，判为PRRSV抗体阴性。
在本发明中，采用该试剂盒对204份来自感染后21天猪检测敏感性为98.5％，对健康猪血清的特异性为98.5％。
下面结合实施例对本发明提供的一种用于捕获PRRSV核衣壳蛋白抗体的融合蛋白3AN及其应用进行详细的说明，但是不能把它们理解为对本发明保护范围的限定。
实施例1
PRRSVN蛋白猪源单个B细胞抗体的制备与鉴定
利用商品化的PRRS弱毒疫苗和实验室自制流行毒株疫苗免疫猪。同时纯化全病毒抗原，并用生物素标记病毒粒子；在第八次免疫后采集0922#猪的静脉血，分离外周血中的单核细胞(PBMCs)，以生物素标记的PRRSV为捕获抗原，通过流式细胞分选技术，从PBMCs中分选出抗原特异性抗体分泌单个B细胞。通过单个B细胞抗体基因扩增技术，获得猪IgG抗体重链和轻链可变区(VH和VL)基因序列，然后将可变区基因序列插入到含有猪IgG抗体恒定区的pcDNA3.4真核载体中，构建出完整的抗体表达质粒。将IgG抗体的表达质粒，转染适应悬浮培养的中国仓鼠卵巢细胞(CHO-S)，进行抗体的小量表达，然后用间接免疫荧光实验(IFA)验证小量表达抗体识别PRRSV的特异性。
结果如图1所示，PRRSV VR-2332毒株接种Marc-145细胞后，IFA实验表明9株单抗均能识别PRRSV。
将这9株抗体进行大量表达与纯化。纯化后的抗体通过Western-blot鉴定，发现9株抗体均能特异性与PRRSV的核衣壳蛋白(N蛋白)反应(图2)。将N蛋白进行截短后克隆到pGEX-6P-1载体中，与GST标签融合表达，图3结果表明，C8单抗识别N蛋白的抗原表位位于C端81～110位。
实施例2
融合口蹄疫病毒3A蛋白表位的PRRSV核衣壳蛋白表达纯化
利用基因合成将3AN蛋白的编码序列克隆到pET-28a(+)载体中，转化BL21感受态细胞，挑单克隆后扩大培养至500ml，加入IPTG至终浓度为1mM，诱导表达5h。离心收集菌体，加入50ml冰浴的IB洗涤液(EDTA10mmoXXl/L、Tris-HCl 20mmol/L，pH 7.5、TritonX-1001％)重悬沉淀，超声裂解至菌体完全裂解，离心收集上清。按4:1的比例加入50％的HighAffinity Ni-Charged ResinFF，4℃200r/min孵育过夜，使目的蛋白与Ni-NTA His·Bind发生完全的特异性结合。用pH值8.0的洗涤液(NaH2PO4 50mm/L，NaCl 300mM，Urea 8M，imidazole 10mM)洗涤柱体3次，每次2倍体积，再用0.5倍体积pH值8.0的洗脱液(NaH2PO450mM，NaCl 300mM，Urea 8M，imidazole 250mM)洗脱4次，洗脱液即为纯化后的N蛋白。纯化后的蛋白4℃用透析液(含终浓度0.1M的Tris-HClpH8.0，0.4M的L-Arg，0.2mM的EDTA，0.5mM的L-Glutathione oxidized和2.5mM的Glutathione)透析3次，透析袋中的溶液即为纯化的3AN融合蛋白。图4所示为SDS-PAGE及考马斯亮蓝染色检测3AN融合蛋白。
实施例3
N蛋白竞争ELISA抗体检测试剂盒的制备方法
1.材料
碳酸盐缓冲液(Na2CO3/NaHCO3)及BSA购自SIGMA公司。
BCA蛋白浓度测定试剂盒及生物素标记试剂盒购自Thermo Fisher公司。
辣根过氧化物酶标记亲和素为购买的商品化试剂(Genscript产品)。
PRRSVN蛋白单克隆抗体C8由本实验室采用单个B细胞抗体制备技术研制并保存，其含量为0.25mg/mL。FMDV 3A单克隆抗体3A24由本实验室采用杂交瘤技术研制，其含量为1.95mg/ml。
阴性与阳性对照血清与PRRSVN蛋白与FMDV的3A表位融合蛋白抗原均由本实验室制备保存。
2.试剂的制备及酶标板的包被方法
2.1.抗原间接包被酶标板的制备
将单抗3A24按终浓度0.49μg/mL稀释于碳酸盐缓冲液中，100μL/孔，4℃过夜包被酶标板；用PBST洗涤3次后，加入终浓度0.1μg/mL的3AN融合蛋白抗原，室温孵育2h；用PBST洗涤3次后，加入固相抗原稳定液(含5％～10％蔗糖与1％BSA的PBS缓冲液)，室温℃孵育1h，吸弃上清，吹干酶标板后，加干燥剂密封，2～8℃冷藏保存。每个试剂盒包装两块包被抗原的酶标板。
2.2.生物素标记单克隆抗体C8的制备
按如下程序进行：
A1、首先，使用截留量100KDa超滤管把单抗C8置换至PBS缓冲液中，4000r/min离心10min。
A2、取180μL超纯水加入到1mg生物素中，稀释至10mM；分别按1∶10和1∶20比例加入生物素，即分别添加50μL、25μL至500μL抗体中。
A3、4℃冰上放置4h。
A4、用超滤管置换至抗体存储缓冲液中。
A5、加入等体积100％甘油，-70℃保存。
每个试剂盒包括1管(300μL)100倍工作浓度的生物素标记C8抗体。
2.3.100×浓缩辣根过氧化物酶标记亲和素
辣根过氧化物酶(HRP)标记的亲和素为购买的商品化试剂(BioFX)，将酶标亲和素按100倍工作浓度稀释于抗体保存液(Sigma)中，2～8℃保存备用，每个试剂盒装1管(300μL)。
2.2.4.浓缩洗涤液(25×)
于500mL超纯水中加入NaCl 200g、KCl 5g、Na2HPO4·12H2O 72.5g、KH2PO45 g和12.5mL吐温20，调pH值为7.4，补超纯水定容至1000mL，高压灭菌(10Pa，15min)，室温存放备用；每盒装1瓶(50mL)。
2.2.5.底物溶液
单组份3,3′,5,5′-四甲基联苯胺(TMB)底物溶液为购买的商品化试剂(BioFX)，直接分装使用，每盒1瓶(25mL)，保存期4年。
2.2.6.终止液
浓度为0.3mol/L的H2SO4溶液，利用超纯水稀释分析纯浓硫酸配制，每盒1瓶(25mL)。
2.2.7.阳性对照血清
采自PRRSV攻毒后120天康复猪，经检测，N蛋白抗体为阳性，经无菌过滤后加防腐剂分装保存备用。
2.2.8.阴性对照血清
采自PRRSV非免疫健康猪，经检测PRRSVN蛋白抗体为阴性，经过滤除菌后加防腐剂分装保存备用。
2.2.9.其他物品：包括一次性封板膜4片与使用说明书。
2.2.10.血清抗体检测程序
检测程序：将2～8℃保存的试剂盒平衡至室温，然后按以下程序操作。
A1.于抗原包被板中，每孔加入30μL血清稀释液，然后依次加入待测血清样本、阴性对照、阳性对照血清样本与PBS对照，每孔20μL，待测样本加1孔，阴性、阳性对照与PBS空白对照各加两孔；然后，用血清稀释液1∶100稀释100倍工作浓度的生物素标记单抗(2.5μg/ml)，每孔50μL，轻振混匀，37℃作用1h；所述血清的最终稀释度优选为1∶5；
A2.用1×PBST洗涤液洗板5次，最后一次拍干；
A3.将HRP标记亲和素按1∶30000稀释至血清稀释液中，加入酶标板，每孔100μL，37℃作用15min；然后同A2洗涤；
A4.每孔加入100μL的底物溶液，37℃作用15min；
A5.加终止液，每孔100μL，轻振混匀，于10min内用酶标仪读450nm吸光值(OD450nm)；
A6.阻断率计算：阻断率(PI)＝(1-样品OD450nm值/空白对照OD450nm值)×100％；
A7.试验成立的条件：空白对照OD450nm值-阳性对照OD450nm值≥1.0，且阳性对照阻断率应不低于70％；阴性对照阻断率应小于25％。
A8.判定标准：是否存在PRRSV抗体，通过计算每个血清样品的PI值判定：PI值大于或等于40％，判为PRRSV抗体阳性；PI值小于40％，判为PRRSV抗体阴性。
实施例4
N蛋白竞争ELISA抗体检测方法的建立
1方法
1.1.PRRSV 3AN融合蛋白抗原与抗体最适工作浓度的确定
将单抗3A24分别以3.9μg/mL、1.95μg/mL、0.98μg/mL、0.49μg/mL、0.24μg/mL和0.12μg/mL包被酶标板，然后捕获不同浓度(0.1μg/mL、0.2μg/mL、0.4μg/mL和0.8μg/mL)的3AN融合蛋白抗原，加入不同稀释度(0.25μg/mL、0.13μg/mL和0.06μg/mL和0.03μg/mL)的生物素标记单克隆抗体C8与固定浓度的HRP标记亲和素，进行方阵滴定。按实施例2中的反应程序检测阳性、阴性对照血清样本与PBS空白对照。计算出阴性、阳性对照血清阻断率(PI)，按照阳性对照血清与阴性对照血清差异最大的反应条件，为抗原与抗体的最适工作浓度。
PI＝(1-阳性对照血清OD450nm值/空白对照血清OD450nm值)×100％
1.2.最佳血清稀释度的确定
在确定抗原与抗体的最适工作浓度之后，将标准阴、阳性血清按1：5、1：10和1：20三个稀释度，进行竞争ELISA检测。对获得的阻断率进行比较，取阴、阳性血清的阻断率差异最大的稀释度作为最佳血清稀释度。
1.3.加入血清后反应时间的选择
设定了以下几个反应条件：①37℃反应30min；②37℃反应45min；③37℃反应60min。对各反应条件下的阳性对照血清阻断率进行对比分析，取阻断率达最大时的最短作用时间为最佳反应条件。
1.4.判定标准确定
根据背景清楚的临床健康的非免疫猪血清与感染猪血清样本阻断率的检测结果，来确定本方法的判定标准；共检测65份非免疫健康猪血清，感染后猪血清204份，统计感染动物与健康动物血清样本阻断率分布区间，绘制ROC曲线，确定本竞争ELISA的判定标准。
1.5.本方法的特异性和敏感性检测
根据确定的判定标准，分析非免疫健康猪血清和感染猪血清样本的检测结果确定本方法的特异性和敏感性。
2.结果
2.1.抗原抗体的工作浓度
通过对3AN融合蛋白抗原及3A24单抗梯度稀释后测定阳性血清和阴性血清的阻断率，如图5所示，当3AN融合蛋白抗原浓度为0.1μg/ml，C8单抗浓度为0.13μg/ml时阳性血清和阴性血清的差异最大，因此确定3AN融合蛋白抗原的最适浓度为0.1μg/ml，C8单抗的最适浓度为0.13μg/ml。
将3A蛋白单抗3A24进行梯度稀释后包被酶标板，捕获0.1μg/ml 3AN蛋白间接包被酶标板，分别检测阳性血清和阴性血清的阻断率。如图6所示，当3A24的工作浓度为0.98μg/ml、0.49μg/ml、0.24μg/ml时阳性血清与阴性血清的阻断率差异均较大，为节约成本，确定3A24单抗的最适工作浓度为0.24μg/ml。
2.2.最佳血清稀释度的确定
各稀释度阳性标准血清及阴性血清阻断率如图7所示，血清1∶5稀释获得的阴、阳性血清的阻断率差异最大，能够满足对检测敏感性的要求，因此确定血清最佳稀释度为1∶5。
2.3加入血清后反应时间的确定
加入阳性血清后，对反应不同时间的标准阳性血清阻断率进行对比分析(见图8)，结果显示，37℃反应1h阳性对照血清阻断率最高，因此确定反应时间为37℃反应1h。
2.4结果判定标准的确定
A.阳性及阴性判定标准(临界值)的确定
根据感染猪血清与健康猪血清的检测结果(图9)，绘制ROC曲线(图10)，并根据ROC曲线确定最佳临界值为40％。因此确定判定标准为：阻断率不低于40％判为阳性，如阻断率小于40％判为阴性。非免疫背景下，样品阳性结果，说明该动物存在PRRSV感染的情况，应该采取相应的防控措施。根据以下公式计算测试样本的阻断率(PI)，PI＝(1-测试样本OD450nm值/空白对照平均OD450nm值)×100％。
B.特异性及敏感性判定结果
检测临床健康非免疫猪血清及感染猪血清后，以检测阴性血清数量与总健康血清数的比值计算特异性，以检测阳性血清数与感染动物血清总数的比值计算敏感性。从结果(表1)可以看出本方法对204份来自感染猪血清的样本检测敏感性为98.5％，对健康猪血清的特异性为98.5％。
表1.N蛋白阻断ELISA抗体特异性及敏感性检测结果
实施例5
以3A24为包被抗体，捕获3AN融合蛋白抗原后形成包被酶标板，其中C8单抗的浓度为0.13μg/ml，3AN的终浓度0.1μg/mL，3A24单抗的包被浓度为0.24μg/ml。具体按照实施例3记载的N蛋白竞争ELISA抗体检测试剂盒的制备方法制备试剂盒。
对比例1
以C8单抗为包被抗体，捕获N蛋白抗原后形成包被酶标板，以D11单抗为检测抗体，其浓度为2μg/ml，N蛋白的终浓度1μg/mL，D11单抗的工作浓度为2μg/ml。具体按照实施例3记载的N蛋白竞争ELISA抗体检测试剂盒的制备方法制备试剂盒。
其中单克隆抗体D11包括氨基酸序列如SEQ ID NO：5(GLVQPGGSLRLSCVASGFTLSNCAMTWVRQAPGKGLEWLAVINIRGDRSAYADSVRGRFTISKDDSQNTAYLEMKTLRVEDTARYYCKTADKAFPSESIQMDVWGPGVEVVASS)所示的重链可变区基因HV2和氨基酸序列如SEQ ID NO：6(WVPGARCAIQVTQSPASLAASLGDTVSITCRASLSMSNYLAWYQQKPGKTPRRLIYYASTLESGVPSRFKGRGVGTEFTLTISGLQAEDVATYYCLQYWSAPWTFGQGSKVELKR)所示的轻链可变区基因LV2。所述单克隆抗体D11参考实施例1中单克隆抗体制备方法进行体外重组制备得到。N蛋白抗原的氨基酸序列如SEQ IDNO：7所示(MPNNNGKQQKKKKGNGQPVNQLCQMLGKIIAQQNQSRGKGPGKKNRKKNPEKPHFPLATEDDVRHHFTPSERQLCLSSIQTAFNQGAGTCALSDSGRISYTVEFSLPTQHTVRLIRATASPSAHHHHHH)。
实施例6
将实施例5和对比例1制备的试剂盒分别在37℃储存7天进行老化实验，实验结束后，取出处理的试剂盒和新鲜制备的试剂盒分别测定N蛋白阳性血清、阴性样本，并设置空白对照组，统计各组检测的OD值，并绘制柱状图。
结果见图11和图12。图11为以3A24为包被抗体，捕获3AN融合蛋白抗原，包被酶标板。由图11可知，C8为检测抗体，对比新鲜包被的酶标板和37℃储存7天的酶标板检测阳性血清、阴性血清及空白对照的OD值，无明显差异，说明3A24抗体捕获抗原3AN融合蛋白，C8作为检测抗体的组合较稳定。图12以C8为包被抗体，捕获N蛋白抗原，包被酶标板。D11为检测抗体，对比新鲜包被的酶标板和37℃储存7天的酶标板检测阳性血清、阴性血清及空白对照的OD值差异。发现37℃储存7天后阴性血清及空白对照检测OD值下降明显，说明C8抗体捕获抗原N蛋白，D11作为检测抗体的组合不稳定。
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。
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<110> 中国农业科学院兰州兽医研究所
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Claims (8)
1.一组基于竞争法检测猪繁殖与呼吸综合征病毒核衣壳蛋白抗体的抗原抗体组合物，其特征在于，包括融合蛋白3AN和PRRSV N蛋白抗体C8；
所述融合蛋白3AN的氨基酸序列如SEQ ID NO：3所示；
所述PRRSV N蛋白抗体C8包括氨基酸序列如SEQ ID NO：1所示的重链可变区HV1和氨基酸序列如SEQ ID NO：2所示的轻链可变区LV1。
2.根据权利要求1所述抗原抗体组合物，其特征在于，所述抗原抗体组合物还包括口蹄疫病毒3A蛋白的单克隆抗体3A24。
3.权利要求1或2所述抗原抗体组合物在制备免疫检测猪繁殖与呼吸综合征病毒感染或免疫动物后N蛋白抗体的试剂或试剂盒中的应用。
4.根据权利要求3所述应用，其特征在于，所述免疫检测包括胶体金免疫检测、免疫沉淀检测、荧光免疫检测和/或酶联免疫检测。
5.根据权利要求3所述应用，其特征在于，所述试剂盒包括抗原反应板；所述抗原反应板是预先包被单克隆抗体3A24再结合所述融合蛋白3AN制备得到。
6.根据权利要求3所述应用，其特征在于，所述试剂盒包括100×浓缩生物素标记抗体；所述100×浓缩生物素标记抗体为生物素标记的所述PRRSV N蛋白抗体C8。
7.根据权利要求6所述应用，其特征在于，所述PRRSV N蛋白抗体C8的标记浓度为0.06～0.25μg/mL；
所述100×浓缩生物素标记抗体的工作浓度为1：10000～30000。
8.根据权利要求3所述应用，其特征在于，所述试剂盒还包括以下组分：100×浓缩酶标亲和素、25倍浓缩洗涤液、血清稀释液、底物溶液、终止液、阳性对照血清和阴性对照血清。
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