CN114891095B - 一种用于检测prrsv抗原的纳米抗体对、试剂盒及其应用 - Google Patents
一种用于检测prrsv抗原的纳米抗体对、试剂盒及其应用 Download PDFInfo
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Abstract
本发明公开了一种用于检测PRRSV抗原的纳米抗体对、试剂盒及其应用,属于生物技术检测技术领域。为了提供一种特异性检测PRRS病毒的抗体对。本发明提供了一种用于检测PRRSV抗原的Nb12抗体和N35抗体,Nb12抗体的可变区的氨基酸序列如SEQ ID NO:7‑9,N35抗体可变区序列如SEQ ID NO:10‑12。抗体对能特异性识别PRRSV抗原,最低检测1.0×103TCID50/100μL病毒。
Description
技术领域
本发明属于生物技术检测技术领域,具体涉及一种用于检测PRRSV抗原的纳米抗体对、试剂盒及其应用。
背景技术
猪繁殖与呼吸综合征(Porcine reproductive and respiratory syndrome,PRRS)是由PRRS病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)引起的病毒性传染病,该病在临床上主要引起母猪流产以及各年龄段猪的呼吸系统疾病,给养猪业造成不可挽回的经济损失。
目前,PRRSV主要通过q-PCR检测猪血液病毒感染情况及通过ELISA试剂盒检测抗体水平来判断猪是否感染病毒。q-PCR方法需要离心机及荧光定量PCR仪提取病毒RNA并进行扩增,需要专业人员进行操作;该方法还存在因引物、反应体系或敏感性问题,导致漏检或假阳性结果。但是,关于纳米抗体在PRRSV病毒抗原诊断方法中的应用未见相关研究报道,也未有商品化产品。因此,筛选与病毒保守的N蛋白反应的纳米抗体,并应用纳米抗体和HRP偶联纳米抗体建立双抗体夹心ELISA检测方法,无需抗体标记和使用二抗,简化了生产工艺并降低了生产成本,广谱性、高灵敏度高、特异性检测血液及组织液病毒,具有非常广阔的应用前景。
发明内容
本发明的目的是为了提供一种特异性检测PRRS病毒的纳米抗体对。
本发明提供了一种用于检测PRRSV抗原的纳米抗体对所述抗体对为Nb12纳米抗体和Nb35纳米抗体,所述Nb12纳米抗体的可变区的CDR-1的氨基酸序列如SEQ ID NO.7所示,所述Nb12纳米抗体的可变区的CDR-2的氨基酸序列如SEQ ID NO.8所示,所述Nb12纳米抗体的可变区的CDR-3的氨基酸序列如SEQ ID NO.9所示;
所述Nb35纳米抗体的可变区的CD-1的氨基酸序列如SEQ ID NO.10所示,所述Nb35纳米抗体的可变区的CDR-2的氨基酸序列如SEQ ID NO.11所示,所述Nb35纳米抗体的可变区的CDR-3的氨基酸序列如SEQ ID NO.12所示。
进一步地限定,所述Nb12纳米抗体的氨基酸序列如SEQ ID NO.15所示。
进一步地限定,所述Nb35纳米抗体的氨基酸序列如SEQ ID NO.16所示。
进一步地限定,所述Nb12纳米抗体的核苷酸序列如SEQ ID NO.13所示。
进一步地限定,所述Nb35纳米抗体的核苷酸如SEQ ID NO.14所示。
进一步地限定,Nb12纳米抗体与辣根过氧化物酶组成融合蛋白。
本发明提供了上述的纳米抗体对在制备用于检测PRRSV抗原的试剂盒的应用。
进一步地限定,所述试剂盒的检测方法包括酶联免疫法、化学发光检测法、蛋白质印迹检测法或者免疫组化法。
有益效果:本发明提供了一种纳米抗体对:Nb12纳米抗体和Nb35纳米抗体,能特异性识别PRRSV抗原,最低检测1.0×103TCID50/100μL病毒,Nb12纳米抗体与HRP融合蛋白表达的制备方法,用于检测PRRSV抗原,不需要标记和使用HRP标记的二抗,可以简化生产工艺,降低生产成本,具有很好的市场转化前景。
附图说明
图1为纳米抗体氨基酸序列比对结果;
图2为ELISA方法检测表达纳米抗体与PRRSV N蛋白ELISA反应,其中,横坐标是组别,纵坐标是OD450nm。
图3为验证PRRSV-N-Nb12-HRP融合蛋白的表达分泌以及亲和力和特异性结果图。
图4为纳米抗体对敏感性检测结果,其中,横坐标是组别,纵坐标是OD450nm。
图5为纳米抗体对广谱性检测结果,其中,横坐标是组别,纵坐标是OD450nm。
图6为纳米抗体对特异性检测结果,其中,横坐标是组别,纵坐标是OD450nm。
图7为载体连接示意图。
具体实施方式
PRRSV HuN4株记载来源于陶冶,王刚,刘永刚,等.高致病性PRRSV HuN4株不同代次病毒对仔猪胸腺损伤的研究[J].中国预防兽医学报,2014,36(5):4.
PRRSV-1株、PRRSV-2的Lineage1、3、5、8分支毒(L1、L3、L5和L8)为本实验室分离培养,经测序鉴定后分类,记载在下述的文章中:
1.Two novel recombinant porcine reproductive and respiratory syndromeviruses belong to sublineage 3.5originating from sublineage 3.2..Wen-LiZhang,Hong-Liang Zhang,Hu Xu,Yan-Dong Tang,Chao-Liang Leng,Jin-Mei Peng,QianWang,Tong-Qing An,Xue-Hui Cai,Jing-Hui Fan,Zhi-Jun Tian,Transbound EmergDis.2019,66(6):2592-2600.
2.Molecular epidemiology of PRRSV:Aphylogenetic perspective.MangShia,Tommy Tsan-Yuk Lama,Chung-Chau Hona,Raymond Kin-Hei Huia,Kay S.Faabergb,Trevor Wennblomc,Michael P.Murtaughd,Tomasz Stadejeke,Frederick Chi-ChingLeunga,Virus Research,2010 1547–17.
实施例1.纳米抗体的制备与筛选的方法
1.PRRSV HuN4株的增殖与纯化
(1)当Marc-145细胞长至单层且铺满培养瓶底部时,将104TCID50 PRRSV HuN4株病毒液(-80℃冻存)接种到15mL含2%FBS DMEM培养液的T175细胞瓶中,37℃,5%CO2培养箱中孵育2h。
(2)孵育完成后,补加2%FBS DMEM至50mL,轻轻混匀后继续培养,待细胞出现明显的CPE时收取病毒上清液。
(3)收取的病毒上清液进行粗离,4000rpm,4℃,离心30min,去除病毒上清液中细胞碎片。
(4)用0.45μm滤膜对病毒液进行过滤,过滤后进行超速离心,40000rpm,4℃,离心4h。离心后沉淀物用6mL PBS重悬,4℃保存备用。
(5)层析柱用灭菌水洗涤干净,将凝胶层析柱填料Sepharose 6FF缓慢注入,注意防止气泡产生。
(6)用2个柱体积的PBS洗涤,流速1mL/min。
(7)当填料Sepharose 6FF平衡完成后,将浓缩后的病毒样品缓慢注射到上样阀中。
(8)用1mL/min的流速洗脱蛋白,连续监测洗脱过程中OD280 nm值。
(9)当OD280 nm值上升时开始收取样品,下降到10mAu以下且不再发生变化时,终止收取样品。
(10)利用凝胶层析方法纯化上述获得的样品得到纯化后PRRSV。
2.纯化后PRRSV的鉴定
(1)取步骤1纯化后PRRSV加入Loading Buffe后在沸水中煮样10min。
(2)各取30μL上样于凝胶孔中,电泳完成后,用考马斯亮蓝染液染色,检测蛋白条带。
(3)或当电泳完成后,将蛋白转移至PVDF膜上。
(4)用5%脱脂乳室温封闭2h。
(5)TBST洗3次,N蛋白的单克隆抗体(Anti-PRRSV-N protein抗体上海钰博生物科技有限公司,货号YB--23941R)的细胞培养上清室温孵育1h。
(6)TBST洗3次,加入Dylight 800标记的山羊抗鼠IgG(1:10000稀释),室温作用1h。
(7)TBST洗涤后,通过扫膜成像系统观察,收集样品为PRRSV病毒颗粒。
3.羊驼免疫
将凝胶层析方法纯化的PRRSV免疫羊驼,每两周1次,共免疫6次,第一次使用弗氏完全佐剂,其余,佐剂与病毒1:1混合乳化5次全部使用弗式不全佐剂。
4.噬菌体文库的构建
(1)第6次免疫后4天,采血,分离羊驼外周血淋巴细胞并提取总RNA,利用的试剂盒为Plus Mini Kit;
(2)将RNA反转录成cDNA,以此为模板进行巢式PCR扩增,引物序列表2所示,并通过PstI、NotI酶切位点连入pCANTAB 5E噬菌体展示载体获得连接产物。
表2 VHH片段扩增及检测引物
名称 | 引物序列(5’-3’) |
Call001 | GTGGTCCTGGCTGCTCTWCTACAA(SEQ ID NO.1) |
Call002 | GGTACGTGCTGTTGAACTGTTCC(SEQ ID NO.2) |
Alpaca FOR Pst I | CAGGTGCAGCTGCAGATGGCCGAGGTGCAGCTGGTGSAG(SEQ ID NO.3) |
VHH FOR Pst I | CAGGTGCAGCTGCAGATGGAGTCDGGGGGAGRCT(SEQ ID NO.4) |
VHH REV | CTAGTGCGGCCGCTGAGGAGACGGTGACCYGGGT(SEQ ID NO.5) |
p5E-FOR | AATACGCAAACCGCCTCTCC(SEQ ID NO.6) |
注:S代表G或C;R代表A或G。W代表A/T,D代表A或G或T,Y代表C或T
(3)将连接产物电转至TG1感受态细胞中,活化后涂于LB-AMP琼脂平板,37℃过夜培养,收集菌苔,制成甘油菌在-80℃保存。
(4)利用引物p5E-FOR,VHH REV,通过菌落PCR检测所建文库的阳性率,并测定库容大小及多样性,检测结果阳性率为96%,库容量为1.7×108克隆。
5.抗PRRSV N蛋白的特异性纳米抗体的筛选
M13KO7辅助噬菌体感染纳米抗体文库,拯救得到羊驼纳米抗体基因噬菌体文库。利用噬菌体展示技术淘选的PRRSV特异性纳米抗体。
5.1重组噬菌体文库的拯救
(1)取100μL制备的重组噬菌体文库,加入100mL 2×YT/AMP-GLU培养基,培养至OD600nm值为0.6~0.8。
(2)加入100μL M13K07(滴度:1.5×1013PFU/mL),约20个MOI的感染复数,37℃静置感染30min。
(3)3800g室温离心10min,沉淀加入到200mL 2×YT/AMP-KAN培养基,37℃200rpm过夜培养。
(4)3800g,4℃离心30min,将上清分装到5个50mL的离心管中,40mL/管,再加入冰上提前预冷的PEG/NaCl溶液,10mL/管,混匀后冰上静置沉淀2h。
(5)3800g,4℃离心30min,各用2mL PBS重悬沉淀。
(6)12000g,4℃离心20min,上清分装到10个1.5mL的EP管中,1mL/管,各加入250μL预冷的PEG/NaCl溶液,混匀后冰上静置2h。
(7)12000g,4℃离心10min,所得沉淀用1mL PBS重悬,4℃保存备用
5.2抗原包被:将纯化的PRRSV每孔4μg包于96孔酶标板,同时选取一个孔直接加入PBS作为无抗原对照孔,4℃包被过夜;用2.5%脱脂奶粉封闭
5.3淘选富集
ELISA板中加入噬菌体37℃孵育1h;用PBST洗涤5次,洗去不结合的噬菌体,加入新鲜配制的0.1M三乙胺100μL每孔,洗脱与PRRSV特异性结合的噬菌体,即P0代洗脱产物。将洗脱产物感染处于对数期生长的大肠杆菌TG1,生产并纯化噬菌体用于下一轮的筛选。经过3轮筛选,富集阳性克隆。
6.抗PRRSV N蛋白的特异性纳米抗体的鉴定
随机挑取96个单菌落进行测序,比对纳米抗体CDR3高变区,发现共筛选出13株抗PRRSV的特异性纳米抗体,结果如图1所示。对13株纳米抗体诱导表达的粗提物进行间接ELISA鉴定其与PRRSV反应性,具体步骤如下:
挑取阳性单菌落,37℃,200rpm过夜培养;分别取上述菌液10μL转接到24孔板的TB培养基(1mL/孔),培养至对数期。;每孔加入终浓度为1mM IPTG,37℃200rpm诱导10h;3800g,4℃离心10min,菌体置于-20℃冷冻30min;待恢复室温后,加入PBS重悬菌体,500μL/孔,250rpm,4℃振荡30min;3800g,4℃离心10min,上清即为重组纳米抗体粗提物。
将重组纳米抗体粗提物加入PRRSV包被的ELISA板(400ng/孔)及PBS包被的ELISA板中,100μL/孔,37℃孵育1h。加入兔抗E-tag多抗(1:2000稀释),100μL/孔,37℃孵育1h。加入HRP标记的山羊抗兔IgG(1:10000稀释),100μL/孔,37℃孵育1h。
加入TMB显色液,100μL/孔,37℃反应15min。
显色完成后加入1M盐酸终止反应,100μL/孔,测定OD450nm值。
根据重组纳米粗提物的间接ELISA检测结果,结果如图2所示,将13株纳米抗体进行原核表达和后续研究。
其中用于检测PRRSV双抗体夹心LISA抗体对的两株纳米抗体,命名为PRRSV-N-Nb12,PRRSV-N-Nb35。Nb12纳米抗体的可变区的CDR-1的氨基酸序列如SEQ ID NO.7所示(GFTFSQHHMS),所述Nb12纳米抗体的可变区的CDR-2的氨基酸序列如SEQ ID NO8所示(GIGTFGQINYADSAKG),所述Nb12纳米抗体的可变区的CDR-3的氨基酸序列如SEQ ID NO.9所示(YTSGRRY);
Nb35纳米抗体的可变区的CD-1的氨基酸序列如SEQ ID NO.10所示(GFSSNTYVTR),所述Nb35纳米抗体的可变区的CDR-2的氨基酸序列如SEQ ID NO.11所示(TLPGAGTARYADSVRG),所述Nb35纳米抗体的可变区的CDR-3的氨基酸序列如SEQ ID NO.12所示(EGYGRNI)。
Nb12纳米抗体的核苷酸序列如SEQ ID NO.13所示,PRRSV-N-Nb12:
ATGGCCGAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTGCACCCTGGGGGGTCTCTGAGGCTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTCAGCATCACATGAGCTGGTACCGGCAGGCTCCAGGAAAGCAGCGCGAGTTGGTCGCAGGTATTGGTACTTTCGGTCAAATAAATTATGCGGACTCCGCGAAGGGCCGATTCACCATTTCCAGAGACAACGCCAAGAACACGGTGTCTCTGCAAATGAACAGCCTGAGTCCTGAGGACACGGCCGTCTATCAGTGTTCTGCATACACATCGGGCCGGCGTTATTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA
Nb35纳米抗体的核苷酸如SEQ ID NO.14所示,PRRSV-N-Nb35:
ATGGCCGAGGTGCAGCTGGTGCAGTCTGGGGGAGGCTTGGTGCAGCCTGGGGGGTCTCTGAGGCTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTCAGCATCACATGAGCTGGTACCGGCAGGCTCCAGGAAAGCAGCGCGAGTTGGTCGCAGGTATTGGTACTTTCGGTCAAATAAATTATGCGGACTCCGCGAAGGGCCGATTCACCATTTCCAGAGACAACGCCAAGAACACGGTGTCTCTGCAAATGAACAGCCTGAGTCCTGAGGACACGGCCGTCTATCAGTGTTCTGCATACACATCGGGCCGGCGTTATTGGGGCCAGGGGACCCAGGTCACCGTCTCCTCA
Nb12纳米抗体的氨基酸序列如SEQ ID NO.15所示,PRRSV-N-Nb12:MAQVQLQMAEVQLVQSGGGLVQPGGSLRLSCAASGFTFSQHHMSWYRQAPGKQRELVAGIGTFGQINYADSAKGRFTISRDNAKNTVSLQMNSLSPEDTAVYQCSAYTSGRRYWGQGTQVTVSSAAAGAPVPYPDPLEPRAA
Nb35纳米抗体的氨基酸序列如SEQ ID NO.16所示,PRRSV-Nb35:
MAEVQLVQSGGGLVHPGGSLRLSCAASGFTFSQHHMSWYRQAPGKQRELVAGIGTFGQINYADSAKGRFTISRDNAKNTVSLQMNSLSPEDTAVYQCSAYTSGRRYWGQGTQVTVSSAAAGAPVPYPDPLEPRAA
实施例2.纳米抗体与辣根过氧化物酶融合蛋白的制备
1.改造载体:将PRRSV-N-Nb12基因序列与HRP序列连接到PEGFP-N2(载体,按照图7展示的连接顺序进行连接,在Nb12两端保留酶切位点Pst I(CTGCAG)和NotI(GCGGCCGC),测序鉴定后,获得阳性质粒pEGFP-Nb12-HRP。HRP序列(如SEQ ID NO.17所示)。
2.表达蛋白
待6孔板中HEK 293T细胞密度达到80%时换液,加入2mL 2% FBS DMEM。取100μL无血清DMEM对4μg质粒(pEGFP-Nb12-HRP)稀释,加入9μL转染试剂PEI,混匀后室温静置15min。将上述转染体系缓慢加入六孔板,轻轻晃匀后37℃培养6~8h换液。37℃继续培养48~60h。收集细胞分泌表达上清液,经离心浓缩后,即获得PRRSV-N-Nb12-HRP融合蛋白。
3.通过ELISA验证PRRSV-N-Nb12-HRP融合蛋白的表达分泌以及亲和力和特异性,结果如图3所示,其中图A是特异性,图B是亲和力。PRRSV-N-Nb12-HRP融合蛋白成功在HEK293T细胞中的表达,细胞上清中的融合蛋白可特异性的与PRRSV N蛋白结合,Nb12-HRP融合蛋白1:80稀释仍可与PRRSV结合。
实施例3.基于纳米抗体的夹心ELISA方法的建立
1.Nb35最佳包被量与Nb12-HRP最佳稀释度的确定
捕获抗体Nb35的包被浓度分别为20μg/孔、10μg/孔、5μg/孔、2μg/孔、1μg/孔和0.5μg/孔,检测抗体Nb12-HRP分别加入原液、1:1、1:2、1:5、1:10和1:20稀释。.通过棋盘法确定Nb35包被浓度为10μg/孔,Nb12-HRP加入原液时,P/N值最高,为28.909(表1所示)。
表1
2.获得的试剂盒的成分是:样品稀释液:含3% BSA,0.5%triton X-100的PBS溶液(PH 7.5),Nb12-HRP抗体,阳性对照:PRRSV培养液,阴性对照:阴性猪血清。
3.试剂盒的使用方法:
(1)将待检测的抗原稀释后加入到包被ELISA板中,孵育1小时,PBST洗板3次,
(2)将Nb12-HRP抗体加入到ELISA板,孵育1小时,PBST洗板3次,;
(3)在ELISA板中加入显色液避光显色15min,加入3M浓H2SO4终止反应;
(4)观察ELISA板的颜色变化,读取OD450数值,计算S/P值,S/P大于0.13判为阳性,S/P≤0.13判为阴性:
4产品质量研究
4.1.敏感性检测
分别将PRRSV HuN4病毒上清液稀释至1.0×105TCID50/100μL,5.0×104TCID50/100μL,2.5×104TCID50/100μL,5.0×103TCID50/100μL,2.5×103TCID50/100μL和1.0×103TCID50/100μL进行检测,根据结果评价ELISA方法的敏感性,结果如图4所示,可以检测到1.0×103TCID50/100μL病毒。
4.2.广谱性检测
分别对PRRSV的多种毒株进行检测,选择4株PRRSV病毒,包括PRRSV-1、PRRSV-2-L1、PRSV-2-L3、PRRSV-2-L5和PRRSV-2-L8,结果如图5所示,该ELISA方法可以检测不同分支的PRRSV病毒。
4.3夹心ELISA方法与荧光定量PCR方法的符合率比较
通过检测临床样品,包括40份组织样品和50份血清样品,对建立的夹心ELISA方法和荧光定量PCR方法进行比较,结果如表2所示,两者的总符合率为86.6%。
表2夹心ELISA与荧光定量PCR方法检测PRRSV临床样品的一致性比较
4.4.特异性检测
为了评价建立的夹心ELISA方法的特异性,分别对猪的其他常见病毒进行检测,包括CSFV(猪瘟病毒)、PCV(猪圆环病毒)、PEDV(猪流行性腹泻病毒)、PPV(猪细小病毒)和PRV(伪狂犬病病毒),结果如图6所示,该方法与其他猪病毒均无反应,特异性好。
SEQUENCE LISTING
<110> 中国农业科学院哈尔滨兽医研究所(中国动物卫生与流行病学中心哈尔滨分中心)
<120> 一种用于检测PRRSV抗原的纳米抗体对、试剂盒及其应用
<160> 17
<170> PatentIn version 3.5
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<213> 人工合成
<400> 9
Tyr Thr Ser Gly Arg Arg Tyr
1 5
<210> 10
<211> 10
<212> PRT
<213> 人工合成
<400> 10
Gly Phe Ser Ser Asn Thr Tyr Val Thr Arg
1 5 10
<210> 11
<211> 16
<212> PRT
<213> 人工合成
<400> 11
Thr Leu Pro Gly Ala Gly Thr Ala Arg Tyr Ala Asp Ser Val Arg Gly
1 5 10 15
<210> 12
<211> 7
<212> PRT
<213> 人工合成
<400> 12
Glu Gly Tyr Gly Arg Asn Ile
1 5
<210> 13
<211> 351
<212> DNA
<213> 人工合成
<400> 13
atggccgagg tgcagctggt gcagtctggg ggaggcttgg tgcaccctgg ggggtctctg 60
aggctctcct gtgcagcctc tggattcacc ttcagtcagc atcacatgag ctggtaccgg 120
caggctccag gaaagcagcg cgagttggtc gcaggtattg gtactttcgg tcaaataaat 180
tatgcggact ccgcgaaggg ccgattcacc atttccagag acaacgccaa gaacacggtg 240
tctctgcaaa tgaacagcct gagtcctgag gacacggccg tctatcagtg ttctgcatac 300
acatcgggcc ggcgttattg gggccagggg acccaggtca ccgtctcctc a 351
<210> 14
<211> 351
<212> DNA
<213> 人工合成
<400> 14
atggccgagg tgcagctggt gcagtctggg ggaggcttgg tgcagcctgg ggggtctctg 60
aggctctcct gtgcagcctc tggattcacc ttcagtcagc atcacatgag ctggtaccgg 120
caggctccag gaaagcagcg cgagttggtc gcaggtattg gtactttcgg tcaaataaat 180
tatgcggact ccgcgaaggg ccgattcacc atttccagag acaacgccaa gaacacggtg 240
tctctgcaaa tgaacagcct gagtcctgag gacacggccg tctatcagtg ttctgcatac 300
acatcgggcc ggcgttattg gggccagggg acccaggtca ccgtctcctc a 351
<210> 15
<211> 142
<212> PRT
<213> 人工合成
<400> 15
Met Ala Gln Val Gln Leu Gln Met Ala Glu Val Gln Leu Val Gln Ser
1 5 10 15
Gly Gly Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala
20 25 30
Ala Ser Gly Phe Thr Phe Ser Gln His His Met Ser Trp Tyr Arg Gln
35 40 45
Ala Pro Gly Lys Gln Arg Glu Leu Val Ala Gly Ile Gly Thr Phe Gly
50 55 60
Gln Ile Asn Tyr Ala Asp Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg
65 70 75 80
Asp Asn Ala Lys Asn Thr Val Ser Leu Gln Met Asn Ser Leu Ser Pro
85 90 95
Glu Asp Thr Ala Val Tyr Gln Cys Ser Ala Tyr Thr Ser Gly Arg Arg
100 105 110
Tyr Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Ala Ala Ala Gly
115 120 125
Ala Pro Val Pro Tyr Pro Asp Pro Leu Glu Pro Arg Ala Ala
130 135 140
<210> 16
<211> 135
<212> PRT
<213> 人工合成
<400> 16
Met Ala Glu Val Gln Leu Val Gln Ser Gly Gly Gly Leu Val His Pro
1 5 10 15
Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser
20 25 30
Gln His His Met Ser Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu
35 40 45
Leu Val Ala Gly Ile Gly Thr Phe Gly Gln Ile Asn Tyr Ala Asp Ser
50 55 60
Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val
65 70 75 80
Ser Leu Gln Met Asn Ser Leu Ser Pro Glu Asp Thr Ala Val Tyr Gln
85 90 95
Cys Ser Ala Tyr Thr Ser Gly Arg Arg Tyr Trp Gly Gln Gly Thr Gln
100 105 110
Val Thr Val Ser Ser Ala Ala Ala Gly Ala Pro Val Pro Tyr Pro Asp
115 120 125
Pro Leu Glu Pro Arg Ala Ala
130 135
<210> 17
<211> 933
<212> DNA
<213> 人工合成
<400> 17
atgcagctga cccccacatt ctacgataac tcttgtccca acgtgtccaa catcgttcgt 60
gacatcatcg tgaatgagct gaggagcgac cctaggatcg ccgccagcat tctgaggctg 120
cacttccacg actgcttcgt gaatggctgc gacgcctcca ttttactgga taacaccacc 180
agctttcgta ccgaaaagga cgctttcggc aacgccaaca gcgctcgtgg cttcagcgtg 240
atcgatcgta tgaaagccgc cgtggaaagc gcttgtcccg gcacagtgtc ttgtgccgat 300
ttattaacca ttgccgccca gcagtccgtg acactggccg gcggacctag ctggagagtg 360
cctctgggtc gtagggattc tttacaagcc tttttagatt tagccaacgc caatttaccc 420
gcccctttct tcactttacc tcagctcaag gacagcttca gaaacgtggg tttaaatagg 480
agcagcgatt tagtggcttt atccggcggc cacacatttg gcaagagcca gtgtcgtttc 540
attatggatc gtctgtacaa tttcagcaac accggtttac ccgaccccac actgaacacc 600
acctatctcc agactttaag aggtttatgc cctttaaacg gcaatctgtc cgctttagtg 660
gatttcgact tacgtacccc cacaatcttc gacaacaaat actacgtgaa tttagaggag 720
cagaagggtt taatccagag cgaccaagaa ctgttttcca gccccgatgc caccgacacc 780
atccctctgg tgaggagctt cgccaactcc acccagacct tcttcaacgc ctttgtggag 840
gccatggatc gtatgggcaa cattacccct ctgaccggca cccaaggtca gattcgtagg 900
aactgtaggg tggtgaatag caacagcgat tta 933
Claims (6)
1.一种用于检测PRRSV抗原的纳米抗体对,其特征在于,所述抗体对为Nb12纳米抗体和Nb35纳米抗体,所述Nb12纳米抗体的可变区的CDR-1的氨基酸序列如SEQ ID NO.7所示,所述Nb12纳米抗体的可变区的CDR-2的氨基酸序列如SEQ ID NO.8所示,所述Nb12纳米抗体的可变区的CDR-3的氨基酸序列如SEQ ID NO.9所示;
所述Nb35纳米抗体的氨基酸序列如SEQ ID NO.16所示。
2.根据权利要求1所述的纳米抗体对,其特征在于,所述Nb12纳米抗体的氨基酸序列如SEQ ID NO.15所示。
3.根据权利要求1所述的纳米抗体对,其特征在于,所述Nb12纳米抗体的核苷酸序列如SEQ ID NO.13所示。
4.根据权利要求1所述的纳米抗体对,其特征在于,Nb12纳米抗体与辣根过氧化物酶组成融合蛋白。
5.权利要求1-4任一项所述的纳米抗体对在制备用于检测PRRSV抗原的试剂盒的应用。
6.根据权利要求5所述的应用,其特征在于,所述试剂盒的检测方法包括酶联免疫法、化学发光检测法、蛋白质印迹检测法或者免疫组化法。
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