CN112876561B - 一种用于检测犬瘟热病毒的抗体对及其应用 - Google Patents
一种用于检测犬瘟热病毒的抗体对及其应用 Download PDFInfo
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Abstract
本发明公开了一种用于检测犬瘟热病毒的抗体对及其应用,属于生物技术领域。一种用于检测犬瘟热病毒的抗体对,抗体1A4重链CDR‑H1‑CDR‑H3即SEQ ID NO:1‑3,抗体1A4轻链CDR‑L1‑CDR‑L3即SEQ ID NO:4‑6;抗体1B2重链CDR‑H1‑CDR‑H3即SEQ IDNO:7‑9;抗体1B2轻链CDR‑L1‑CDR‑L3即SEQ ID NO:10‑12。抗体对能特异性识别犬瘟热病毒,在抗原试纸条实验中最低检测1000个TCID50/mL犬瘟热病毒,在双抗体夹心法ELISA实验中最低检测400个TCID50/mL犬瘟热病毒,识别特异性高,且不与其他病毒反应。
Description
技术领域
本发明属于生物技术领域,具体涉及一种用于检测犬瘟热病毒的抗体对及其应用。
背景技术
犬瘟热(Canine Distemper)是由犬瘟热病毒(Canine distemper virus,CDV)感染,会导致一种伴有免疫抑制和全身性感染的疾病。犬只感染后会引起呼吸道消化道以及中枢神经的症状,其感染数组相当的广泛,包括熊猫狗,水貂,狐狸,狼等,陆生肉食兽,以及鲸鱼海豹等水生哺乳类。犬瘟热病毒属于副粘病毒科,麻疹病毒属成员,具有囊膜结构,不分节段的负链RNA病毒。病毒基因组全长约15690bp,病毒颗粒多以球状存在,位于蛋白囊膜内的核蛋白长度为600~800纳米,直径为18纳米,早在1995年已有报道指出麻疹病毒与犬瘟热病毒在宿主身上造成持续性感染的原因,与病毒复制时,装配和病毒出芽释放有关,尤其是F蛋白和H蛋白扮演着重要的角色。以PAGE进行电泳分析F蛋白大小约为63kDa,F蛋白和H蛋白除了可自行组装成病毒囊膜外,还介导病毒的细胞受体侵入细胞内进行复制。犬瘟热的临床症状取决于病毒的毒性、环境、宿主年龄与免疫状况,超过50%的感染为亚临床型,常见的症状包括精神萎靡,发热,上呼吸道感染,且双侧眼鼻分泌物,会由浆液型转化为粘液型溶性病,水咳嗽及呼吸困难;感染犬只可发生角膜结膜炎。有报道指出患病耐过的犬只,可造成持续性嗅觉丧失症,所有年龄的犬只皆易感犬瘟热病毒,尤其是在3~6个月的幼犬,由于缺乏母源抗体,感染初期症状温和,出现食厌食症,食欲减退,呕吐腹泻,粪便会呈水样,接着会有里急后重与肠套叠现象发生,液体流失会造成严重的脱水和消瘦,因此感染全身性症状的犬只经常突然死亡。
随着当前宠物诊疗业的高速发展,基于免疫反应的犬瘟热病原的诊断技术越来越得到重视(徐刚.犬瘟热病毒单克隆抗体的筛选与双抗体夹心ELISA的建立[D].西北农林科技大学,2019.),而单克隆抗体是诊断抗原的重要试剂,目前已经研发出具备中和作用的单克隆抗体(CN 102618503B)(毕振威,徐立波,夏兴霞,梁霆,王永山.鉴别犬瘟热病毒强弱毒株中和单克隆抗体的制备[J].江苏农业科学,2020,48(20):178-182.)但是缺乏研制犬瘟热配对单抗的相关报道,而配对的单抗组可以形成检测抗原的三明治结构,是研制夹心法ELISA和抗原胶体金试纸条的关键;本研究通过纯化犬瘟热病毒,筛选到高亲和的单克隆抗体2株1A4和1B2,经免疫印迹检测,其识别犬瘟热蛋白大小约为63kDa,推测为识别犬瘟热病毒F蛋白,经过测试,该配对抗体可用于夹心法ELISA试剂或者胶体金试剂研发。发明内容
本发明的目的是为了提供一种特异性检测犬瘟热病毒的试剂,本发明提供了一种用于检测犬瘟热病毒的抗体对,所述抗体对为1A4和1B2,所述抗体1A4重链的可变区的CDR-H1的氨基酸序列如SEQ ID NO:1所示,CDR-H2的氨基酸序列如SEQ ID NO:2所示,CDR-H3的氨基酸序列如SEQ ID NO:3所示;所述抗体1A4轻链的可变区的CDR-L1的氨基酸序列如SEQID NO:4所示,CDR-L2的氨基酸序列如SEQ ID NO:5所示,CDR-L3的氨基酸序列如SEQ IDNO:6所示;所述抗体1B2重链的可变区的CDR-H1的氨基酸序列如SEQ ID NO:7所示,CDR-H2的氨基酸序列如SEQ ID NO:8所示,CDR-H3的氨基酸序列如SEQ ID NO:9所示;所述抗体1B2轻链的可变区的CDR-L1的氨基酸序列如SEQ ID NO:10所示,CDR-L2的氨基酸序列如SEQ IDNO:11所示,CDR-L3的氨基酸序列如SEQ ID NO:12所示。
进一步地限定,所述抗体1A4的轻链可变区的CDR-L1的基因序列如SEQ ID NO:16所示,所述抗体1A4的轻链可变区的CDR-L2的基因序列如SEQ ID NO:17所示,所述抗体1A4的轻链可变区的CDR-L3的基因序列如SEQ ID NO:18所示。
进一步地限定,所述抗体1B2的重链可变区的CDR-H1的基因序列如SEQ ID NO:19所示,所述抗体1A4的重链可变区的CDR-H2的基因序列如SEQ ID NO:20所示,所述抗体1A4的重链可变区的CDR-H3的基因序列如SEQ ID NO:21所示。
进一步地限定,所述抗体1B2的轻链可变区的CDR-L1的基因序列如SEQ ID NO:22所示,所述抗体1B2的轻链可变区的CDR-L2的基因序列如SEQ ID NO:23所示,所述抗体1B2的轻链可变区的CDR-L3的基因序列如SEQ ID NO:24所示。
进一步地限定,所述抗体1A4为捕捉抗体,所述抗体1B2为检测抗体。
本发明还提供了一种上述用于检测犬瘟热病毒的抗体对在制备检测犬瘟热病毒的试剂盒中的应用。
有益效果:本发明提供的抗体对能够检测犬瘟热病毒,在抗原试纸条实验中能够检测100倍稀释的CDV病毒,约为1000个TCID50/mL;在双抗体夹心法ELISA实验中,能够检测400个TCID50/mL经过检测本发明的抗体对是针对犬瘟热病毒囊膜由F蛋白,F蛋白是囊膜的纤突部分的重要组成,而且该部分直接与感染细胞接触,是病毒感染机体的重要结构,F蛋白的抗血清能中和病毒,抗F蛋白的单抗与病毒之间有高度的亲和性,识别特异性强。
附图说明
图1为犬瘟热单抗纯化检测,其中,1是故蛋白质Marker,2是犬瘟热纯化单抗1A4,3是犬瘟热纯化单抗1B2;
图2为为夹心法ELISA检测犬瘟热病毒的临界值400TCID50/ml,其中横坐标是犬瘟热病毒半数感染量(病毒含量),纵坐标是吸光度值;
图3为夹心法ELISA检测不同种类病毒结果,其中横坐标是犬瘟热病毒及其相关病毒,纵坐标是吸光度值;
图4为犬瘟热病毒间接免疫荧光实验,1A4和1B2显示出阳性结果,其中A是1A4,B是1B2,C是对照(未感染犬瘟热病毒的Vero细胞);
图5为犬瘟热病毒免疫印迹实验,1A4和1B2显示出阳性结果,其中A是采用抗体1A4,B是采用抗体1B2,M是marker;
图6为犬瘟热病毒胶体金试纸条结构图;
图7为犬瘟热病毒胶体金试纸条与PCR检测敏感性对比,其中A是犬瘟热病毒胶体金试纸条检测敏感性实验结果,B是PCR检测敏感性结果,其中的1是CDV原液的滴度为1×104TCID50/ml,2是CDV原液的滴度为1×103TCID50/ml,3是CDV原液的滴度为1×102TCID50/ml,4是CDV原液的滴度为1×101TCID50/ml,5是CDV原液的滴度为1×100TCID50/ml,control是未感染犬瘟热病毒的Vero细胞,M是marker(DNA DL2000)。
具体实施例
犬瘟热野毒株CDV-PS记载在施鹏飞,程悦宁,罗国良,王建科,程世鹏,易立.犬瘟热病毒CDV-PS株致病力及基因进化分析[J].特产研究,2019,41(03):17-20。
Vero细胞(非洲绿猴肾细胞)购自中国科学院细胞库,徐汇区岳阳路320号,上海200031。
CPV(犬细小病毒)记载在施鹏飞,程悦宁,冯二凯,罗国良,王振军,易立.CPV-2c型犬细小病毒分离及其VP2序列分析[J].特产研究,2020,42(01):11-14+20。
FPV(猫瘟病毒)记载在施鹏飞,程悦宁,冯二凯,罗国良,王振军,易立.CPV-2c型犬细小病毒分离及其VP2序列分析[J].特产研究,2020,42(01):11-14+20。
CCV(犬冠状病毒)记载在杨艳玲,程世鹏,任林柱.貂源冠状病毒与2019新型冠状病毒的遗传进化关系[J].中国动物检疫,2020,37(04):21-26。
实施例1.
一、杂交瘤制备
1.犬瘟热抗原的制备:将犬瘟热野毒株CDV-PS接种于Vero细胞(非洲绿猴肾细胞)。当细胞发生病变5日后收毒。将病变的细胞培养皿-80℃反复冻融。1000转/分离心后去除细胞碎片,留取上清。病毒上清经硫酸铵沉淀后,10000转/分离心取沉淀,沉淀用EDTA溶液重新悬浮。上清液再次10000转/分离心30分钟取沉淀。沉淀用PBS重新悬浮即为纯化后的病毒。利用分光光度计,测定病毒浓度,至于-80℃冰箱保存备用。
2.动物免疫:用纯化的犬瘟热野毒株CDV-PS作为免疫原。选4只6~8周龄雌性BALB/c小鼠作为免疫动物制备单抗。首免采用等体积的弗氏完全佐剂乳化抗原,10分钟完全乳化后,皮下免疫100微升/只。每隔7天用弗氏不完全佐剂乳化抗原再次免疫。
表1犬瘟热病毒ELISA效价结果
三免后的第7天断尾采血,测定血清抗体效价,应用犬瘟热病毒ELISA方法对3免后的小鼠进行效价检测,结果如表1所示,其中效价为大于最大OD/2的最小OD读数所对应的稀释度确定为最终犬冠状病毒血清效价。结果显示犬瘟热血清ELISA效价最高的小鼠为4号。选取血型效价最高的小鼠4号。
3.细胞融合:在超净台上铺好用酒精彻底喷洒过的吸水纸,将免疫好的小鼠放在上面。用一副直剪和直镊剪开小鼠的表皮,换一副弯剪和弯镊从小鼠的脾脏下面靠近脾脏的位置剪开内皮,使脾脏完全暴露。最后用弯剪和弯镊剪下脾脏(注意尽量剔除脂肪),放到细胞筛上。用注射器内芯轻轻地将脾和淋巴结充分碾碎(密切关注脾脏的组织弹性是否良好),将碾碎后的脾和淋巴结悬浊液全部吸入15ml离心管里,1500转离心5min,离心后去掉上清,将离心好的sp2/0-脾细胞(约3×107个)混合物上清倒掉,上清尽量倒干净将离心管底小心地在手心上磕打,使两种细胞充分的混匀。与此同时,助手用烧杯装来37℃的温水放入超净台中,将枪头伸入到管底靠近细胞,60-90s内加入1ml的PEG,然后120S内加入2ml的1640培养基,接着120S内缓慢加入8ml的无血清的1640培养基。加完无血清的IMDM后,细胞应均匀粒状分散)。从温水中取出离心管,盖好离心(1000rpm/min,5min)。离心后加入5ml饲养细胞,40ml DMEM+HAT分3盘,96孔板,每孔150μl,37度培养。
4.杂交瘤的筛选:HAT完全培养基培养7-10天后,全部更换为HT完全培养基,融合14-16天后,吸取100μL至事先已包被抗原(纯化的犬瘟热病毒CDV-PS)的ELISA板中,同时补充100μL HT完全培养基至96孔板。
ELISA测试结果为阳性者,进行亚克隆单株化,将融合后的杂交瘤细胞进行ELISA筛选,确定阳性细胞株,步骤如下:
1)用包被液稀释“犬瘟热野毒株CDV-PS”,终浓度为0.5mg/ml,100ul/孔,4℃,过夜;后用洗液洗涤3次。2)1%BSA封闭液封闭,200ul/孔,37℃孵箱,2h;后用洗液洗涤3次。3)加入一抗(细胞培养上清)、阴性对照(SP2/0培养上清)、空白对照(PBS)、阳性对照(阳性血清PBS 1000倍稀释),均为100ul/孔,37℃孵箱,1h;后用洗液洗涤3次。4)加入PBS稀释20000倍的二抗,100ul/孔,37℃孵箱,1h;取出后用洗液洗涤3次。5)显色,显色液100ul/孔,显色时间为5min左右。6)每孔加入50ul终止液终止。
融合后共筛选呈阳性的杂交瘤细胞孔9个,其中免疫效果最强的2个分别命名为1A4和1B2,其上清ELISA效价OD达到1.213和1.178。
5.单克隆抗体的制备与纯化:小鼠腹水抗体使用G蛋白亲和纯化。方法如下:将填料装载到层析柱中,用缓存液(20mM PBS,300mM NaCl,pH7.4-8.5)将腹水稀释约5倍,上柱进行纯化。上样后,用平衡缓冲液(20mM PBS,300mM NaCl,pH7.4-8.5)将蛋白未结合部分除去;然后加入洗脱缓冲液(0.1M柠檬酸钠,pH4.0),将结合蛋白洗脱下来,最后用中和缓冲液(1M tris-HCl,pH9.0)进行PH值调整。
将5倍体积纯化抗体和1体积的buffer蛋白混匀后煮沸5min。离心后上清上样。上样:样品10μL;蛋白Marker 5μL。电泳条件:95伏,电流约75mA,电泳2h。电泳结束后,取出电泳胶,置入染色液中1h,摇床缓慢摇晃。染色完全后,取出放入脱水液中,摇床缓慢摇晃1-2h。脱色完全后取出观察结果,结果如图1所示。
6.抗体鉴定和分型:羊抗鼠IgG(北京中杉金桥生物技术有限公司)稀释至0.5μg/m1包被96孔酶标板(100μL/孔),于37℃包被2小时,用PBST洗净拍干后,每孔加入200μl封闭液(含2%BSA和3%庶糖的PBS),37℃孵育1h。倾空液体,用PBST清洗3次。再加入1:5000稀释的待检单抗腹水(50μL/孔),37℃孵育1小时,同样洗净拍干后,分别加入1:1000稀释羊抗鼠IgG1、IgG2a、IgG2b、IgG3、IgM、IgA、Igк、Igд各亚类血清(50μL/孔)(Southern Biotech公司,货号0202-1),37℃作用1小时,洗净拍干后,加入1:5000稀释的HRP-兔抗羊IgG(100μL/孔),室温(15~25℃)作用15分钟,加TMB底物于孔中,37℃避光显色5~10分钟(100μL/孔),加2M H2SO4终止显色(100μL/孔),读取OD450nm值,选择读值明显高于其它孔的抗体类型作为单抗亚类。
结果如表2所示,将筛选出来的2株阳性细胞株进行亚类鉴定,最后得IgG类型的阳性杂交瘤细胞株。1A4细胞株为IgG2b,κ链,1B2细胞株为IgG1,κ链。1A4单抗IgG2b,κ值最高;1B2单抗IgG1,κ值最高,因此,1A4细胞株亚型为IgG2b,κ链,1B2细胞株亚型为IgG1,κ链。
表2单克隆抗体1A4和1B2亚类鉴定
7.单抗亲和力测定:利用非竞争性ELISA测定抗体的亲和力,包被抗原为纯化好的犬瘟热病毒,按1、0.5、0.25、0.125μg/mL包被酶标板,每孔100μL,4℃包被过夜;加入1%BSA,每孔150μL,37℃封闭2小时;PBST洗涤后,按确定的单抗浓度,将单抗从5μg/mL开始进行倍比稀释,以抗体浓度(moL/L)的对数值为横坐标,以其对应的OD450值为纵坐标,在一个坐标系中做出4条S形曲线。找出S曲线的顶部,设定为ODmax。在曲线中分别找出4条曲线各自50%ODmax对应的抗体浓度。按4个浓度两两一组,根据公式计算单抗的亲和常数。
Ka=(n-1)/2(n[Ab']t-[Ab]t)
n为每组中两个包被抗原浓度的倍数,[Ab']t和[Ab]t分别为每组中两个50%ODmax对应的抗体浓度(moL/L)。
单克隆抗体亲和常数测定显示犬瘟热病毒单克隆抗体1A4的Ka平均值为6.67×107M-1、1B2的Ka平均值为5.0×107M-1。
8.单抗中和活性测定。
采用固定病毒-稀释血清法测定各株单克隆抗体的中和活性:将100TCID50稀释的犬瘟热病毒与等体积杂交瘤细胞的培养上清液(2倍比连续稀释)或腹水(10倍比连续稀释)混合均匀,置37℃温箱内作用1小时,取该病毒-抗体混合液接种于96孔细施板中(100μL/孔),均匀铺入Vero细胞,置37℃5%CO2温箱培养数日,逐日观察细胞病变,同步设抗犬瘟热病毒阳性血清、阴性血清及正常Vero细胞作为对照。单克隆抗体中和活性测定1A4培养上清液中抗体的中和效价为28,腹水的中和效价为104,1B2单克隆抗体均没有中和活性。
9.犬瘟热病毒配对抗体夹心结构的确认:为了确认犬瘟热病毒能形成配对的三明治结构,我们采用夹心ELISA方法进行确认。首先1A4被当做捕捉抗体,包被在ELISA板上,采用封闭液进行封闭,然后加入梯度稀释的CDV抗原,洗涤掉未结合的部分后,加入1B2检测抗体(400ng/孔),洗涤掉未结合部分后,加入二抗然后显色。具体操作步骤如下:
(1)包被:犬瘟热病毒单抗1A4与包被液按1:50比例稀释(800ng/孔),100ul/孔,37℃恒温箱孵育1小时,洗板机清洗三次。
(2)封闭:5%脱脂乳+1%BSA进行封闭,每孔250ul,4℃封闭过夜,洗板机清洗3次。
(3)CDV样品100ul,使用100/200/400的比例稀释,37℃恒温箱孵育1小时,洗板机清洗三次。
(4)二抗1:10000稀释100μl,37℃恒温箱孵育1小时,洗板机清洗三次。
(5)显色:TMB 100μl室温3min;2M H2SO4终止液50μl。
结果如图2显示,本方法能够检测出等比例稀释的CDV病毒,换算成病毒滴度,本方法最低检测线值为400TCID50/ml。注CDV原液的滴度为1×105TCID50/ml。
10.配对抗体特异性检测:为了检测犬瘟热单抗1A4/1B2配对抗体的特异性,即只能识别CDV病毒而不与其他病毒进行反应,分别测试了不同的CDV毒株以及犬细小病毒、圆环病毒等对照,结果如图3显示,犬瘟热配对单抗1A4/1B2,均能识别CDV不同毒株,而不能识别犬细小病毒、圆环病毒和对照水。注明:CDV-Rockborn,DV3,CDV-Onderstepoort,CDV-PS,CDV-BT and CDV-R为不同来源的CDV毒株(记载在李爽.犬瘟热病毒P蛋白单克隆抗体的制备及其抗原表位的筛选[D].中国农业科学院,2019.),这里为了测试配对抗体的特异性。
二、抗体检测
1.间接免疫荧光试验:将Vero细胞以2×105个/ml~3×105个/ml的细胞悬液加入到96孔板中,100μl/孔。犬瘟热病毒稀释至100TCID50,100μl/孔,加到细胞中,同时在以下病毒的易感细胞中分别加入一定稀释倍数的犬细小病毒以及犬腺病毒,100μl/孔。同时设立空白细胞对照,37℃5%CO2细胞培养箱培养48小时。在病毒感染细胞48小时后,弃掉病毒液,80%冷丙酮固定细胞、200μl/孔,室温条件下固定30分钟。PBST洗板,100μl/孔,洗板3次,每次3分钟,随后将孔内液体拍净。将杂交瘤细胞培养上清进行2倍倍比稀释,50倍、100倍、200倍、400倍···819200倍,共15个稀释梯度,依次加到96孔板中,50μl/孔,37℃温箱孵育1小时。PBST洗板,100μl/孔,洗板3次,每次3分钟,随后将孔内液体拍净。二抗为FITC标记兔抗鼠IgG,二抗稀释液(FITC标记兔抗鼠IgG 1:200倍稀释,伊文思蓝1:300倍稀释),加到96孔板细胞内,37℃温箱孵育1小时。PBST洗板,100μl/孔,洗板3次,结果显示1A4和1B2均能与CDV毒株特异性结合。通过的倍比稀释间接免疫荧光结果如图4所示,抗体稀释6400倍时,仍然可以检测到荧光信号。
2.免疫印迹实验:将CPV-PS野毒株加入5×loading buffer,煮沸10min,进行12%SDS-PAGE电泳;通过半干转膜仪在300mA的恒流下转膜1h,将蛋白转移至NC膜上;将PVDF膜封闭:5%脱脂乳封闭过夜,或者37℃2h;一抗孵育1h,PBS洗3遍,每次5min;二抗孵育0.5h,PBS洗3遍,每次5min;配制好DAB显色液,将PVDF膜放入,避光显色5-8min。结果如图5显示1A4和1B2均能与CDV毒株特异性结合,结合大小显示其约为63kDa,与犬瘟热病毒F蛋白大小类似,可推断其为抗犬瘟热F蛋白的单克隆抗体。
3.犬瘟热配对单抗1A4/1B2制备检测CDV抗原的胶体金试纸条:
以PVC背板为支撑物,在其上分别贴有样品垫1、金标垫2、硝酸纤维膜5、吸水垫6,其中金标垫采用聚酯膜,经含有1%BSA和1%吐温-20的PBS处理后,将制备好的胶体金标记犬瘟热病毒单抗1A4按照40μL/30cm喷涂见图6的示意图金标垫2部分,完毕,37℃烘干2h。硝酸纤维膜5上包被有两条线:分别为质控线4和检测线3,其中检测线包被物为犬瘟热病毒单抗1B2浓度为1.6mg/mL,质控线包被物为纯化的兔抗鼠抗体浓度为2.1mg/mL,然后用Biodot划膜仪喷涂于硝酸纤维膜上,组装好后大板用切条机切成7-8mm的裸条备用。
4.犬瘟热病毒试纸条检测能力检验:为了检测犬瘟热病毒试纸条检测能力,我们将CDV原液的滴度为1×105TCID50/ml进行10倍倍比稀释,分别使用试纸条和PCR的方法进行验证,结果如图7显示,本试纸条基本能检测100倍稀释的CDV病毒,约为103TCID50/ml,而PCR能检测到101TCID50/ml。
三、单克隆抗体1A4和1B2的氨基酸序列
选取杂交瘤细胞的总RNA,采用逆转录试剂盒得到cDNA,以cDNA为模板扩增1A4和1B2的可变区CDR的基因,并经过测序结果进行氨基酸翻译分析。
结果表明,1A4重链的可变区的CDR-H1的氨基酸序列如SEQ ID NO:1所示,对应编码的核酸序列为ggcaccagcggctttacctgg如SEQ ID NO:13所示;CDR-H2的氨基酸序列如SEQID NO:2所示,对应编码的核酸序列为aacccgagcagcggctatattttt如SEQ ID NO:14所示;CDR-H3的氨基酸序列如SEQ ID NO:3所示,对应编码的核酸序列为gcgagcggccgctggggcagccatgtggatttt如SEQ ID NO:15所示;所述抗体1A4轻链的可变区的CDR-L1的氨基酸序列如SEQ ID NO:4所示,对应编码的核酸序列为ctggtggcgccgaccggcatgagc如SEQ ID NO:16所示;CDR-L2的氨基酸序列如SEQ ID NO:5所示,对应编码的核酸序列为gcgaccgcgagcgcg如SEQ ID NO:17所示;CDR-L3的氨基酸序列如SEQ ID NO:6所示,对应编码的核酸序列为gcgggccgccagagctatattgataaaagcagcaccaccgcgtat如SEQ ID NO:18所示;
抗体1B2重链的可变区的CDR-H1的氨基酸序列如SEQ ID NO:7所示,对应编码的核酸序列为aaaagccagaccattctg如SEQ ID NO:19所示;CDR-H2的氨基酸序列如SEQ ID NO:8所示,对应编码的核酸序列为catccggtggcggcgtatgattat如SEQ ID NO:20所示;CDR-H3的氨基酸序列如SEQ ID NO:9所示,对应编码的核酸序列为ccggataacgtgaaagataacagctataccattagcacc如SEQ ID NO:21所示;所述抗体1B2轻链的可变区的CDR-L1的氨基酸序列如SEQ ID NO:10所示,对应编码的核酸序列为cagagcaaaaccaacttt如SEQ ID NO:22所示;CDR-L2的氨基酸序列如SEQ ID NO:11所示,对应编码的核酸序列为tggagcgcgccgacc如SEQID NO:23所示;CDR-L3的氨基酸序列如SEQ ID NO:12所示,对应编码的核酸序列为ggcgcgcgcggcgatatggaaaaaagcgaaaccaactatagcattgcg如SEQ ID NO:24所示。
SEQUENCE LISTING
<110> 中国农业科学院特产研究所
<120> 一种用于检测犬瘟热病毒的抗体对及其应用
<130>
<160> 24
<170> PatentIn version 3.5
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<213> 1A4重链的可变区的CDR-H1的氨基酸序列
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Gly Thr Ser Gly Phe Thr Trp
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Gly Ala Arg Gly Asp Met Glu Lys Ser Glu Thr Asn Tyr Ser Ile Ala
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ggcaccagcg gctttacctg g 24
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catccggtgg cggcgtatga ttat 24
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<213> 1B2的CDR-L3的基因序列
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ggcgcgcgcg gcgatatgga aaaaagcgaa accaactata gcattgcg 48
Claims (7)
1.一种用于检测犬瘟热病毒的抗体对,其特征在于,所述抗体对为1A4和1B2,所述抗体1A4重链的可变区的CDR-H1的氨基酸序列如SEQ ID NO:1所示,CDR-H2的氨基酸序列如SEQID NO:2所示,CDR-H3的氨基酸序列如SEQ ID NO:3所示;所述抗体1A4轻链的可变区的CDR-L1的氨基酸序列如SEQ ID NO:4所示,CDR-L2的氨基酸序列如SEQ ID NO:5所示,CDR-L3的氨基酸序列如SEQ ID NO:6所示;所述抗体1B2重链的可变区的CDR-H1的氨基酸序列如SEQID NO:7所示,CDR-H2的氨基酸序列如SEQ ID NO:8所示,CDR-H3的氨基酸序列如SEQ IDNO:9所示;所述抗体1B2轻链的可变区的CDR-L1的氨基酸序列如SEQ ID NO:10所示,CDR-L2的氨基酸序列如SEQ ID NO:11所示,CDR-L3的氨基酸序列如SEQ ID NO:12所示。
2.根据权利要求1所述的抗体对,其特征在于,所述抗体1A4的重链可变区的CDR-H1的基因序列如SEQ ID NO:13所示,所述抗体1A4的重链可变区的CDR-H2的基因序列如SEQ IDNO:14所示,所述抗体1A4的重链可变区的CDR-H3的基因序列如SEQ ID NO:15所示。
3.根据权利要求1所述的抗体对,其特征在于,所述抗体1A4的轻链可变区的CDR-L1的基因序列如SEQ ID NO:16所示,所述抗体1A4的轻链可变区的CDR-L2的基因序列如SEQ IDNO:17所示,所述抗体1A4的轻链可变区的CDR-L3的基因序列如SEQ ID NO:18所示。
4.根据权利要求1所述的抗体对,其特征在于,所述抗体1B2的重链可变区的CDR-H1的基因序列如SEQ ID NO:19所示,所述抗体1B2的重链可变区的CDR-H2的基因序列如SEQ IDNO:20所示,所述抗体1B2的重链可变区的CDR-H3的基因序列如SEQ ID NO:21所示。
5.根据权利要求1所述的抗体对,其特征在于,所述抗体1B2的轻链可变区的CDR-L1的基因序列如SEQ ID NO:22所示,所述抗体1B2的轻链可变区的CDR-L2的基因序列如SEQ IDNO:23所示,所述抗体1B2的轻链可变区的CDR-L3的基因序列如SEQ ID NO:24所示。
6.根据权利要求1所述的抗体对,其特征在于,所述抗体1A4为捕捉抗体,所述抗体1B2为检测抗体。
7.权利要求1-6任意一项所述的抗体对在制备检测犬瘟热病毒的试剂盒中的应用。
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