CN116769021A - 一种针对非洲马瘟病毒vp7蛋白的单克隆抗体及用途 - Google Patents
一种针对非洲马瘟病毒vp7蛋白的单克隆抗体及用途 Download PDFInfo
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Abstract
本发明属于免疫学和体外诊断技术领域,具体涉及一种针对非洲马瘟病毒VP7蛋白的单克隆抗体及制备方法。本发明通过将重组表达的非洲马瘟病毒VP7蛋白免疫BALB/c小鼠,筛选获得一种针对非洲马瘟病毒VP7蛋白的单克隆抗体,并通过反转录PCR技术成功获得了编码该株抗体重链及轻链可变区的核苷酸序列。所述单克隆抗体与重组表达的VP7蛋白具有良好的亲和反应能力。利用该单克隆抗体建立了非洲马瘟病毒竞争ELISA抗体检测方法,为有效防控非洲马瘟传入我国提供了技术基础。
Description
技术领域
本发明属于免疫学和体外诊断技术领域,具体涉及一种针对非洲马瘟病毒VP7蛋白的单克隆抗体及制备方法与用途。
背景技术
非洲马瘟是由非洲马瘟病毒(African horse sickness virus,AHSV)引起马、驴、骡等马属动物的一种非接触性的急性或亚急性传染病。马对非洲马瘟最易感,幼龄马的易感性最高,驴、骡次之,斑马感染后一般无症状,可能是其储存宿主,在该病的传播过程中扮演重要角色。该病主要通过库蠓叮咬传播。非洲马瘟的临床症状可分为4种类型,肺型:潜伏期为3~5天,典型特征表现为呼吸道症状,鼻孔有泡沫样分泌物,可在出现临床症状的数小时内死亡。心型:最初表现为发热,随后出现面颊、眼眶、颈部和胸部等部位水肿,最后心力衰竭而亡。混合型:肺型和心型混和存在,常于死后剖检发现。发热型:临床症状不明显,主要存在于驴和斑马等动物。世界动物卫生组织(WOAH)将其列为必须通报的疫病,我国将其列为一类动物疫病。
非洲马瘟病毒属于呼肠孤病毒科环状病毒属,其基因组由10个大小不同的双链RNA片段组成,共编码7种结构蛋白(VP1-VP7)和4种非结构蛋白(NS1-NS4),其中VP2是血清型特异性抗原,而VP7蛋白是血清群特异性抗原。已发现非洲马瘟病毒至少存在9个血清型,基于VP7蛋白的ELISA方法可用于检测非洲马瘟病毒群特异性抗体,适合大规模样品的检测和流行病学调查。
目前检测非洲马瘟抗体的方法主要包括竞争ELISA、病毒中和试验、琼脂凝胶免疫扩散反应和免疫荧光试验。目前国际上通用的是竞争ELISA方法,然而竞争抗体是该试剂盒的关键成分,本发明筛选获得一种针对AHSV VP7蛋白的单克隆抗体及其重链和轻链可变区序列,并利用该抗体作为竞争抗体建立了AHSV竞争ELISA抗体检测方法。
发明内容
针对上述技术问题,本发明提供了一种针对非洲马瘟病毒VP7蛋白的单克隆抗体,所述的单克隆抗体具有效价高、亲和力强、特异性好的特点,能够特异性结合非洲马瘟病毒VP7蛋白。具体包括以下内容:
第一方面,本发明提供了一种针对非洲马瘟病毒VP7蛋白的单克隆抗体,所述单克隆抗体包括抗体重链和抗体轻链;
所述单克隆抗体重链的可变区CDR包括氨基酸序列如SEQ ID NO.5所示的CDR1、氨基酸序列如SEQ ID NO.6所示的CDR2和氨基酸序列如SEQ ID NO.7所示的CDR3;
所述单克隆抗体轻链的可变区CDR包括氨基酸序列如SEQ ID NO.8所示的CDR1、氨基酸序列如SEQ ID NO.9所示的CDR2和氨基酸序列如SEQ ID NO.10所示的CDR3。
优选地,所述抗体重链的可变区的氨基酸序列如SEQ ID NO.1所示,所述抗体轻链的可变区的氨基酸序列如SEQ ID NO.3所示。
第二方面,本发明提供了一种核酸,所述核酸编码上述第一方面所述单克隆抗体的抗体重链和抗体轻链。
优选地,编码所述抗体重链的可变区的基因序列如SEQ ID NO.2所示,编码所述抗体轻链的可变区的基因序列如SEQ ID NO.4所示。
第三方面,本发明提供了上述第一方面所述的单克隆抗体在制备检测非洲马瘟病毒的试剂,或试纸条,或试剂盒中的应用。
第四方面,本发明提供了一种免疫偶联物,其特征在于,所述免疫偶联物包括:
(i)上述第一方面所述的单克隆抗体;
(ii)和选自下组的偶联部分:可检测标记物、药物、金纳米颗粒/纳米棒、纳米磁粒、病毒外壳蛋白或VLP、或其组合。
第五方面,本发明提供了一种非洲马瘟病毒ELISA检测试剂盒,其特征在于,所述试剂盒包括上述第一方面所述的单克隆抗体。
优选地,所述试剂盒包括酶标板、非洲马瘟病毒抗原、封闭液、稀释液、权利要求1或2所述的单克隆抗体、酶标二抗、洗涤液、显色剂、终止液。
优选地,所述非洲马瘟病毒抗原选自非洲马瘟病毒VP7重组蛋白。
优选地,所述封闭液为含5%脱脂奶粉的PBST缓冲液;
优选地,所述稀释液为0.01M PBS pH7.2;
优选地,所述洗涤液为PBST缓冲液。
优选地,所述酶标二抗为HRP标记的山羊抗鼠二抗。
第六方面,本发明提供了一种上述第一方面所述的单克隆抗体在以非疾病诊断为目的非洲马瘟病毒体外检测中的应用。
第七方面,本发明提供了一种以非疾病诊断为目的非洲马瘟病毒ELISA检测方法,所述方法包括以下步骤:
(1)包被:用CBS缓冲液将表达的AHSV VP7重组蛋白稀释为0.5μg/mL,100μL/孔包被到酶标板中,4℃包被过夜;用PBST缓冲液洗板5次;所述CBS缓冲液为0.05M碳酸盐-碳酸氢盐缓冲液,pH9.6;
(2)封闭:用含5%脱脂奶粉的PBST缓冲液封闭酶标板,200μL/孔,37℃孵育1h;用PBST缓冲液洗板5次;
(3)检测:将非洲马瘟阳性血清、标准阳性血清和标准阴性血清加入到酶标板内,100μl/孔,37℃孵育1h,用PBST缓冲液洗板5次;
(4)然后加入1:2000稀释的上述第一方面所述的单克隆抗体,37℃孵育1h,用PBST缓冲液洗板5次;所述稀释液为0.01M PBS pH7.2;
(5)加酶标二抗:将1:40000稀释的HRP标记的山羊抗鼠二抗加入到酶标板中,100μL/孔,37℃孵育1h;用PBST缓冲液洗板5次;所述稀释液为0.01M PBS pH7.2;
(6)显色:用TMB显色液避光显色,100μL/孔,37℃孵育10min;加入终止液100μl/孔,读取OD450的值;
判定方法:阻断率(BP)=(阴性对照OD450-样品OD450)/(阴性对照OD450-阳性对照OD450)×100%;阳性对照小于0.2,阴性对照大于2.0检测方法成立;当BP≤45%时,结果为阴性;当BP≥50%时,结果为阳性;当45%<BP<50%时,结果可疑。
本发明的有益效果是:①本发明提供了一种抗非洲马瘟病毒VP7蛋白的单克隆抗体;②免疫印迹试验表明其特异性的和结构蛋白VP7反应;③而且所述单克隆抗体能特异性的与感染细胞中的非洲马瘟病毒结合,能够用于非洲马瘟病毒的检测与诊断;④本发明利用所述单克隆抗体建立了非洲马瘟病毒ELISA检测试剂盒及检测方法,相较于现有抗体检测方法,本发明所述方法具有更好的灵敏度和特异性,适用于非洲马瘟病毒的检测与诊断。
附图说明
图1抗AHSV VP7单克隆抗体分子量及纯度的鉴定;
图2抗AHSV VP7单克隆抗体的免疫荧光检测;
图3抗AHSV VP7单克隆抗体的免疫印迹检测。
具体实施方式
下面详细描述本发明的实施例,需要说明的是下面描述的实施例是示例性的,仅用于解释本发明,而不能理解为对本发明的限制。另外,如果没有明确说明,在下面的实施例中所采用的所有试剂均为市场上可以购得的,或者可以按照文本或已知的方法合成的,对于没有列出的反应条件,均为本领域技术人员容易获得的。
实施例1针对非洲马瘟病毒VP7蛋白单克隆抗体的制备
1、小鼠免疫
取6只6-8周龄雌性BALB/c小鼠,用表达的AHSV VP7重组蛋白对其进行免疫。重组VP7蛋白与等量的弗氏完全佐剂乳化,背部皮下注射,50μg/只。初次免疫后第14天、28天用等量的弗氏不完全佐剂与VP7重组蛋白充分乳化,皮下注射。利用间接ELISA检测小鼠的血清抗体效价,当抗体水平达到要求后,对小鼠进行加强免疫,免疫3d后无菌采取小鼠脾细胞,然后与SP2/0细胞融合。
2、细胞融合
将准备好的SP2/0细胞和小鼠脾细胞按照1:5的比例混合,加入20mL不完全培养基,1000rpm/min离心10min,弃去上清。用手指轻轻击打离心管底部,使沉淀的细胞分散,在37℃的水浴中,用1mL 37℃预温的50% PEG进行细胞的融合,边加边轻轻搅拌,静止1min,用20mL的DMEM培养基终止融合,1000rpm离心5min,用HAT培养基重悬融合细胞,接种于96孔细胞培养板中,将96孔板放在37℃5% CO2培养箱进行培养。
3、杂交瘤细胞的筛选和亚克隆
融合后前7天用HAT培养基,第7-14天用HT培养基,第14天以后用普通的完全培养基,当克隆的面积达到1/3-1/2培养孔的面积,此时即对所有克隆生长孔的培养基进行检测。对检测出特异性抗体阳性孔的细胞,及时进行克隆化。亚克隆采用倍比稀释法,亚克隆7天后,再利用间接ELISA的方法检测培养上清,选取细胞团单一的阳性孔继续进行亚克隆,重复次过程3次,筛选出稳定的杂交瘤细胞株。
间接ELISA检测的具体方法为:
(1)包被:用CBS缓冲液(0.05M碳酸盐-碳酸氢盐缓冲液,pH9.6)将AHSV VP7重组蛋白稀释成0.5μg/mL、100μL/孔包被到酶标板中,4℃包被过夜。用PBST缓冲液洗板5次。
(2)封闭:用含5%脱脂奶粉的PBST缓冲液封闭酶标板,200μL/孔,37℃孵育1h。用PBST缓冲液洗板5次。
(3)检测:将待检测的杂交瘤细胞培养上清加入到酶标板中,100μL/孔,同时用正常细胞的细胞培养上清做阴性对照,37℃孵育1h,用PBST缓冲液洗板5次。
(4)加酶标二抗:将1:40000稀释(稀释液为0.01M PBS pH7.2)的HRP标记的山羊抗鼠二抗加入到酶标板中,100μL/孔,37℃孵育1h。用PBST缓冲液洗板5次。
(5)显色:用TMB显色液避光显色,100μL/孔,37℃孵育10min;加入终止液100μL/孔,读取OD450的值。
OD450的值结果如表1所示,结果表明,该杂交瘤细胞(命名为1H4细胞株)与重组VP7蛋白的反应性良好。
表1OD450检测结果
组别 | OD450 |
1H4细胞株 | 3.58005 |
阴性对照 | 0.0757 |
4、小鼠腹水抗体的制备
准备2只6-8周龄雌性BALB/c小鼠,腹腔注射液体石蜡,0.5mL/只。7天后,接种106个步骤3获得的杂交瘤细胞,观察小鼠,约7-10天观察到小鼠的腹部明显膨大,用手触摸时,皮肤有紧张感,即可抽取腹水。将收集的腹水离心,除去沉淀,收集上清。然后利用ProteinG柱对腹水进行纯化获得高效价和高纯度的1H4单克隆抗体。纯化后的单克隆抗体浓度为2mg/mL。
具体纯化步骤:
(1)层析柱预处理:10倍柱体积的去离子水冲洗3-5遍,再用10倍柱体积的PBS冲洗3-5遍。
(2)上样:腹水用PBS稀释后,022μL滤膜过滤。
(3)洗杂:PBS冲洗至检测无蛋白流出为止(G250不变蓝)。
(4)抗体洗脱:0.1M PH3.0甘氨酸洗脱,收集洗脱物并用G250检测洗脱产物至不变蓝。
(5)PH值调节:饱和碳酸钠调节洗脱产物PH至中性。
(6)样品浓缩:10kDa超滤管,超滤浓缩至1-5mL左右。
实施例2抗非洲马瘟病毒VP7蛋白单克隆抗体的鉴定
1、抗AHSV VP7单克隆抗体分子量及纯度的鉴定
采用SDS-PAGE法进行抗体分子量及纯度鉴定;将实施例1中纯化后的单克隆抗体制样,然后进行SDS-PAGE,每孔上样10μL,电泳完毕后,用考马斯亮蓝溶液染色1h,然后脱色。结果如图1所示。结果表明,单克隆抗体纯化成功,纯度大于95%,可满足应用需求。
2、抗AHSV VP7单克隆抗体效价的测定
利用间接ELISA测定单克隆抗体的效价,用AHSV VP7重组蛋白作为包被抗原,浓度为0.5μg/mL,100μL/孔,4℃过夜包被;PBST洗板3次;加入5%脱脂奶粉进行封闭,200μL/孔,37℃孵育2h,PBST洗板3次;分别加入100μL倍比稀释(稀释液为0.01M PBS pH7.2)的待测样品,37℃孵育1h,PBST洗板3次;加入1:10000稀释(稀释液为0.01M PBS pH7.2)的HRP标记的山羊抗鼠二抗,100μL/孔,37℃孵育40min,PBST洗板5次;加入底物溶液90μL/孔,37℃避光显色10-15min,加入终止液50μL/孔。读取每孔的OD450值,按阳性抗体与阴性抗体比值(P/N)大于2.1判定。结果如表2所示,该单克隆抗体的效价可达1:1024000,即其稀释浓度为1.953ng/mL。
表2抗AHSV VP7单克隆抗体效价的测定的OD450值
一抗浓度 | OD450 |
1ug/mL(1:2000) | 4.0883 |
0.5ug/mL(1:4000) | 2.8676 |
0.25ug/mL(1:8000) | 2.9268 |
0.125ug/mL(1:16000) | 2.709 |
62.5ng/mL(1:32000) | 2.0886 |
31.25ng/mL(1:64000) | 1.4443 |
15.625ng/mL(1:128000) | 0.9321 |
7.813ng/mL(1:256000) | 0.5376 |
3.906ng/mL(1:512000) | 0.2770 |
1.953ng/mL(1:1024000) | 0.1695 |
阴性抗体 | 0.0378 |
3、抗AHSV VP7单克隆抗体的免疫荧光检测
用制备的抗AHSV VP7单克隆抗体对转染了表达AHSV VP7的重组质粒(pcDNA3.1-AHSV VP7,采用常规手段即可制备)的BSR细胞进行免疫荧光分析。将BSR细胞进行爬片,用脂质体3000将重组质粒pcDNA3.1-AHSV VP7转染到BSR细胞内,37℃,培养24h,弃去细胞培养上清,用4%多聚甲醛固定10min,PBS洗3次,再用0.5% Triton X-100作用10min,PBS洗3次后,用3% BSA封闭1h,吸弃旧液后,加入实施例1制备的抗AHSV VP7单克隆抗体(1:3000),4℃过夜孵育,然后用Hoechst 33342染核10min,PBS洗3次,加入山羊抗鼠IgG(Alexa488)室温避光孵育1h,封片后观察并收集图像。
检测结果如图2所示,转染了重组质粒pcDNA3.1-AHSV VP7的BSR细胞显示绿色荧光,而转染空载体pcDNA3.1的细胞未出现荧光,结果表明抗AHSV VP7单克隆抗体能够和真核细胞表达的VP7蛋白特异性结合。
4、抗AHSV VP7单克隆抗体的免疫印记检测
用表达的AHSV VP7重组蛋白制样进行免疫印迹,一抗用实施例1制备的抗AHSVVP7单克隆抗体(1:3000),室温孵育2h,PBST洗3次,每次10分钟,二抗用HRP标记的山羊抗鼠(1:10000),室温孵育1h,PBST洗3次,最后显色观察。
结果如图3所示,在免疫印迹实验中本申请制备的抗AHSV VP7单克隆抗体能够和重组VP7蛋白发生特异性反应。
5、单克隆抗体重链和轻链可变区基因的克隆及分析
(1)单克隆抗体重链和轻链可变区的扩增
将实施例1制备的分泌抗AHSV VP7单克隆抗体的1H4杂交瘤细胞用TRIzol裂解法提取其总RNA,然后通过反转录合成cDNA,然后以cDNA为模板进行PCR扩增,以获得抗体的重链和轻链可变区核苷酸序列。
测序结果显示,抗AHSV VP7单克隆抗体的重链可变区的核苷酸序列为:GAGGTGCAGTTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGGGTCCCTGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGGTATGGCATGTCTTGGGTTCGCCAGACTCCAGACAAGAGGCTGGAGTTGGTCGCAATAATAAAAAATGATGGTCGTAGTTCCTATTATCCAGACAGTGTGAAGGGCCGATTCACCATCTACAGAGACAATGCCAAGAACACCCTGTACCTGCAAATGAGCACTCTGAAGTCTGAGGACACAGCCATGTATCACTGTGCCTACGGCAGTAGCCTCTACTGGTACTTCGATGTCTGGGGCGCAGGGACCACGGTCACCGTCTCCTCA(SEQ ID NO.2所示);
抗AHSV VP7单克隆抗体重链可变区的氨基酸序列为:EVQLVESGGGLVQPGGSLKLS CAASGFTFSRYGMSWVRQTPDKRLELVAIIKNDGRSSYYPDSVKGRFTIYRDNAKNTLYLQ MSTLKSEDTAMYHCAYGSSLYWYFDVWGAGTTVTVSS(SEQ ID NO.1所示);
抗AHSV VP7单克隆抗体轻链可变区的核苷酸序列为:GATGTTTTGATGACCCAAACT CCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGAGCATTGTACATAGTAATGGAAACACCTATTTAGAATGGTACCTGCAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGCCAGGTTCAGTGGCAGTGGATCAGGGACAGGTTTCACACTCAAGATCAGCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAAGGTTCACATGTTCCTCCGACGTTCGGTGGAGGCACCAAACTGGAAATCAAA(SEQ ID NO.4所示);
抗AHSV VP7单克隆抗体轻链可变区氨基酸序列为:DVLMTQTPLSLPVSLGDQASISC RSSQSIVHSNGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPARFSGSGSGTGFTLKISRVEAEDLGVYYCFQGSHVPPTFGGGTKLEIK(SEQ ID NO.3所示)。
(2)CDR区的确定
将测序所得的抗AHSV VP7单克隆抗体的重链和轻链可变区的序列进行分析,重链可变区的3个CDR区氨基酸序列分别为:
CDR1:RYGMS(SEQ ID NO.5所示);
CDR2:IIKNDGRSSYYPDSVKG(SEQ ID NO.6所示);
CDR3:GSSLYWYFDV(SEQ ID NO.7所示);
轻链可变区的3个CDR区氨基酸序列分别为:
CDR1:RSSQSIVHSNGNTYLE(SEQ ID NO.8所示);:
CDR2:KVSNRFS(SEQ ID NO.9所示);
CDR3:FQGSHVPPT(SEQ ID NO.10所示)。
实施例3基于抗AHSV VP7单克隆抗体的非洲马瘟竞争ELISA诊断方法的建立
把制备的非洲马瘟VP7重组蛋白4℃包被过夜,浓度为0.5μg/mL,100μl/孔。非洲马瘟标准阳性血清和标准阴性血清由本实验室保存,制备的抗AHSV VP7单克隆抗体作为竞争抗体建立诊断方法。
具体检测方法:
(1)包被:用CBS缓冲液(0.05M碳酸盐-碳酸氢盐缓冲液,pH9.6)将表达的AHSV VP7重组蛋白稀释为0.5μg/mL,100μL/孔包被到酶标板中,4℃包被过夜;用PBST缓冲液洗板5次。
(2)封闭:用含5%脱脂奶粉的PBST缓冲液封闭酶标板,200μL/孔,37℃孵育1h;用PBST缓冲液洗板5次。
(3)检测:将非洲马瘟阳性血清、标准阳性和标准阴性血清加入到酶标板内,100μl/孔,37℃孵育1h,用PBST缓冲液洗板5次。
(4)然后加入1:2000稀释(0.01M PBS pH7.2)的实施例1制备的抗AHSV VP7单克隆抗体,37℃孵育1h,用PBST缓冲液洗板5次。
(5)加酶标二抗:将1:40000稀释(0.01M PBS pH7.2)的HRP标记的山羊抗鼠二抗加入到酶标板中,100μL/孔,37℃孵育1h;用PBST缓冲液洗板5次。
(6)显色:用TMB显色液避光显色,100μL/孔,37℃孵育10min;加入终止液100μl/孔,读取OD450的值。
判定方法:阻断率(BP)=(阴性对照OD450-样品OD450)/(阴性对照OD450-阳性对照OD450)×100%;阳性对照小于0.2,阴性对照大于2.0,检测方法成立。当BP≤45%时,结果为阴性;当BP≥50%时,结果为阳性;当45%<BP<50%时,结果可疑。
1、敏感性实验
把非洲马瘟阳性血清按照1:2、1:4、1:8、1:16进行连续稀释,以评估非洲马瘟竞争ELISA检测方法的灵敏度。结果如表3所示,当血清样品进行1:16稀释时,检测结果仍为阳性,表明,利用本申请所述的单克隆抗体1H4建立的非洲马瘟竞争ELISA检测方法具有良好的敏感性。
表3非洲马瘟竞争ELISA检测方法的敏感性结果
OD450 | BP | |
1:2 | 0.2 | 99.4% |
1:4 | 0.487 | 88.3% |
1:8 | 0.881 | 73.1% |
1:16 | 1.23 | 59.8% |
2、符合率实验
用本实施例建立的竞争ELISA实验方法检测本实验室保存的AHSV阳性血清5份(1-5)和AHSV阴性血清5份(6-10),结果如表4所示,表明,利用本申请所述的单克隆抗体1H4建立的非洲马瘟竞争ELISA方法检测结果的符合率达到100%。
表4非洲马瘟竞争ELISA检测方法的符合率结果
样品 | OD450 | BP |
1 | 0.5452 | 84.2% |
2 | 0.3182 | 93.1% |
3 | 0.1554 | 99.5% |
4 | 0.3167 | 94.9% |
5 | 1.1153 | 64.1% |
6 | 2.2916 | 18.7% |
7 | 2.2963 | 18.5% |
8 | 2.7539 | 0.8% |
9 | 2.216 | 21.6% |
10 | 2.6319 | 5.6% |
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。
Claims (10)
1.一种特异性结合非洲马瘟病毒VP7蛋白的单克隆抗体,其特征在于,所述单克隆抗体包括抗体重链和抗体轻链;
所述抗体重链的可变区CDR包括氨基酸序列如SEQ ID NO.5所示的CDR1、氨基酸序列如SEQ ID NO.6所示的CDR2和氨基酸序列如SEQ ID NO.7所示的CDR3;
所述抗体轻链的可变区CDR包括氨基酸序列如SEQ ID NO.8所示的CDR1、氨基酸序列如SEQ ID NO.9所示的CDR2和氨基酸序列如SEQ ID NO.10所示的CDR3。
2.如权利要求1所述的单克隆抗体,其特征在于,所述抗体重链的可变区的氨基酸序列如SEQ ID NO.1所示,所述抗体轻链的可变区的氨基酸序列如SEQ ID NO.3所示。
3.一种核酸,其特征在于,所述核酸编码权利要求1或2所述单克隆抗体的抗体重链和抗体轻链。
4.如权利要求3所述的核酸,其特征在于,编码所述抗体重链的可变区的基因序列如SEQ ID NO.2所示,编码所述抗体轻链的可变区的基因序列如SEQ ID NO.4所示。
5.如权利要求1或2所述的单克隆抗体在制备检测非洲马瘟病毒的试剂,或试纸条,或试剂盒中的应用。
6.一种免疫偶联物,其特征在于,所述免疫偶联物包括:
(i)如权利要求1或2所述的单克隆抗体;
(ii)和选自下组的偶联部分:可检测标记物、药物、金纳米颗粒/纳米棒、纳米磁粒、病毒外壳蛋白或VLP、或其组合。
7.一种非洲马瘟病毒ELISA检测试剂盒,其特征在于,所述试剂盒包括权利要求1或2所述的单克隆抗体。
8.如权利要求7所述的ELISA检测试剂盒,其特征在于,所述试剂盒包括酶标板、非洲马瘟病毒抗原、封闭液、稀释液、权利要求1或2所述的单克隆抗体、酶标二抗、洗涤液、显色剂、终止液。
9.如权利要求7所述的ELISA检测试剂盒,其特征在于,所述非洲马瘟病毒抗原选自非洲马瘟病毒VP7重组蛋白。
10.如权利要求1或2所述的单克隆抗体在以非疾病诊断为目的的非洲马瘟病毒体外检测中的应用。
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