CN110144007B - 抗h7n9禽流感病毒血凝素蛋白单克隆抗体zju79-01及其应用 - Google Patents
抗h7n9禽流感病毒血凝素蛋白单克隆抗体zju79-01及其应用 Download PDFInfo
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- CN110144007B CN110144007B CN201910420945.1A CN201910420945A CN110144007B CN 110144007 B CN110144007 B CN 110144007B CN 201910420945 A CN201910420945 A CN 201910420945A CN 110144007 B CN110144007 B CN 110144007B
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- monoclonal antibody
- avian influenza
- influenza virus
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- hemagglutinin protein
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Abstract
本发明属于生物技术领域,涉及抗H7N9禽流感病毒血凝素蛋白单克隆抗体的制备及应用。一种抗H7N9禽流感病毒血凝素蛋白单克隆抗体ZJU79‑01,抗体的重链氨基酸序列如SEQ ID No.1,轻链氨基酸序列如SEQ ID No.2所示,该单克隆抗体亚型为IgG1、κ型,能与禽流感病毒血凝素蛋白抗原特异结合。本发明的优点在于提供了一种抗H7N9禽流感病毒血凝素蛋白的单克隆抗体。制备方法简单易行,更为重要的是该方法制备的单克隆抗体可以有多种用途,如对临床和实验室的H7N9禽流感样本进行定性诊断,而且可以快速便捷的进行检测。
Description
技术领域
本发明属于生物技术领域,涉及抗H7N9禽流感病毒血凝素蛋白单克隆抗体的制备及应用,是利用细胞工程、抗体工程技术,获得分泌抗血凝素蛋白的单克隆抗体的杂交瘤细胞系,通过同品系的小鼠诱导腹水,制备抗血凝素蛋白的单克隆抗体ZJU79-01,鉴定为IgG1、κ型,再通过亲和纯化、电泳、免疫等技术实现对该抗体的应用。
背景技术
新型H7N9禽流感于2013年3月底在中国上海和安徽率先发现,到目前为止,H7N9禽流感在中国已出现5波疫情,并且在加拿大、马来西亚和澳大利亚也有人感染H7N9的报道。值得令人注意的是,从2016年10月开始的第五波H7N9疫情中观察到感染人数急剧增加,感染范围向西部省份蔓延,以及出现人感染高致病性H7N9禽流感病例。H7N9禽流感对人的致病性强和死亡率高,感染者有一半以上会发生重症肺炎,且死亡率高达30%以上。其次,该病毒对家禽致病性弱,家禽患病不易被发现,从而不能做到对人的及时防护处理。目前治疗H7N9禽流感方法主要是神经氨酸酶抑制剂(奥司他韦)。而早期治疗成为关键,在症状出现超过5天后才开始抗病毒治疗时,死亡风险增加。因此,快速而准确的诊断对降低病死率至关重要。
目前H7N9感染的诊断方法主要包括病毒分离鉴定、血清学方法和分子生物学方法,如逆转录酶聚合酶链反应和实时定量聚合酶链反应。但是上述方法都需要特殊设备和条件,对于某些条件落后的地区并不适合。因而开发一种快速、灵敏、廉价的H7N9病毒检测产品势在必行,这能促进更早更广泛的发现H7N9感染,减少重症病例发生,降低病死率。
综上所述,开发H7N9禽流感病毒单克隆抗体,用于快速灵敏检测方法的建立迫在眉睫。基于以上背景,本项目选定血凝素蛋白为靶抗原,采用融合杂交瘤技术建立稳定分泌抗血凝素蛋白单克隆抗体的杂交瘤细胞系,并大量制备、纯化和鉴定这些单克隆抗体。该单克隆抗体的成功获得,为建立新型的H7N9禽流感病毒病诊断方法——基于免疫学技术的诊断奠定物质基础。同时对疾病发病机理、预后及疗效判定等方面的研究起到重要作用。
本发明用到杂交瘤细胞技术。该技术将免疫小鼠的B淋巴细胞与骨髓瘤细胞融合,以建立分泌均质抗体的杂交瘤细胞系,也称为单克隆抗体技术。该技术涉及到动物免疫、细胞培养、细胞融合、细胞克隆培养和免疫测定等一系列方法。
发明内容
为了解决以上问题,本发明提供了一种可以快速灵敏检测病毒的抗H7N9禽流感病毒血凝素蛋白单克隆抗体。
本发明的目的是提供一种抗H7N9禽流感病毒血凝素蛋白单克隆抗体,能识别H7N9禽流感病毒。该单克隆抗体亚型为IgG1、κ型,命名为ZJU79-01,能特异性识别禽流感病毒的血凝素蛋白。
一种抗H7N9禽流感病毒血凝素蛋白单克隆抗体ZJU79-01,抗体的重链氨基酸序列如SEQ ID No.2(DNA序列如SEQ ID No.1),轻链氨基酸序列如SEQ ID No.4(DNA序列如SEQID No.3)所示,该单克隆抗体亚型为IgG1、κ型,能与禽流感病毒血凝素蛋白抗原特异结合。
SEQ ID No.1
Heavy chain:DNA sequence(417bp)
Signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
ATGAAATGCAGCTGGGTCATCTTCTTCCTGATGGCAGTGGTTACAGGGGTCAATTCAGAGGTTCAGCTGCAGCAGTCTGGGGCTGAACTTGTGAGGCCAGGGGCCTTAGTCAAGTTGTCCTGCAAAGCTTCTGGCTTCAGCTTTAAAGACTACTTTATGCACTGGGTGAGGCAGAGGCCTGAACAGGGCCTGGAGTGGATTGGTTGGATTGATCCTGAAAAAGATAATACTATGTATGACCCGAAATTCCAGGACAAGGCCAGTATTACAACAGACACATCCTCCAACACAGCCTACCTGCATCTCAGCAGCCTGACATCTGAGGACACTGCCGTCTATTACTGTGCTAGGGGGGCTACGGTCTATTACTATACTCTGGACTACTGGGGTCAGGGAACATCAGTCGCCGTCTCCTCA
SEQ ID No.2
Heavy chain:Amino acid sequence(139AA)
Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
MKCSWVIFFLMAVVTGVNSEVQLQQSGAELVRPGALVKLSCKASGFSFKDYFMHWVRQRPEQGLEWIGWIDPEKDNTMYDPKFQDKASITTDTSSNTAYLHLSSLTSEDTAVYYCARGATVYYYTLDYWGQGTSVAVSS
SEQ ID No.3
Light chain:DNA sequence(381bp)
Signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
ATGGAGTCTCATACTCAGGCCTTTGTATTCGCGCTTCTCTGGTTGTCTGGTGTTGATGGAGACATTGTGATGACCCAGTCTCAAAAATTCATGTCCACATCAGTAGGAGACAGGGTCAGTGTCACCTGCAAGGCCAGTCAGAATGTGGTTACTACTGTAGCCTGGTATCAACAGAAACCTGGACAATCTCCTAAACTACTGATTTACTCGGCATCCAATCGATACACTGGAGTCCCTGATCGCTTCACTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCCCTGTACAGTCTGAAGACCTGGCAGATTATTTCTGCCAGCAATATACCAGCTATCCTCTCACGTTCGGAGGGGGGACCAAGCTGGAAATAAGA
SEQ ID No.4
Light chain:Amino acid sequence(127AA)
Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
MESHTQAFVFALLWLSGVDGDIVMTQSQKFMSTSVGDRVSVTCKASQNVVTTVAWYQQKPGQSPKLLIYSASNRYTGVPDRFTGSGSGTDFTLTISPVQSEDLADYFCQQYTSYPLTFGGGTKLEIR
所述单克隆抗体由杂交瘤细胞产生。
所述杂交瘤细胞是由免疫的BALB/C小鼠脾淋巴细胞和小鼠骨髓瘤细胞SP2/0经融合、筛选、克隆、传代和反复冻存、复苏后获得的杂交瘤细胞系ZJU79-01,能稳定分泌抗H7N9禽流感病毒血凝素蛋白的单克隆抗体ZJU79-01。
本发明的第二个目的提供抗H7N9禽流感病毒血凝素蛋白单克隆抗体的制备方法,通过以下步骤和技术方案实现:
(1)动物的免疫:选择6周龄的BALB/C小鼠,以纯化的H7N9禽流感病毒血凝素蛋白对小鼠进行免疫。血凝素蛋白由H7N9禽流感病毒减毒株接种鸡胚,培养并收获病毒液,经甲醛灭活、纯化、裂解及再纯化等方法,以磷酸缓冲液稀释制备获得。
(2)小鼠骨髓瘤细胞的培养:培养小鼠骨髓瘤细胞SP2/0并使之保持良好的生长状态用于细胞融合。
(3)细胞融合:采用聚乙二醇融合法。以BALB/C小鼠腹腔巨噬细胞作为饲养细胞,在融合前一天,接种BALB/C小鼠腹腔巨噬细胞于96孔培养板,含20%牛血清的次黄嘌呤-鸟嘌呤-磷酸核糖转移酶培养基培养一天。将(1)中备好的小鼠处死,获取脾脏淋巴细胞。收集(2)中的小鼠骨髓瘤细胞。将上述两种细胞混合离心,然后以聚乙二醇介导细胞融合。融合后的细胞适当稀释,接种至饲养细胞培养板,适当条件培养。
(4)杂交瘤细胞的筛选:将上述培养物在次黄嘌呤-磷酸核糖转移酶选择性培养基中培养。在细胞集落长到大小合适时,吸取细胞培养上清液做抗体鉴定,筛选阳性克隆。
(5)杂交瘤细胞的克隆化:以有限稀释法克隆杂交瘤细胞,将稀释到一定密度的细胞接种至96孔板,使每孔只有一个细胞生长。形成细胞集落的孔取培养上清液做酶联免疫吸附实验,鉴定阳性克隆。重复有限稀释克隆若干次,直到杂交瘤细胞的阳性孔率达到100%。将克隆化后的杂交瘤细胞扩大培养做抗体鉴定及理化性状分析。
(6)单抗腹水的诱导:在接种杂交瘤细胞前一周,给BALB/C小鼠腹腔注射石蜡油每只0.5毫升,然后每只接种5×106个阳性杂交瘤细胞,10天后收集腹水离心,测定抗体效价,并纯化单克隆抗体。
(7)单克隆抗体的纯化:利用Protein G亲和纯化法纯化腹水中的单克隆抗体。
本发明得到一个产生抗H7N9禽流感病毒血凝素蛋白单克隆抗体杂交瘤系,即ZJU79-01,ZJU79-01杂交瘤细胞系经4次克隆化,持续培养六月余,分泌抗体稳定。该细胞株经液氮冻存,复苏后生长良好,抗体分泌未见衰退。酶联免疫吸附实验间接法测得ZJU79-01培养上清效价为1:320,腹水效价分别为1:25600。经单克隆抗体免疫球蛋白亚型分析显示该杂交瘤细胞产生的抗体类型为IgG1、κ型。
本发明提供产生单克隆抗体的杂交瘤细胞,它是由免疫的BALB/C小鼠脾细胞和小鼠骨髓瘤细胞SP2/0经融合、筛选、克隆、传代和反复冻存、复苏后获得的小鼠杂交瘤细胞系ZJU79-01,能稳定分泌抗H7N9禽流感病毒血凝素蛋白的单克隆抗体ZJU79-01。
本发明的另一个目的是提供该单克隆抗体ZJU79-01在含有H7N9禽流感病毒的体液、尿囊液或其他环境样本中的检测,通过制备胶体金免疫层析试纸条实现。
抗H7N9禽流感病毒血凝素蛋白的单克隆抗体ZJU79-01在制备H7N9禽流感病毒检测试产品中的应用,单克隆抗体,能识别H7N9禽流感病毒。该单克隆抗体亚型为IgG1、κ型,命名为ZJU79-01,能特异性识别禽流感病毒的血凝素蛋白。
一种胶体金免疫层析试纸条,包含有权利要求1或2所述的抗H7N9禽流感病毒血凝素蛋白的单克隆抗体ZJU79-01。
胶体金免疫层析试纸条在检测H7N9禽流感病毒中的应用。
本发明的优点在于提供了一种抗H7N9禽流感病毒血凝素蛋白的单克隆抗体。制备方法简单易行,更为重要的是该方法制备的单克隆抗体可以有多种用途,如对临床和实验室的H7N9禽流感样本进行定性诊断,而且可以快速便捷的进行检测。
说明书附图
图1为单克隆抗体ZJU79-01的免疫球蛋白亚型分析。
图2为单克隆抗体ZJU79-01(胶体金试纸条)检测H7N9禽流感病毒的灵敏度。
具体实施方式
下面结合具体实施例,进一步阐述本发明。应理解,这些实施例仅用于说明本发明而不用于限制本发明的范围。
实施例1.抗H7N9禽流感病毒血凝素蛋白的单克隆抗体的制备方法
(1)小鼠的免疫:首次免疫,将纯化的H7N9禽流感病毒血凝素全蛋白与佐剂按等体积混合均匀,总体积600微升。每只BALB/C小鼠0.1毫升(含H7N9禽流感病毒血凝素蛋白抗原30微克),大腿内侧肌肉注射。第21天按同样方式加强免疫一针。第35天采微量尾血进行酶联免疫吸附实验测定,抗体滴度达到1:128000,随即尾静脉注射加强毫升免疫一次,于3天后进行细胞融合。
(2)小鼠骨髓瘤细胞SP2/0的培养:把来自BALB/C小鼠的SP2/0骨髓瘤细胞株以含10%牛血清DMEM培养基培养传代,在含5%二氧化碳饱和37℃孵箱中培养。融合前一天传代以保证融合时细胞进入对数生长期。
(3)细胞融合:以BALB/C小鼠腹腔巨噬细胞作为饲养细胞,在融合前一天,接种BALB/c小鼠腹腔巨噬细胞于96孔培养板,含20%牛血清的次黄嘌呤-鸟嘌呤-磷酸核糖转移酶培养基培养一天。次日取(1)中小鼠脾脏,采用压力注水法分离脾细胞,离心洗涤细胞2次后用培养液重悬。收集(2)中SP2/0细胞,离心,洗涤2次后用培养液重悬,作为待融合的SP2/0细胞。以1×108个免疫小鼠脾淋巴细胞与2×107个小鼠骨髓瘤细胞SP2/0混合,在聚乙二醇作用下融合。两种细胞混合后洗涤一次,离心弃上清,轻弹管壁悬开细胞,用37℃预温的聚乙二醇0.9毫升于90秒内逐滴加入细胞沉淀中,其间轻轻振摇离心管,但勿吹打,静置1分钟,然后按先慢后快的原则,于第1分钟内加完1毫升无血清DMEM,于第2分钟内加完2毫升无血清DMEM,于第3分钟内加完7毫升无血清DMEM,以后1分钟内逐渐加入37℃预温的无血清DMEM培养基40毫升。1000转每分钟低速离心10分钟。然后加入培养基,分别接种到加有饲养细胞的96孔培养板,一般每次融合的细胞铺2块板,置细胞孵箱中培养。
(4)杂交瘤细胞的筛选:每隔4天换一半培养液(含次黄嘌呤-鸟嘌呤-磷酸核糖转移酶)一次,10天后改用含次黄嘌呤-磷酸核糖转移酶培养液。融合后的杂交瘤细胞在含次黄嘌呤-磷酸核糖转移酶的选择性培养液中大约持续培养两周。吸取培养上清做酶联免疫吸附实验,筛选阳性克隆。采用酶联免疫吸附实验间接法筛选阳性杂交瘤克隆。主要步骤:①0.01摩尔每升pH9.6碳酸盐缓冲液稀释H7N9血凝素蛋白,浓度20纳克/孔,分别于96孔酶标板加入0.1毫升每孔,4℃过夜;②0.01摩尔每升pH7.4磷酸盐缓冲液(含吐温20)洗板三次;③用2%牛血清白蛋白0.01摩尔每升pH 7.4的磷酸盐缓冲液封闭2小时;④同上洗板;⑤加入杂交瘤培养上清,0.1毫升每孔,同时设阳性对照(免疫小鼠血清)、阴性对照(SP2/0培养上清液)和空白对照,室温反应2小时;⑥洗板;⑦加1:6000稀释的辣根过氧化物酶标记的羊抗小鼠IgG,0.1毫升每孔,室温反应1小时;⑧洗板;⑨加入底物室温避光反应5分钟;⑩2摩尔每升硫酸终止反应;450纳米测定其光密度值,以测定值除以阴性≥2.1为阳性。
(5)杂交瘤细胞的克隆化:杂交瘤细胞的克隆化培养按有限稀释法进行,选择抗体检测阳性的杂交瘤孔细胞作适当增殖后,准确计数细胞。用完全DMEM培养基稀释成10个每毫升的细胞悬液接种到已有饲养细胞的96孔培养板中,每孔0.1毫升,10天后观察细胞生长情况,并检测上清液中抗体水平,选择5个抗体滴度最高的,呈单个克隆细胞生长的培养孔,作再次有限稀释。此方法可重复多次,直至单克隆孔抗体检测阳性率为100%。
(6)诱生腹水:在接种杂交瘤细胞前一周,给BALB/C小鼠腹腔注射石蜡油每只0.5毫升,然后每只接种5×106个阳性杂交瘤细胞,10天后收集腹水测定抗体效价。
(7)单克隆抗体的纯化:采用亲和纯化法(Protein G交联的Sepharose)纯化腹水中单克隆抗体。①腹水用冷结合缓冲液稀释3倍后,于4℃10000转每分钟离心15分钟去除沉淀物。②将预装有Sepharose-Protein G的亲和纯化柱用10倍柱床体积的结合缓冲液充分流洗。③将稀释的腹水上柱,控制流速10滴每分钟。④将流穿的腹水重复上柱一次。⑤用20倍柱床体积的结合缓冲液充分洗涤,直至流穿液280纳米吸光值小于0.01。⑥用洗脱缓冲液洗脱结合的单克隆抗体,控制流速10滴每分钟,收集洗脱液于预加有0.1毫升的磷酸钾缓冲液(PH7.9,0.5摩尔/升)的收集管中,每管收集0.5毫升含抗体的洗脱液,共收集毫升20管以上。⑦于280纳米检测每管洗脱液的吸光度,并收集吸光值大于0.2的洗脱液。⑧将收集的洗脱液置于透析卡中,并于0.1摩尔每升PH7.4的磷酸盐缓冲液中透析。每隔6小时换液一次,共透析24小时。⑨将透析后的抗体溶液稀释后,于280纳米测蛋白含量。⑩将纯化的抗体分装于小管中,置于低温冰箱备用。
(8)单克隆抗体的亚型鉴定:采用Bio-Rad公司的小鼠单克隆抗体免疫球蛋白分型试剂盒分析。将纯化的单抗作适当稀释后进行检测,操作严格按试剂盒说明书进行。试验结果为ZJEU79-01杂交瘤细胞分泌的单克隆抗体为IgG1、κ型。
结果见附图1。
实施例2.用该单克隆抗体进行H7N9禽流感病毒的定性检测
本发明制备的抗H7N9禽流感病毒血凝素蛋白单克隆抗体可以用来定性检测H7N9禽流感病毒,鉴定方法可以通过下述方法实现:
H7N9免疫层析胶体金检测试纸条:
(1)胶体金溶液制备:取200毫升玻璃瓶1个,加入超纯水49.5毫升,而后加入1%氯金酸0.5毫升,配制成50毫升0.01%氯金酸水溶液,加热煮沸后在持续搅拌的条件下一次性加入1%柠檬酸三钠溶液1.8毫升,继续搅拌加热,当溶液的颜色完全变为透明的紫红色时,维持5分钟后停止加热,补水至原体积,冷却至室温,4℃避光保存备用;
(2)抗体的预处理:将纯化得到的ZJU79-01抗体用0.01摩尔每升磷酸盐缓冲液稀释成1毫克每毫升和0.1毫克每毫升,过0.22微米滤膜;
(3)胶体金标记物条件优化:取9个离心管,每个离心管中加入步骤(1)中的胶体金溶液1毫升,依次加入0微升、5微升、10微升、15微升、20微升、25微升、30微升、40微升、50微升的0.1摩尔每升碳酸钾溶液,混匀,室温静置1小时;依次从每个离心管中取100微升液体置于96孔板中,再分别加入3微升的1毫克每毫升的ZJU79-01抗体,混匀,室温静置15分钟;每孔中加入20微升10%的氯化钠溶液,混匀,室温下静置2小时,观察胶体金颜色,保持红色的最低pH即为胶体金溶液的最佳pH。取最佳pH值胶体金溶液600微升,分别加至离心管,每管100微升;依次加2微升、4微升、6微升、8微升、10微升、12微升的0.1毫克每毫升抗体,混匀,静置15分钟;再加入20微升10%的氯化钠溶液,混匀,室温下静置2小时,观察胶体金颜色,保持红色的最小蛋白量为该抗体最低稳定蛋白量,将此基础上增加20%,即为金标抗体最适蛋白量;
(4)胶体金标记物的制备:取步骤(1)中的胶体金溶液20毫升,根据步骤(3)中选定的最优条件,电磁搅拌器250转每分钟搅拌,逐滴加入2毫升的1毫克每毫升抗体,反应10分钟;逐滴加入2毫升10%牛血清白蛋白,继续搅拌反应10分钟;金标抗体溶液4℃,12000转每分钟离心30分钟,弃上清,收集沉淀,将沉淀用金标抗体稀释液定容至2毫升,制备成ZJU79-01抗体胶体金标记物;
(5)胶体金膜的制备:将载体玻璃纤维素膜浸泡在步骤(4)中的胶体金标记物溶液中,室温自然晾干或者37℃烘干3小时,备用;
(6)包被膜的制备:将羊抗鼠IgG抗体、抗H7N9禽流感病毒的另一株单抗稀释成1毫克每毫升,分别划在硝酸纤维素膜的质控线和检测线上,制备成包被膜,37℃包被2小时后,室温自然晾干或者37℃烘干;
(7)样品垫前处理:将样品垫处理液均匀涂布在玻璃纤维素膜上,在空气湿度低于60%的条件下,室温自然晾干;
(8)检测卡的组装:塑料胶板自上而下依次粘贴样品垫、胶体金膜、包被膜和吸水纸,组装成试纸条,切割成一定宽度的长条,备用;
(9)确定胶体金试纸条检测H7N9禽流感病毒的灵敏度:将H7N9禽流感病毒稀释至27、26、25、24、23、22、21血凝素单位,将100微升的待检样品滴加到试纸条样品垫区,观察质控线和检测线是否出现红色条带。结果判定:质控线出现红色条带说明试纸条有效,否则无效。检测线出现红色条带说明样品中含有H7N9禽流感病毒,否则不含有H7N9禽流感病毒。检测结果表明,胶体金试纸条检测H7N9禽流感病毒的灵敏度为2个单位血凝素单位。
结果见附图2。
(10)胶体金试纸条与其他检测方法的对比实验:采用临床上具有广泛使用的H7N9流感病毒荧光定量聚合酶链式反应检测试剂盒(上海之江生物有限公司)进行对比分析,对临床样本进行检测分析,胶体金试纸条的特异性达到了97.9%,胶体金试纸条检测方法具有快速、特异、便捷等优点,具有了良好的临床应用前景。
应理解,本发明是结合最佳实施例进行描述的,然而在阅读了本发明的上述内容后,本领域技术人员可以对本发明作各种改动或修改,这些等价形式同样落于本申请所附权利要求书所限定的范围。
序列表
<110> 浙江大学医学院附属第一医院
<120> 抗H7N9禽流感病毒血凝素蛋白单克隆抗体ZJU79-01及其应用
<130> 21-2019-0802
<140> 2019104209451
<141> 2019-05-20
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 417
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atgaaatgca gctgggtcat cttcttcctg atggcagtgg ttacaggggt caattcagag 60
gttcagctgc agcagtctgg ggctgaactt gtgaggccag gggccttagt caagttgtcc 120
tgcaaagctt ctggcttcag ctttaaagac tactttatgc actgggtgag gcagaggcct 180
gaacagggcc tggagtggat tggttggatt gatcctgaaa aagataatac tatgtatgac 240
ccgaaattcc aggacaaggc cagtattaca acagacacat cctccaacac agcctacctg 300
catctcagca gcctgacatc tgaggacact gccgtctatt actgtgctag gggggctacg 360
gtctattact atactctgga ctactggggt cagggaacat cagtcgccgt ctcctca 417
<210> 2
<211> 139
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Lys Asp Tyr Phe Met His Trp Val Arg Gln Arg Pro Glu Gln Gly Leu
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Glu Trp Ile Gly Trp Ile Asp Pro Glu Lys Asp Asn Thr Met Tyr Asp
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Pro Lys Phe Gln Asp Lys Ala Ser Ile Thr Thr Asp Thr Ser Ser Asn
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Thr Ala Tyr Leu His Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val
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Tyr Tyr Cys Ala Arg Gly Ala Thr Val Tyr Tyr Tyr Thr Leu Asp Tyr
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Trp Gly Gln Gly Thr Ser Val Ala Val Ser Ser
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<210> 3
<211> 381
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atggagtctc atactcaggc ctttgtattc gcgcttctct ggttgtctgg tgttgatgga 60
gacattgtga tgacccagtc tcaaaaattc atgtccacat cagtaggaga cagggtcagt 120
gtcacctgca aggccagtca gaatgtggtt actactgtag cctggtatca acagaaacct 180
ggacaatctc ctaaactact gatttactcg gcatccaatc gatacactgg agtccctgat 240
cgcttcactg gcagtggatc tgggacagat ttcactctca ccatcagccc tgtacagtct 300
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gggaccaagc tggaaataag a 381
<210> 4
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<213> 人工序列(Artificial Sequence)
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Met Glu Ser His Thr Gln Ala Phe Val Phe Ala Leu Leu Trp Leu Ser
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Gly Val Asp Gly Asp Ile Val Met Thr Gln Ser Gln Lys Phe Met Ser
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Thr Ser Val Gly Asp Arg Val Ser Val Thr Cys Lys Ala Ser Gln Asn
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Val Val Thr Thr Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro
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Lys Leu Leu Ile Tyr Ser Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp
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Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser
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Pro Val Gln Ser Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln Tyr Thr
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Ser Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Arg
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Claims (6)
1.一种抗H7N9禽流感病毒血凝素蛋白单克隆抗体ZJU79-01,抗体的重链氨基酸序列如SEQ ID No.2,轻链氨基酸序列如SEQ ID No.4所示,该单克隆抗体亚型为IgG1、κ型,能与禽流感病毒血凝素蛋白抗原特异结合。
2.根据权利要求1所述的单克隆抗体ZJU79-01,其特征在于:所述单克隆抗体由杂交瘤细胞产生。
3.根据权利要求2所述的单克隆抗体ZJU79-01,其特征在于:所述杂交瘤细胞是由免疫的BALB/C小鼠脾淋巴细胞和小鼠骨髓瘤细胞SP2/0经融合、筛选、克隆、传代和反复冻存、复苏后获得的杂交瘤细胞系ZJU79-01,能稳定分泌抗H7N9禽流感病毒血凝素蛋白的单克隆抗体ZJU79-01。
4.权利要求1或2所述的抗H7N9禽流感病毒血凝素蛋白的单克隆抗体ZJU79-01在制备H7N9禽流感病毒检测产品中的应用。
5.一种胶体金免疫层析试纸条,其特征在于:包含有权利要求1或2所述的抗H7N9禽流感病毒血凝素蛋白的单克隆抗体ZJU79-01。
6.权利要求5所述胶体金免疫层析试纸条在非诊断目的检测H7N9禽流感病毒中的应用。
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