CN114426576A - 抗h3n2流感病毒核蛋白单克隆抗体zju-np-a3及其在检测中的应用 - Google Patents
抗h3n2流感病毒核蛋白单克隆抗体zju-np-a3及其在检测中的应用 Download PDFInfo
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- CN114426576A CN114426576A CN202210045302.5A CN202210045302A CN114426576A CN 114426576 A CN114426576 A CN 114426576A CN 202210045302 A CN202210045302 A CN 202210045302A CN 114426576 A CN114426576 A CN 114426576A
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Abstract
本发明提供抗H3N2流感病毒核蛋白单克隆抗体ZJU‑NP‑A3及其在检测中的应用。一种抗H3N2流感病毒核蛋白单克隆抗体，能识别H3N2流感病毒。该单克隆抗体亚型为IgG2a、κ型，命名为ZJU‑NP‑A3，能特异性识别流感病毒的核蛋白。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。对该单克隆抗体进行进一步的理化性状分析和鉴定，并建立了该单克隆抗体通过免疫荧光实验检测流感病毒核蛋白的方法。本发明为临床H3N2流感病毒感染的辅助诊断提供了有效工具，并可推广应用于多种检测技术以及临床和实验研究。
Description
技术领域
本发明属于生物技术领域，涉及抗H3N2流感病毒核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，获得分泌抗核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，制备抗核蛋白的单克隆抗体ZJU-NP-A3，鉴定为IgG2a、κ型，再通过亲和纯化、免疫等技术实现对该抗体的应用。
背景技术
流行性感冒病毒由于其超强的传染性和变异性成为第一个实行全球监测的疾病。1968年，H3N2流感病毒引发了全球的大流行,此后该病毒一直在人群中呈季节性的流行并不断发生着变异，自1999年至2016年，世界卫生组织(World Health Organization,WHO)公布的流感疫苗组分中H3N2毒株更新了10次，正是这种不断突变特点，导致了疫苗株与流行株不匹配，给防控策略的制定带来了巨大困扰。因此，快速准确的诊断对降低病死率至关重要。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。
对于甲型流感病毒不同亚型的检测，目前已有多种方法，基于检测标靶物的不同，这些方法又分为病毒分离检测，抗原、抗体检测等。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、灵敏的H3N2病毒检测产品势在必行，能促进更早更广泛的发现H3N2病毒感染，减少重症病例发生，降低病死率。
综上所述，开发H3N2流感病毒单克隆抗体，用于快速灵敏检测方法的建立是十分有意义的。基于以上背景，本项目选定核蛋白为靶抗原，采用融合杂交瘤技术建立稳定分泌抗核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H3N2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
本发明的目的是提供一种抗H3N2流感病毒核蛋白单克隆抗体，能识别H3N2流感病毒。该单克隆抗体亚型为IgG2a、κ型，命名为ZJU-NP-A3，能特异性识别流感病毒的核蛋白。
SEQ ID No.1
Heavy chain:DNA sequence(360bp)
Signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
CAGATCCAGTTGGCGCAGTCTGGACCTGAACTGAAGAAGCCTGGAGAGACAGTCAGGATCTCCTGCAAGGCTTCTGGGTATACCTTCACATCTGCTGGAATACAGTGGGTGCAAAAGATGCCAGGAAAGGGTTTGAAGTGGATTGGCTGGATAAACACCCACTCTGAATTGCCAAAATATGCAGAAGACTTCAAGGGACGGTTTGCCTTCTCTTTGGAAACCTCTGCCAGCACTGCATATTTACAGATAAGCAACCTCAAAAATGAAGACACGGCTACGTTTTTCTGTGCGAGAAACTTTGGTAACTACCCCTATGCTATGGACTTCTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCA
SEQ ID No.2
Heavy chain:Amino acid sequence(120AA)
Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
QIQLAQSGPELKKPGETVRISCKASGYTFTSAGIQWVQKMPGKGLKWIGWINTHSELPKYAEDFKGRFAFSLETSASTAYLQISNLKNEDTATFFCARNFGNYPYAMDFWGQGTSVTVSS
SEQ ID No.3
Light chain:DNA sequence(321bp)
Signal sequence-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
GACATCTTGCTGACTCAGTCTCCAGCCATCCTGTCTGTGAGTCCAGGAGAAAGAGTCAGTTTCTCCTGCAGGGCCAGTCAGAACATTGGCACAAGCATACATTGGTATCAGCAAAGAACAAATGGTTCTCCAAGGCTTCTCATAAAGTATGCTTCTGATTCAATCTCTGGGATCCCTTCCAGGTTTAGTGGCAGTGGATCAGGGACAGATTTTACTCTTAGCATCAACAGTGTGGAGTCTGAAGATATTGCAGATTATTACTGTCAACAAAGTAATAACTGGCCGCTCACGTTCGGTGCTGGGACCAAGCTGGAGCTGAAA
SEQ ID No.4
Light chain:Amino acid sequence(107AA)
Signal peptide-FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4
DILLTQSPAILSVSPGERVSFSCRASQNIGTSIHWYQQRTNGSPRLLIKYASDSISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQSNNWPLTFGAGTKLELK
本发明的第二个目的提供抗H3N2流感病毒核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H3N2流感病毒核蛋白对小鼠进行免疫。
(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)单克隆抗体腹水的诱导：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。
(8)本发明得到一个产生抗H3N2流感病毒核蛋白单克隆抗体杂交瘤系，即ZJU-NP-A3，ZJU-NP-A3杂交瘤细胞系经5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ZJU-NP-A3腹水纯化后抗体对H3N2核蛋白的亲和力可达10纳克每毫升。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IgG2a。
本发明提供产生单克隆抗体的杂交瘤细胞系，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ZJU-NP-A3，能稳定分泌抗H3N2流感病毒核蛋白的单克隆抗体ZJU-NP-A3。
本发明的另一个目的是提供该单克隆抗体ZJU-NP-A3在含有H3N2流感病毒及其他不同流感病毒亚型的检测的应用，通过免疫荧光实验实现。
本发明还提供了一种H3N2流感病毒检测产品，包含单克隆抗体ZJU-NP-A3。
本发明的优点在于提供了一种抗H3N2流感病毒核蛋白的单克隆抗体。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H3N2流感样本进行定性诊断。
说明书附图
图1为单克隆抗体ZJU-NP-A3的免疫球蛋白亚型分析。
图2为单克隆抗体ZJU-NP-A3检测H3N2流感病毒的亲和力。
图3为单克隆抗体ZJU-NP-A3检测H3N2流感病毒的特异性。
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H3N2流感病毒核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H3N2流感病毒核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H3N2流感病毒核蛋白抗原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H3N2核蛋白，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PH7.4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。将纯化的单克隆抗体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ZJU-NP-A3杂交瘤细胞分泌的单克隆抗体为IgG2a、κ型。
结果见附图1。
实施例2.用该单克隆抗体对H3N2核蛋白亲和力进行检测
本发明制备的抗H3N2流感病毒核蛋白单克隆抗体可以与H3N2核蛋白特异性反应并具有较好的亲和力：
使用间接ELISA法分析单克隆抗体的亲和力
(1)用包被缓冲液稀释纯化的H1N1或H3N2病毒核蛋白，稀释至0.2微克/毫升，96孔酶标板中每孔加入100微升核蛋白稀释液，4℃包被过夜；
(2)用0.01摩尔每升，pH7.4的磷酸盐缓冲液(含吐温20)洗板三遍，每孔加入封闭缓冲液200微升，室温下封闭2小时；
(3)用同上的磷酸盐缓冲液将封闭液洗去，洗板三遍；
(4)取腹水纯化后的ZJU-NP-A3抗体，用磷酸盐缓冲液将抗体的起始浓度稀释至10微克/毫升，取微升稀释后抗体加入96孔板第1列，于第2-12列加入100微升磷酸盐缓冲液，将抗体进行2倍倍比稀释。将磷酸盐缓冲液、正常小鼠血清和免疫小鼠血清用作空白对照、阴性对照和阳性对照，室温下孵育2小时；
(5)用含吐温20的磷酸盐缓冲液洗板三遍，加入1:5000稀释的辣根过氧化物酶标记的羊抗小鼠二抗，每孔加入100微升，室温下孵育1小时；
(6)同上方法洗板三遍，每孔加入100微升显色液，避光显色5分钟；
(7)每孔加入终止液100微升终止反应，酶标仪450纳米处测定吸光度值。
结果判定：以阳性值/阴性值(P/N)≥2.1标准判读阳性。检测结果表明，该单克隆抗体与H1N1与H3N2核蛋白均具有较好亲和力，与H1N1的亲和力为40纳克/毫升而对H3N2则可达10纳克/毫升。
结果见附图2。
实施例3.用该单克隆抗体对H3N2核蛋白特异性进行检测
使用免疫荧光实验观察该单克隆抗体的特异性
(1)将犬肾传代细胞提前一天以接种在24孔板中，待细胞长至70％左右开始实验；
(2)取出铺好细胞的细胞板，弃去培养上清，用磷酸盐缓冲液洗一遍，备用；
(3)用病毒稀释液稀释病毒(H1N1、H3N2、H9N2、H10N7、H6N1、乙型流感病毒)，稀释后的病毒液感染细胞(感染复数为0.5)，将感染的细胞置于37℃，5％二氧化碳条件培养两小时；
(4)取出细胞板，弃去病毒液，用磷酸盐缓冲液洗细胞2遍，注意沿孔壁缓慢加入以免将细胞冲下，洗好后于每孔中加入200微升的病毒培养液，37℃，5％二氧化碳孵箱培养16小时；
(5)于镜下观察细胞病变情况，取出细胞板，弃去培养上清，磷酸盐缓冲液洗细胞2遍；
(6)每孔中加入200微升4％多聚甲醛固定细胞，室温下固定30分钟后，加入磷酸盐缓冲液并将板置于水平摇床上以60转/分的速度洗板3遍，每次3分钟；
(7)用磷酸盐缓冲液配置含0.5％聚乙二醇辛基苯基醚的细胞通透液通透细胞，室温下通透30分钟，用同样方法洗3遍；
(8)用含3％牛血清白蛋白溶液封闭，室温下封闭1小时，弃去封闭液；
(9)用磷酸盐缓冲液将ZJU-NP-A3单克隆抗体稀释至10微克/毫升，同时将无关同型抗体和磷酸盐缓冲液设置为同型对照和阴性对照，每孔加入200微升，4℃孵育过夜后加入磷酸盐缓冲液置于水平摇床以60转/分的速度洗板3遍，每次3分钟；
(10)用1％牛血清白蛋白溶液稀释羊抗鼠荧光二抗至5微克/毫升，每孔加入200微升，37℃避光孵育90分钟，用以上方法洗板3遍；
(11)以1:100稀释比稀释4’,6-二脒基-2-苯基吲哚溶液，对细胞核进行染色，室温下避光孵育10分钟，洗板3次，方法同上；
(12)在荧光显微镜下观察实验结果。
结果判定：观察到绿色荧光即为阳性。检测结果表明，该单克隆抗体能与H3N2核蛋白具有较好的检测能力。
结果见附图3。
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H3N2流感病毒核蛋白单克隆抗体ZJU-NP-A3及其在检测中的应用
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Claims (8)
1.一种抗H3N2流感病毒核蛋白单克隆抗体ZJU-NP-A3，该单克隆抗体亚型为IgG2a、κ型，能与H3N2流感病毒核蛋白特异结合。
2.根据权利要求1所述的单克隆抗体ZJU-NP-A3，其特征在于：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。
3.根据权利要求1或2所述的单克隆抗体ZJU-NP-A3，其特征在于：所述抗体由杂交瘤细胞产生。
4.根据权利要求3所述的单克隆抗体ZJU-NP-A3，其特征在于：产生该单克隆抗体的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ZJU-NP-A3，能稳定分泌抗H3N2流感病毒核蛋白的单克隆抗体ZJU-NP-A3。
5.权利要求1或2所述的抗H3N2流感病毒核蛋白的单克隆抗体ZJU-NP-A3在制备H3N2流感病毒检测产品中的应用。
6.根据权利要求5所述的应用，所述检测产品通过免疫荧光实验和酶联免疫吸附试验验证。
7.根据权利要求5所述的应用，所述抗体可以与H3N2流感病毒的核蛋白反应。
8.一种H3N2流感病毒检测产品，其特征在于：包含有权利要求1或2所述的单克隆抗体ZJU-NP-A3。
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