CN111434682B - 抗h7n9全人源单克隆抗体7t33及其制备方法与应用 - Google Patents
抗h7n9全人源单克隆抗体7t33及其制备方法与应用 Download PDFInfo
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Abstract
本发明涉及抗H7N9全人源单克隆抗体7T33及其制备方法与应用。利用记忆B细胞PCR方法快速筛选全人源单克隆抗体7T33，不含任何鼠源成分。本发明抗体可靶向结合H7N9病毒的血凝素HA，具有显著地抗H7N9病毒感染的中和活性；本发明抗体不发生抗鼠抗抗体等毒副作用，具有更好的生物相容性，更适合和更有潜力成为治疗流感病毒的大分子药物。
Description
技术领域
本发明属于免疫学领域，具体地涉及一种抗H7N9全人源单克隆抗体7T33及其制备方法与应用。
背景技术
在2015年全球十大畅销药中，有6个是全人源或人源化单克隆抗体药物。排名第一的是艾伯维公司治疗关节炎的抗TNFa单克隆抗体Humira，这是一个全人源单克隆抗体，已经是连续3年销售额100亿以上的药王。从1986年第一个单克隆抗体药物上市开始，单抗药物经历了鼠源单抗药物(如Orthoclone OKT3)、嵌合单抗药物(Rituximab)、人源化单抗药物(Herceptin)和全人源单抗药物(Humira)等阶段。由于人体出现抗鼠抗体反应(HAMA)，鼠源单抗药物、嵌合单抗药物已经逐渐被淘汰，目前占据市场的单克隆抗体药物全都是人源化单克隆抗体药物。与国际先进的人源抗体生产技术相比，深圳乃至全中国都有很大差距，主要表现在人源抗体药物领域的创新能力薄弱，自主研发的品种少，目前还没有原创人源化单克隆抗体药物上市的报道，庞大的抗体药物市场被国外药企占领。我国要改变落后局面，争夺消费潜力巨大的国内外抗体药物市场，亟需攻克全人源单克隆抗体技术。
人源单克隆抗体在治疗炎症、癌症特别是流行性感冒方面具有高特异性的显著疗效。流行性感冒是由流感病毒引起的传染性疾病，严重威胁人类健康。全球每年约有10亿人受季节性流感病毒感染，其中有25-50万人死亡。H7N9病毒是一种流感病毒，对传统的抗病毒药金刚烷胺(amantadine)和金刚烷乙胺(rimantadine)有耐药性，目前尚没有有效治疗手段。H7N9病毒在入侵细胞时需要依赖病毒自身表达的特定分子与人细胞上的受体结合，才能感染细胞，并进一步扩增。中和病毒的人源抗体是人B淋巴细胞产生的某些特异抗体，能够与病毒表面的抗原结合，从而阻止该病毒黏附靶细胞受体，防止病毒侵入细胞，能够高效防治H7N9流行性感冒。
发明内容
一方面，本发明涉及抗H7N9全人源单克隆抗体7T33或来源于该单克隆抗体的能够中和H7N9病毒的生物活性片段。
另一方面，本发明涉及编码所述抗H7N9全人源单克隆抗体7T33或来源于该单克隆抗体的能够特异性结合H7N9的生物活性片段的基因及含该基因的载体或细胞。
另一方面，本发明涉及产生所述抗H7N9全人源单克隆抗体7T33的方法。
另一方面，本发明涉及包含所述的抗H7N9全人源单克隆抗体7T33或来源于该单克隆抗体的能够特异性结合H7N9的生物活性片段的药物组合物。
另一方面，本发明涉及本发明所述的抗H7N9全人源单克隆抗体7T33或来源于该单克隆抗体的能够特异性结合H7N9的生物活性片段或所述药物组合物的应用。
另一方面，本发明涉及一种检测H7N9病毒的试剂盒。
具体地，一方面，本发明提供了一种抗H7N9全人源单克隆抗体7T33或来源于该单克隆抗体的能够特异性结合H7N9的生物活性片段，其中，该抗体的重轻链CDR1、CDR2及CDR3区的氨基酸序列分别如下所示：
重链CDR1:GTIFYSTE；
重链CDR2:MNPESGEY；
重链CDR3:AYGNAECSAGGSCTNWFGEP；
轻链CDR1:RLRSTT；
轻链CDR2:GKN；
轻链CDR3:NSREYSGTHLV。
在一些实施方式中，该抗体的重链可变区氨基酸序列如SEQ ID NO:2所示，或该序列经替换、缺失或添加一个或几个氨基酸形成的具有同等功能的氨基酸序列；和/或
该抗体的轻链可变区氨基酸序列如SEQ ID NO:4所示，或该序列经替换、缺失或添加一个或几个氨基酸形成的具有同等功能的氨基酸序列。
在一些实施方式中，该抗体的重链氨基酸序列如SEQ ID NO:6所示，或该序列经替换、缺失或添加一个或几个氨基酸形成的具有同等功能的氨基酸序列；和/或
该抗体的轻链氨基酸序列如SEQ ID NO:8所示，或该序列经替换、缺失或添加一个或几个氨基酸形成的具有同等功能的氨基酸序列。
经ELISA实验验证，本发明所述抗H7N9全人源单克隆抗体7T33可以靶向结合H7N9病毒的血凝素HA，亲和力为5.43×10-9M；在病毒感染细胞模型中，其IC50值仅为12.84uM左右。本发明所述抗体为全人源性单克隆抗体，相比鼠源抗体，全人源抗体的基因完全来源于人的基因，没有其他种属的成分，在人体内不发生抗鼠抗抗体等毒副作用，具有更好的生物相容性，更适合和更有潜力成为治疗流感病毒的大分子药物。
另一方面，本发明提供编码本发明所述的抗H7N9全人源单克隆抗体7T33的基因。在一些实施方式中，所述基因包含编码具有SEQ ID NO:2所示的氨基酸的核苷酸序列，在一些实施方式中，该核苷酸序列如SEQ ID NO:1所示；和/或
所述基因包含编码具有SEQ ID NO:4所示的氨基酸的核苷酸序列，在一些实施方式中，该核苷酸序列如SEQ ID NO:3所示。
在一些体实施方式中，所述基因包含编码具有SEQ ID NO:6的氨基酸的核苷酸序列，在一些实施方式中，该核苷酸序列如SEQ ID NO:5所示；和/或
所述基因包含编码具有SEQ ID NO:8所示的氨基酸的核苷酸序列，在一些实施方式中，该核苷酸序列如SEQ ID NO:7所示。
本发明SEQ ID NO:1～8的序列如序列表所示，其中：
(1)SEQ ID NO:1中第76～99位序列以及SEQ ID NO:5中第124～147位序列：用于编码重链CDR1区序列；
(2)SEQ ID NO:1中第151～174位序列以及SEQ ID NO:5中第199～222位序列：用于编码重链CDR2区序列；
(3)SEQ ID NO:1中第289～348位序列以及SEQ ID NO:5中第337～396位序列：用于编码重链CDR3区序列；
(4)SEQ ID NO:3中第76～93位序列以及SEQ ID NO:7中第124～141位序列：用于编码轻链CDR1区序列；
(5)SEQ ID NO:3中第145～153位序列以及SEQ ID NO:7中第193～201位序列：用于编码轻链CDR2区序列；
(6)SEQ ID NO:3中第262～294位序列以及SEQ ID NO:7中第310～342位序列：用于编码轻链CDR3区序列；
(7)SEQ ID NO:5和SEQ ID NO:7中的第1～48位序列为用于编码信号肽的序列。
(8)SEQ ID NO:2和SEQ ID NO:6中的第26-33位氨基酸序列为重链CDR1序列；SEQID NO:2和SEQ ID NO:6中的第51-58位氨基酸序列为重链CDR2序列；SEQ ID NO:2和SEQIDNO:6中第97-116位氨基酸序列为重链CDR3序列。
(9)SEQ ID NO:4和SEQ ID NO:8中的第26-31位氨基酸序列为轻链CDR1序列；SEQID NO:4和SEQ ID NO:8中的第49-51位氨基酸序列为轻链CDR2序列；SEQ ID NO:4和SEQIDNO:8中的第88-98位氨基酸序列为轻链CDR3序列。
另一方面，本发明提供含如上所述基因的载体。
再一方面，本发明提供含如上所述基因或如上所述载体的细胞。
再一方面，本发明提供一种产生所述抗H7N9全人源单克隆抗体7T33或来源于该单克隆抗体的能够特异性结合H7N9的生物活性片段的方法，该方法包括培养含编码抗H7N9全人源单克隆抗体7T33的重轻链的上述基因或上述载体的基因工程细胞或直接培养上述细胞，收集，纯化得所述抗H7N9全人源单克隆抗体7T33。
现有技术中存在采用噬菌体展示技术制备抗H7N9病毒人源单克隆抗体的方法，尽管该方法具有生产成本低、不经过免疫和细胞融合等繁琐工作的优点，但是其缺点也比较明显，从非免疫抗体库中获得的抗体往往亲和力不足、受外源基因转化率的限制、抗体库的库容量不足以涵盖动物的抗体多样性等。本发明从病人的血液中分离分泌功能抗体的B细胞，然后提取RNA和合成cDNA，从中克隆分泌目的抗体的基因，最后重组和表达全人源单克隆抗体。该技术操作简单快捷，生产的人源抗体具有高亲和力和特异性，此外，可进一步采用改进的从记忆B细胞中分离具有中和病毒功能或杀伤肿瘤功能的本发明所述单克隆抗体技术，更是大大减少了繁琐操作和成本。
另一方面，本发明提供一种药物组合物，其包含本发明所述的抗H7N9全人源单克隆抗体7T33或来源于该单克隆抗体的能够特异性结合H7N9的生物活性片段。
另一方面，本发明提供所述的抗H7N9全人源单克隆抗体7T33或来源于该单克隆抗体的能够特异性结合H7N9的生物活性片段或所述的药物组合物在制备用于治疗由H7N9病毒引起的疾病的药物中的应用。
另一方面，本发明提供一种检测H7N9病毒水平的试剂盒，其含有本发明所述的抗H7N9全人源单克隆抗体7T33或来源于该单克隆抗体的能够特异性结合H7N9的生物活性片段；在一些实施方式中，所述的试剂盒还含有第二抗体和用于检测的酶或荧光或放射标记物，以及缓冲液；所述第二抗体例如为抗本发明所述单克隆抗体7T33的抗抗体。
与现有技术相比，本发明具有如下有益效果：
(1)本发明所述抗H7N9全人源单克隆抗体7T33可以靶向结合H7N9病毒的血凝素HA，具有显著抗H7N9病毒感染的中和活性。
(2)相比鼠源抗体，本发明全人源抗体的基因完全来源于人的基因，没有其他种属的成分，在人体内不发生抗鼠抗抗体等毒副作用，具有更好的生物相容性，更适合和更有潜力成为治疗流感病毒的大分子药物。
(3)相较于现有技术提供的噬菌体展示技术制备抗H7N9病毒人源单克隆抗体的方法，本发明采用的单个B细胞开发抗H7N9病毒的抗体具有操作简单快捷，生产的人源抗体具有高亲和力和特异性等优点。
附图说明
图1为实施例1中NTH-3T3表达CD40L的流式检测结果图。
图2为实施例1中流式细胞仪分选记忆B细胞结果图。
图3为实施例1中ELISA实验结果图。
图4为实施例3中和实验结果图。
具体实施方式
为了对本发明的技术特征、目的和有益效果有更加清楚的理解，现结合具体实施例对本发明的技术方案进行以下详细说明，应理解这些实例仅用于说明本发明而不用于限制本发明的范围。实施例中，各原始试剂材料均可商购获得，未注明具体条件的实验方法为所属领域熟知的常规方法和常规条件，或按照仪器制造商所建议的条件。
实施例1
(1)构建稳定表达CD40L的NTH-3T3细胞系(3T3-CD40L)
利用慢病毒建立3T3-CD40L饲养细胞。构建慢病毒表达载体pLVX-CD40L，转染293T细胞，转染第四天收集病毒上清液。活化NIH-3T3细胞，培养3代后用慢病毒感染，继续培养并传代3次。利用流式细胞仪进行分选FITC荧光强度在MFI附近的细胞，重新加入至培养瓶中，37℃，5％CO2培养箱中培养和检测，检测结果如图1所示，其是将表达CD40L的3T3细胞和空载体pLVX(带有ZxGreen)转染的3T3细胞分别用带有APC的抗CD40L染色，然后上流式细胞仪分析。结果发现，所有3T3-CD40L饲养细胞都表达CD40L。当细胞长到80％～90％时，消化收集细胞，浓度为每毫升1×107细胞。置于辐射仪中进行5000rads辐射，冻存液重悬细胞，浓度为每毫升3.5×107细胞，分装1ml在冷冻小管，液氮冻存(可以保存2年)。
(2)记忆B细胞的分选和活化
用淋巴分离液分离和冻存曾经感染H7N9病毒的康复病人的PBMC，每管10～50×106细胞，冻存在液氮罐中。配制PBMC流式染色液，其成分如下表1所示
表1 PBMC流式染色液
| 抗体 | 体积(μL) |
| CD19-PE-Cy7 | 0.5 |
| IgM-PE | 1.0 |
| IgA-APC | 2.5 |
| IgD-FITC | 2.5 |
| PBS-1％(wt/vol)BSA | 43.5 |
解冻PBMC，加入上述PBMC流式染色液并在流式细胞仪上分选，结果如图2所示，分选出CD19+IgM-IgA-IgD-的记忆B细胞，细胞纯度需在90％以上，若低于90％，重复分选过程。配制激活B细胞的混合培养基，如下表2所示：
表2
| 组分 | 体积 |
| 完全IMDM培养基 | 336mL |
| IL-2(10,000U mL<sup>-1</sup>) | 3.5mL |
| IL-21(100μg mL<sup>-1</sup>) | 175μL |
| 步骤(1)中得到的3T3-CD40L | 10mL |
将记忆B细胞加入到混合培养基中，混匀后有限稀释在384孔板，每孔1个细胞，体积为50μl，置于37℃，5％CO2培养箱中静置培养。13天后，取上清液进行ELISA。
(3)获得人源单克隆抗体7T33
流感病毒血凝素HA是病毒包膜表面柱状抗原，能与人、鸡、豚鼠等多种红细胞受体结合引起红细胞凝集，具有免疫原性，抗血凝素抗体可以中和流感病毒。本发明中，通过ELISA发现了能够分泌结合H7N9病毒的抗体7T33的B细胞，其分泌的人源单克隆抗体7T33可以靶向结合H7N9病毒的血凝素HA(图3)。
ELISA实验具体操作：
(1)将100ng/100μl的H7N9病毒的HA蛋白(购自ACROBiosystems)包被在96孔酶标板中，每孔100μl；
(2)放置4℃冰箱过夜；
(3)用PBST溶液洗涤三遍，每孔加5％的脱脂奶粉溶液200μl，37℃孵育1小时；
(4)用PBST溶液洗涤三遍，加100μl没有感染病毒的正常人血清(阴性对照)或加感染病毒的病人血清或抗H7N9全人源单克隆抗体7T33，各三个重复；
(5)37℃孵育1小时后用PBST溶液洗涤三遍；
(6)以1:5000稀释带HRP的抗人IgG抗体(abcam)，加入酶标版中，每孔100μl；
(7)37℃孵育1小时后用PBST溶液洗涤三遍；
(8)每孔加100μl TMB底物溶液(Thermo Scientific)，37℃5分钟；
(9)每孔加终止溶液2M硫酸100μl，立刻在酶标仪中450nm波长检测吸光值。其结果如图3所示，ELISA实验表明本发明获得的人源单克隆抗体7T33可以靶向结合H7N9病毒的血凝素HA。
实施例2人源化单克隆抗体7T33基因的克隆、重组、表达和纯化
将实施例1获得的能够分泌结合H7N9病毒的7T33抗体的B细胞进行裂解，取裂解液进行RNA的反转录，获得人源抗体基因的PCR模板cDNA。设计和合成克隆抗体基因的引物，以cDNA为模板克隆抗体的重链和轻链的基因，并且重组在真核细胞293F或HEK293中进行表达和纯化。具体地：
(1)将裂解后的B细胞液转移至96孔板(Eppendorf，030133366)。
(2)反转录体系：150ng随机引物(invitrogen,48190-011),0.5μl 10mM dNTP(Invitrogen,18427-088),1μl 0.1M DTT(Invitrogen,18080-044),0.5％v/v Igepal CA-630(Sigma,I3021-50ML),4U RNAsin(Promega),6U Prime RNAse Inhibitor(Eppendorf)and 50UIII reverse transcriptase(Invitrogen,18080-044)，补DEPC水至14μl/well。
(3)反转录反应程序：42℃，10min；25℃，10min；50℃，60min；94℃，5min。
(4)cDNA保存在-20℃。
(5)引物的设计和合成:
正向引物5′-3′序列(Forward Primer 5′-3′sequence)
重链可变区PCR引物:
5′VH1 CTGCAACCGGTGTACATTCCCAGGTGCAGCTGGTGCAG(SEQ ID NO:9)
5′VH1/5 CTGCAACCGGTGTACATTCCGAGGTGCAGCTGGTGCAG(SEQ ID NO:10)
5′VH3CTGCAACCGGTGTACATTCTGAGGTGCAGCTGGTGGAG(SEQ ID NO:11)
5′VH3-23 CTGCAACCGGTGTACATTCTGAGGTGCAGCTGTTGGAG(SEQ ID NO:12)
5′VH4 CTGCAACCGGTGTACATTCCCAGGTGCAGCTGCAGGAG(SEQ ID NO:13)
5′VH 4-34CTGCAACCGGTGTACATTCCCAGGTGCAGCTACAGCAGTG(SEQ ID NO:14)
5′VH 1-18 CTGCAACCGGTGTACATTCCCAGGTTCAGCTGGTGCAG(SEQ ID NO:15)
5′VH 1-24 CTGCAACCGGTGTACATTCCCAGGTCCAGCTGGTACAG(SEQ ID NO:16)
5′VH3-33 CTGCAACCGGTGTACATTCTCAGGTGCAGCTGGTGGAG(SEQ ID NO:17)
5′VH 3-9 CTGCAACCGGTGTACATTCTGAAGTGCAGCTGGTGGAG(SEQ ID NO:18)
5′VH4-39 CTGCAACCGGTGTACATTCCCAGCTGCAGCTGCAGGAG(SEQ ID NO:19)
5′VH 6-1 CTGCAACCGGTGTACATTCCCAGGTACAGCTGCAGCAG(SEQ ID NO:20)
3′JH 1/2/4/5 TGCGAAGTCGACGCTGAGGAGACGGTGACCAG(SEQ ID NO:21)
3′JH 3 TGCGAAGTCGACGCTGAAGAGACGGTGACCATTG(SEQ ID NO:22)
3′JH 6 TGCGAAGTCGACGCTGAGGAGACGGTGACCGTG(SEQ ID NO:23)
κ轻链可变区PCR产物
5′Vκ1-5 CTGCAACCGGTGTACATTCTGACATCCAGATGACCCAGTC(SEQ ID NO:24)
5′Vκ1-9 TTGTGCTGCAACCGGTGTACATTCAGACATCCAGTTGACCCAGTCT(SEQ ID NO:25)
5′Vκ1D-43 CTGCAACCGGTGTACATTGTGCCATCCGGATGACCCAGTC(SEQ ID NO:26)
5′Vκ2-24 CTGCAACCGGTGTACATGGGGATATTGTGATGACCCAGAC(SEQ ID NO:27)
5′Vκ2-28 CTGCAACCGGTGTACATGGGGATATTGTGATGACTCAGTC(SEQ ID NO:28)
5′Vκ2-30 CTGCAACCGGTGTACATGGGGATGTTGTGATGACTCAGTC(SEQ ID NO:29)
5′Vκ3-11
TTGTGCTGCAACCGGTGTACATTCAGAAATTGTGTTGACACAGTC(SEQ ID NO:30)
5′Vκ3-15 CTGCAACCGGTGTACATTCAGAAATAGTGATGACGCAGTC(SEQ ID NO:31)
5′Vκ3-20 TTGTGCTGCAACCGGTGTACATTCAGAAATTGTGTTGACGCAGTCT(SEQ ID NO:32)
5′Vκ4-1 CTGCAACCGGTGTACATTCGGACATCGTGATGACCCAGTC(SEQ ID NO:33)
3′Jκ1/4 GCCACCGTACGTTTGATYTCCACCTTGGTC(SEQ ID NO:34)
3′Jκ2 GCCACCGTACGTTTGATCTCCAGCTTGGTC(SEQ ID NO:35)
3′Jκ3 GCCACCGTACGTTTGATATCCACTTTGGTC(SEQ ID NO:36)
3′Jκ5 GCCACCGTACGTTTAATCTCCAGTCGTGTC(SEQ ID NO:37)
(6)用KOD-Plus-Neo(TOYOBO，KOD401)试剂盒PCR分别扩增抗体基因的重链和轻链，40μL体系：3.5μL cDNA，20nM混合引物，4μL缓冲液(buffer)，4μL2mM dNTPs，2.4μLMgSO4，1μL KOD。
(7)反应程序：94℃，2min；45个循环：98℃,10s；58℃,30s；68℃,28s。
(8)对扩增产物进行琼脂糖凝胶，结果发现，抗体轻链大小为327bp，重链大小是375bp。
(9)抗体基因重链可变区PCR产物测序结果如SEQ ID NO：1所示序列，其相应的氨基酸序列如SEQ ID NO：2所示序列。抗体基因轻链可变区PCR产物测序结果如SEQ ID NO：3所示序列，其相应的氨基酸序列如SEQ ID NO：4所示序列。
依据所得重轻链可变区序列，设计并委托Invitrogen公司合成抗体基因重链全长H基因，其带有BamH1/EcoR1双酶切位点；及抗体基因轻链全长L基因，其带有Not1/Xho1双酶切位点。
(10)将H基因和pcDNA3.1分别进行BamH1/EcoR1双酶切后相连，形成pcDNA3.1-H载体。
(11)将L基因和pcDNA3.1分别进行Not1/Xho1双酶切后相连，形成pcDNA3.1-L载体。
(12)培养293F细胞。
(13)20μg pcDNA3.1-H(该H基因的核苷酸序列如SEQ ID NO：5所示)载体和10μgpcDNA3.1-L(该L基因的核苷酸序列如SEQ ID NO：7所示)载体共转染293F细胞，培养96小时。
(14)取上清液进行ELISA(ABC是上清液，DEF是阳性对照，GH是阴性对照)；ELISA具体的实验步骤如前所述，ELISA实验结果如下表3和图3所示：
表3
| 数据 | 450nm | 数据 | 450nm |
| A | 1.528 | E | 1.185 |
| B | 1.859 | F | 1.201 |
| C | 1.796 | G | 0.0675 |
| D | 1.114 | H | 0.0554 |
由表3和图3数据可知：上清液中含有能够结合H7N9病毒的抗体。
(15)纯化过程，具体地，全人源单克隆抗体7T33的纯化过程为：
(a)200μg pcDNA3.1-L载体和100μg pcDNA3.1-H载体共转染300ml 293F细胞，培养96小时。
(b)收集上清液，加入proteinA亲和层析柱，用10倍PBS清洗，加入2ml pH3.0，0.1M甘氨酸收集抗体。收集管中加入100μl中和缓冲液(1M Tri-HCL)，以便及时中和洗脱所得抗体液的pH值。
(c)在磷酸盐缓冲液(PBS)中透析，透析完后，近期用的存放在4℃，长期储存在-20℃。共获得500μg 7T33纯抗体，其重链氨基酸序列如SEQ ID NO：6所示，轻链氨基酸序列如SEQ ID NO:8所示。
实施例3纯化后的全人源单克隆抗体7T33的中和实验及抗体亲和力实验
(1)实验目的
使用病毒感染细胞模型(犬肾细胞MDCK)，通过微量中和-ELISA实验评价7T33抗体对H7N9流感病毒的抑制作用和效果，检测抗体抗流感病毒活性。
(2)实验步骤
(2.1)细胞铺板
胰酶消化对数生长期MDCK犬肾细胞，终止后离心收集，吹散均匀，制备单细胞悬液；用细胞培养液将细胞浓度调整至5×104个/ml，接种于96孔细胞培养板，细胞置于37℃、5％CO2培养箱中培养过夜。
(2.2)7T33抗体与H7N9病毒(该病毒A/Anhui/1/2013取自于中国科学院微生物研究所)预处理
7T33抗体设立10个浓度梯度，依次为10-1010倍稀释，各组各浓度均设3个平行孔。
(2.3)病毒感染
弃步骤(2.1)培养的细胞培养上清，PBS洗3遍。将预混的抗体-病毒混合液(以102μg/ml的7T33单克隆抗体依次以10-1010倍稀释，将每个浓度的7T33抗体分别与等体积的100TCID50病毒混合得该混合液)。加入96孔细胞培养板，37℃孵育1h，吸弃混合液，PBS洗2遍。
(2.4)配置维持液
用无血清DMEM中加入终浓度为2μg/ml的TPCK-胰蛋白酶(TPCK-Trypsin)(维持液)。弃去96孔板中的PBS，每孔加入100μl维持液，置于37℃，5％CO2培养箱培养20h。
(2.5)中和实验-ELISA法
(2.5.1)弃去微量培养板中的维持液；
(2.5.2)100μl PBS洗细胞一次；
(2.5.3)弃去PBS(不要让细胞干燥)，加入50μl/孔固定液(体积比为丙酮：无水乙醇＝2：3)；
(2.5.4)覆盖微量培养板，于室温固定细胞10min；
(2.5.5)弃去固定液，用100μl PBS液洗涤细胞，重复洗涤3次(轻轻晃动，避免强烈清洗)，以去除残余的丙酮。
(2.5.6)用5％脱脂奶粉于室温封闭细胞1h，用100μl PBS液洗涤细胞1次；
(2.5.7)用PBS1：2000稀释1抗(商购抗H7N9的NP单克隆抗体)每孔加入稀释后的50μl，室温作用1小时。
(2.5.8)用100μl PBST洗板5次以除去1抗；
(2.5.9)用PBS1:2000稀释2抗(带HRP的抗鼠IgG抗体)，每孔加入50μl，室温作用1小时。
(2.5.10)用100μl PBST洗涤板6次以除去2抗。
(2.5.11)每孔加TMB显色液50μl。
(2.5.12)室温避光放10分钟左右显色后，每孔加2M盐酸50μl终止反应。
(2.5.13)在ELISA测定仪上(450纳米)读出每孔OD值。
(3)统计分析
使用GraphPad Prism 6.0.1对数据进行分析和绘制剂量-效应曲线，并计算IC50。
抑制率计算公式：
抑制率＝[(OD病毒孔-OD阴性细胞对照孔)-(OD药物孔-OD阴性细胞对照孔)]/(OD病毒孔-OD阴性细胞对照孔)×100％。
所得结构如图4所示，从图4中可以看出7T33的IC50＝12.84ug/mL。
从实施例3可以看出，本发明7T33对H7N9有良好的IC50值，证明7T33的中和病毒能力好。
本发明对比了2016年05年10日向中国国家知识产权局递交的申请号为“201610303416.X”、发明名称为“抗H7N9全人源单克隆抗体2L11及其制法与应用”中的单克隆抗体2L11，及2016年05月03日向中国国家知识产权局递交的申请号为“201610288358.8”、发明名称为“抗H7N9全人源单克隆抗体2J17及其制法与应用”中的单克隆抗体2J17，本发明7T33中和活性的IC50为12.84μg/mL，而2L11及2J17抗体没有中和活性。研究组2018年07月24日提交了一件“抗H7N9全人源单克隆抗体hIg311及其制法与应用”的申请(申请号：201810818233.0)，目前该申请尚未公开，本发明是在该申请内容的基础上针对重轻链CDR1、CDR2及CDR3区的氨基酸进行了部分氨基酸的改变，以研究其相关功能。
抗体亲和力检测：
亲和力检测的仪器为PALL的Fortebio。配制200μl 50μg/ml 7T33抗体，结合proteinA传感器120秒，HA抗原配制100nM、50nM、2.5nM、12.5nM和、6.25nM和0nM浓度溶液，结合抗体120秒，解离时间为5分钟，显示7T33对H7N9病毒有较高亲和力，KD＝5.43×10-9M。
最后说明的是：以上实施例仅用于说明本发明的实施过程和特点，而非限制本发明的技术方案，尽管参照上述实施例对本发明进行了详细说明，本领域的普通技术人员应当理解：依然可以对本发明进行修改或者等同替换，而不脱离本发明的精神和范围的任何修改或局部替换，均应涵盖在本发明的保护范围当中。
序列表
<110> 中国科学院深圳先进技术研究院
<120> 抗H7N9全人源单克隆抗体7T33及其制法与应用
<130> GAI18CN6778
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<170> SIPOSequenceListing 1.0
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caagtgcagc tggtggagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtc 60
tcctgcaagg cttctggaac catattctac agtactgaaa tcaactgggt gcgacaggcc 120
actggccaag ggcttgagtg gatgggatgg atgaaccctg aaagtggtga gtacggcttt 180
gcacagaagt tccagggcag agtcaccatg accaggaaca cctccataac cacagcctac 240
atggagctga gcagcctgac ttctgaggac acggccgtgt attactgtgc gtatggaaat 300
gcggaatgta gtgctggtgg tagctgcact aattggttcg gtgaaccctg gggccaggga 360
accctggtca ccgtctcctc a 381
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<212> PRT
<213> 人工序列()
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Gln Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Thr Ile Phe Tyr Ser Thr
20 25 30
Glu Ile Asn Trp Val Arg Gln Ala Thr Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Met Asn Pro Glu Ser Gly Glu Tyr Gly Phe Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asn Thr Ser Ile Thr Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Tyr Gly Asn Ala Glu Cys Ser Ala Gly Gly Ser Cys Thr Asn Trp
100 105 110
Phe Gly Glu Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser
115 120 125
<210> 3
<211> 327
<212> DNA
<213> 人工序列()
<400> 3
tcgtctgagc tgactcagga ccctgctgtg tctgtggcct tgggacagac agtcaggatc 60
acatgccaag gagacagact cagaagcact actgcaagct ggtaccagca gaagccagga 120
caggcccctg tacttgtcat ctatggtaaa aacaaccggc cctcagggat cccagaccga 180
ttctctggct ccagctcagg aaacacagct tccttgacca tcactggggc tcaggcggaa 240
gatgaggctg actattactg taactcccgg gaatacagtg gtactcatct ggtgttcggc 300
ggagggacca agctgaccgt cctagta 327
<210> 4
<211> 109
<212> PRT
<213> 人工序列()
<400> 4
Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln
1 5 10 15
Thr Val Arg Ile Thr Cys Gln Gly Asp Arg Leu Arg Ser Thr Thr Ala
20 25 30
Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr
35 40 45
Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser
50 55 60
Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu
65 70 75 80
Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Glu Tyr Ser Gly Thr His
85 90 95
Leu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Val
100 105
<210> 5
<211> 1422
<212> DNA
<213> 人工序列()
<400> 5
atgggctggt cctgcatcat cctgttcctg gtggccaccg ccaccggcca agtgcagctg 60
gtggagtctg gggctgaggt gaagaagcct ggggcctcag tgaaggtctc ctgcaaggct 120
tctggaacca tattctacag tactgaaatc aactgggtgc gacaggccac tggccaaggg 180
cttgagtgga tgggatggat gaaccctgaa agtggtgagt acggctttgc acagaagttc 240
cagggcagag tcaccatgac caggaacacc tccataacca cagcctacat ggagctgagc 300
agcctgactt ctgaggacac ggccgtgtat tactgtgcgt atggaaatgc ggaatgtagt 360
gctggtggta gctgcactaa ttggttcggt gaaccctggg gccagggaac cctggtcacc 420
gtctcctcag ctagcaccaa gggcccatcg gtcttccccc tggcaccctc ctccaagagc 480
acctctgggg gcacagcggc cctgggctgc ctggtcaagg actacttccc cgaaccggtg 540
acggtgtcgt ggaactcagg cgccctgacc agcggcgtgc acaccttccc ggccgtccta 600
cagtcctcag gactctactc cctcagcagc gtggtgaccg tgccctccag cagcttgggc 660
acccagacct acatctgcaa cgtgaatcac aagcccagca acaccaaggt ggacaagaga 720
gttgagccca aatcttgtga caaaactcac acatgcccac cgtgcccagc acctgaactc 780
ctggggggac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc 840
cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag 900
ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag 960
cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 1020
aatggcaagg agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa 1080
accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc 1140
cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc 1200
agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg 1260
cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag 1320
agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac 1380
cactacacgc agaagagcct ctccctgtct ccgggtaaat ga 1422
<210> 6
<211> 457
<212> PRT
<213> 人工序列()
<400> 6
Gln Val Gln Leu Val Glu Ser Gly Ala Glu Val Lys Lys Pro Gly Ala
1 5 10 15
Ser Val Lys Val Ser Cys Lys Ala Ser Gly Thr Ile Phe Tyr Ser Thr
20 25 30
Glu Ile Asn Trp Val Arg Gln Ala Thr Gly Gln Gly Leu Glu Trp Met
35 40 45
Gly Trp Met Asn Pro Glu Ser Gly Glu Tyr Gly Phe Ala Gln Lys Phe
50 55 60
Gln Gly Arg Val Thr Met Thr Arg Asn Thr Ser Ile Thr Thr Ala Tyr
65 70 75 80
Met Glu Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Tyr Gly Asn Ala Glu Cys Ser Ala Gly Gly Ser Cys Thr Asn Trp
100 105 110
Phe Gly Glu Pro Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala
115 120 125
Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser
130 135 140
Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe
145 150 155 160
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly
165 170 175
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu
180 185 190
Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr
195 200 205
Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg
210 215 220
Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
225 230 235 240
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
245 250 255
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
260 265 270
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
275 280 285
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
290 295 300
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
305 310 315 320
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
325 330 335
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
340 345 350
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
355 360 365
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
370 375 380
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
385 390 395 400
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
405 410 415
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
420 425 430
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
435 440 445
Lys Ser Leu Ser Leu Ser Pro Gly Lys
450 455
<210> 7
<211> 696
<212> DNA
<213> 人工序列()
<400> 7
atgggctggt cctgcatcat cctgttcctg gtggccaccg ccaccggctc gtctgagctg 60
actcaggacc ctgctgtgtc tgtggccttg ggacagacag tcaggatcac atgccaagga 120
gacagactca gaagcactac tgcaagctgg taccagcaga agccaggaca ggcccctgta 180
cttgtcatct atggtaaaaa caaccggccc tcagggatcc cagaccgatt ctctggctcc 240
agctcaggaa acacagcttc cttgaccatc actggggctc aggcggaaga tgaggctgac 300
tattactgta actcccggga atacagtggt actcatctgg tgttcggcgg agggaccaag 360
ctgaccgtcc tagtaaccgt ggccgccccc tccgtgttca tcttcccccc ctccgacgag 420
cagctgaagt ccggcaccgc ctccgtggtg tgcctgctga acaacttcta cccccgggag 480
gccaaggtgc agtggaaggt ggacaacgcc ctgcagtccg gcaactccca ggagtccgtg 540
accgagcagg actccaagga ctccacctac tccctgtcct ccaccctgac cctgtccaag 600
gccgactacg agaagcacaa ggtgtacgcc tgcgaggtta cccaccaggg cctgtcctcc 660
cccgtgacca agtccttcaa ccggggcgag tgctag 696
<210> 8
<211> 215
<212> PRT
<213> 人工序列()
<400> 8
Ser Ser Glu Leu Thr Gln Asp Pro Ala Val Ser Val Ala Leu Gly Gln
1 5 10 15
Thr Val Arg Ile Thr Cys Gln Gly Asp Arg Leu Arg Ser Thr Thr Ala
20 25 30
Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Val Leu Val Ile Tyr
35 40 45
Gly Lys Asn Asn Arg Pro Ser Gly Ile Pro Asp Arg Phe Ser Gly Ser
50 55 60
Ser Ser Gly Asn Thr Ala Ser Leu Thr Ile Thr Gly Ala Gln Ala Glu
65 70 75 80
Asp Glu Ala Asp Tyr Tyr Cys Asn Ser Arg Glu Tyr Ser Gly Thr His
85 90 95
Leu Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Val Thr Val Ala
100 105 110
Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser
115 120 125
Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu
130 135 140
Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser
145 150 155 160
Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu
165 170 175
Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val
180 185 190
Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys
195 200 205
Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 9
<211> 38
<212> DNA
<213> 人工序列()
<400> 9
ctgcaaccgg tgtacattcc caggtgcagc tggtgcag 38
<210> 10
<211> 38
<212> DNA
<213> 人工序列()
<400> 10
ctgcaaccgg tgtacattcc gaggtgcagc tggtgcag 38
<210> 11
<211> 38
<212> DNA
<213> 人工序列()
<400> 11
ctgcaaccgg tgtacattct gaggtgcagc tggtggag 38
<210> 12
<211> 38
<212> DNA
<213> 人工序列()
<400> 12
ctgcaaccgg tgtacattct gaggtgcagc tgttggag 38
<210> 13
<211> 38
<212> DNA
<213> 人工序列()
<400> 13
ctgcaaccgg tgtacattcc caggtgcagc tgcaggag 38
<210> 14
<211> 40
<212> DNA
<213> 人工序列()
<400> 14
ctgcaaccgg tgtacattcc caggtgcagc tacagcagtg 40
<210> 15
<211> 38
<212> DNA
<213> 人工序列()
<400> 15
ctgcaaccgg tgtacattcc caggttcagc tggtgcag 38
<210> 16
<211> 38
<212> DNA
<213> 人工序列()
<400> 16
ctgcaaccgg tgtacattcc caggtccagc tggtacag 38
<210> 17
<211> 38
<212> DNA
<213> 人工序列()
<400> 17
ctgcaaccgg tgtacattct caggtgcagc tggtggag 38
<210> 18
<211> 38
<212> DNA
<213> 人工序列()
<400> 18
ctgcaaccgg tgtacattct gaagtgcagc tggtggag 38
<210> 19
<211> 38
<212> DNA
<213> 人工序列()
<400> 19
ctgcaaccgg tgtacattcc cagctgcagc tgcaggag 38
<210> 20
<211> 38
<212> DNA
<213> 人工序列()
<400> 20
ctgcaaccgg tgtacattcc caggtacagc tgcagcag 38
<210> 21
<211> 32
<212> DNA
<213> 人工序列()
<400> 21
tgcgaagtcg acgctgagga gacggtgacc ag 32
<210> 22
<211> 34
<212> DNA
<213> 人工序列()
<400> 22
tgcgaagtcg acgctgaaga gacggtgacc attg 34
<210> 23
<211> 33
<212> DNA
<213> 人工序列()
<400> 23
tgcgaagtcg acgctgagga gacggtgacc gtg 33
<210> 24
<211> 40
<212> DNA
<213> 人工序列()
<400> 24
ctgcaaccgg tgtacattct gacatccaga tgacccagtc 40
<210> 25
<211> 46
<212> DNA
<213> 人工序列()
<400> 25
ttgtgctgca accggtgtac attcagacat ccagttgacc cagtct 46
<210> 26
<211> 40
<212> DNA
<213> 人工序列()
<400> 26
ctgcaaccgg tgtacattgt gccatccgga tgacccagtc 40
<210> 27
<211> 40
<212> DNA
<213> 人工序列()
<400> 27
ctgcaaccgg tgtacatggg gatattgtga tgacccagac 40
<210> 28
<211> 40
<212> DNA
<213> 人工序列()
<400> 28
ctgcaaccgg tgtacatggg gatattgtga tgactcagtc 40
<210> 29
<211> 40
<212> DNA
<213> 人工序列()
<400> 29
ctgcaaccgg tgtacatggg gatgttgtga tgactcagtc 40
<210> 30
<211> 45
<212> DNA
<213> 人工序列()
<400> 30
ttgtgctgca accggtgtac attcagaaat tgtgttgaca cagtc 45
<210> 31
<211> 40
<212> DNA
<213> 人工序列()
<400> 31
ctgcaaccgg tgtacattca gaaatagtga tgacgcagtc 40
<210> 32
<211> 46
<212> DNA
<213> 人工序列()
<400> 32
ttgtgctgca accggtgtac attcagaaat tgtgttgacg cagtct 46
<210> 33
<211> 40
<212> DNA
<213> 人工序列()
<400> 33
ctgcaaccgg tgtacattcg gacatcgtga tgacccagtc 40
<210> 34
<211> 30
<212> DNA
<213> 人工序列()
<400> 34
gccaccgtac gtttgatytc caccttggtc 30
<210> 35
<211> 30
<212> DNA
<213> 人工序列()
<400> 35
gccaccgtac gtttgatctc cagcttggtc 30
<210> 36
<211> 30
<212> DNA
<213> 人工序列()
<400> 36
gccaccgtac gtttgatatc cactttggtc 30
<210> 37
<211> 30
<212> DNA
<213> 人工序列()
<400> 37
gccaccgtac gtttaatctc cagtcgtgtc 30
Claims (16)
1.抗H7N9全人源单克隆抗体7T33或来源于该单克隆抗体的能够特异性结合H7N9的生物活性片段，其中，所述抗体的重轻链CDR1、CDR2及CDR3区的氨基酸序列分别如下所示：
重链CDR1:GTIFYSTE；
重链CDR2:MNPESGEY；
重链CDR3:AYGNAECSAGGSCTNWFGEP；
轻链CDR1:RLRSTT；
轻链CDR2:GKN；
轻链CDR3:NSREYSGTHLV。
2.根据权利要求1所述的抗H7N9全人源单克隆抗体7T33或来源于该单克隆抗体的能够特异性结合H7N9的生物活性片段，其中，所述抗体的重链可变区氨基酸序列如SEQ ID NO:2所示；和/或
所述抗体的轻链可变区氨基酸序列如SEQ ID NO:4所示。
3.根据权利要求2所述的抗H7N9全人源单克隆抗体7T33或来源于该单克隆抗体的能够特异性结合H7N9的生物活性片段，其中，所述抗体的重链氨基酸序列如SEQ ID NO:6所示；和/或
所述抗体的轻链氨基酸序列如SEQ ID NO:8所示。
4.编码权利要求1所述的抗H7N9全人源单克隆抗体7T33或来源于该单克隆抗体的能够特异性结合H7N9的生物活性片段的基因。
5.根据权利要求4所述的基因，其中，所述基因包含编码具有SEQ ID NO:2所示的氨基酸的核苷酸序列，和/或
所述基因包含编码具有SEQ ID NO:4所示的氨基酸的核苷酸序列。
6.根据权利要求5所述的基因，其中，编码具有SEQ ID NO:2所示的氨基酸的核苷酸序列如SEQ ID NO:1所示；编码具有SEQ ID NO:4所示的氨基酸的核苷酸序列如SEQ ID NO:3所示。
7.根据权利要求4所述的基因，其中，所述基因包含编码具有SEQ ID NO:6所示的氨基酸的核苷酸序列；和/或
所述基因包含编码具有SEQ ID NO:8所示的氨基酸的核苷酸序列。
8.根据权利要求7所述的基因，其中，编码具有SEQ ID NO:6所示的氨基酸的核苷酸序列如SEQ ID NO:5所示；编码具有SEQ ID NO:8所示的氨基酸的核苷酸序列如SEQ ID NO:7所示。
9.一种载体，其包含权利要求4-8任一项所述的基因。
10.一种细胞，其含有权利要求4-8任一项所述基因或含有权利要求9所述的载体。
11.一种产生权利要求1-3任一项所述抗H7N9全人源单克隆抗体7T33或来源于该单克隆抗体的能够特异性结合H7N9的生物活性片段的方法，该方法包括：培养含编码抗H7N9全人源单克隆抗体7T33的重轻链的权利要求4-8任一项所述的基因或权利要求9所述的载体的基因工程细胞，或直接培养权利要求10所述的细胞；收集，纯化得所述抗H7N9全人源单克隆抗体7T33。
12.一种药物组合物，其包含权利要求1-3任一项所述的抗H7N9全人源单克隆抗体7T33或来源于该单克隆抗体的能够特异性结合H7N9的生物活性片段。
13.权利要求1-3任一项所述的抗H7N9全人源单克隆抗体7T33或来源于该单克隆抗体的能够特异性结合H7N9的生物活性片段、或权利要求12所述的药物组合物在制备用于治疗由H7N9病毒引起的疾病的药物中的应用。
14.一种检测H7N9病毒水平的试剂盒，其含有权利要求1-3任一项所述的抗H7N9全人源单克隆抗体7T33或来源于该单克隆抗体的能够特异性结合H7N9的生物活性片段。
15.根据权利要求14所述的检测H7N9病毒水平的试剂盒，所述的试剂盒还含有：第二抗体和用于检测的酶或荧光或放射标记物，以及缓冲液。
16.根据权利要求15所述的检测H7N9病毒水平的试剂盒，所述第二抗体为抗权利要求1-3任一项所述单克隆抗体7T33的抗抗体。
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