CN110483637B - 一种抗禽流感病毒神经氨酸酶n2单克隆抗体、编码基因及其应用 - Google Patents
一种抗禽流感病毒神经氨酸酶n2单克隆抗体、编码基因及其应用 Download PDFInfo
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- CN110483637B CN110483637B CN201910659588.4A CN201910659588A CN110483637B CN 110483637 B CN110483637 B CN 110483637B CN 201910659588 A CN201910659588 A CN 201910659588A CN 110483637 B CN110483637 B CN 110483637B
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Abstract
本发明公开了一种抗禽流感病毒神经氨酸酶N2单克隆抗体、编码基因及其应用，属于生物医学技术领域。抗禽流感病毒神经氨酸酶N2单克隆抗体特异性地识别禽流感病毒神经氨酸酶蛋白N2，具有抑制神经氨酸酶活性和中和病毒能力。本发明还克隆了抗禽流感病毒神经氨酸酶N2单克隆抗体的轻、重链可变区基因序列和氨基酸序列，确认了基因序列和氨基酸序列的唯一性。本发明涉及的单克隆抗体、可变区基因及氨基酸序列可用于N2的禽流感病毒的诊断和治疗药物研发等，具有广泛的临床应用价值。
Description
技术领域
本发明属于生物医学技术领域，涉及特异性识别禽流感病毒神经氨酸酶N2、具有抑制神经氨酸酶活性功能和中和病毒能力的单克隆抗体及其轻、重链可变区基因以及应用。
背景技术
禽流感是由A型流感病毒引起禽类的一种从呼吸系统到严重全身败血症等多种症状的急性高度接触性传染病。A型流感病毒基因组分为八个节段组成，包括PB2、PB1、PA、HA、NP、NA、M和NS。血凝素(HA)和神经氨酸酶(NA)是位于流感病毒囊膜表面的糖蛋白，是病毒亚型和毒株分类的重要依据。目前已知A型流感病毒有16个HA亚型(H1～H16)，10个NA亚型，在流感病毒复制过程中容易发生重组产生新的亚型毒株。H9N2亚型禽流感病毒在禽类中广泛分布，为流感病毒的变异和重组提供了有利的条件。H9N2亚型流感病毒对禽类呈现低致病力，感染禽时仅表现轻微的呼吸道症状。产蛋禽感染则表现产蛋下降，甚至产蛋完全停止。但是，H9N2亚型流感病毒容易与其他致病菌和病毒发生协同作用，造成混合感染和继发感染使得发病率和死亡率显著提升。此外，H9N2亚型流感病毒可以通过病毒受体位点突变，从而突破宿主屏障感染哺乳动物和人。同时，已有报道H9N2亚型流感病毒为高致病性流感病毒提供内部基因。因此，H9N2流感病毒对人类生产和自身健康都有着潜在威胁。
NA具有神经氨酸酶活性，可以水解细胞表面受体特异性糖蛋白的N-2酰基神经氨酸，利于流感病毒粒子的成熟和释放。目前人类临床上常用抗流感病毒药物主要为干扰病毒M2蛋白功能的金刚烷胺,以及包括奥司他韦、扎那米韦、帕拉米韦和拉尼那米韦在内的神经氨酸酶抑制剂。然而，随着抗流感病毒药物的广泛应用，药物的副作用和病毒耐药问题日趋严重。因此，研制具有抑制H9N2流感病毒神经氨酸酶活性的中和抗体，不但能够为流感病毒的诊断提供新方法，也将为设计预防和治疗N2亚型流感病毒感染的药物提供新思路。
发明内容
为了克服上述缺陷，本发明提供了一种抗禽流感病毒神经氨酸酶N2单克隆抗体、编码基因及其应用，获得了该抗体轻、重链可变区基因序列和氨基酸序列。该抗体代号为N2-QW，N2-QW单克隆抗体具有神经氨酸酶抑制效果。N2-QW单克隆抗体具有体外抑制病毒复制能力。
为了实现上述发明目的，本发明采用的技术方案为：一种抗禽流感病毒神经氨酸酶N2单克隆抗体，该抗体包含：重链和轻链；
所述的重链和轻链均包括可变区，该可变区包括互补决定区；
所述重链的互补决定区CDR1、CDR2和CDR3分别用HCDR1、HCDR2、HCDR3表示；
所述轻链的互补决定区CDR1、CDR2和CDR3分别用LCDR1、LCDR2、LCDR3表示，其特征在于，HCDR1、HCDR2、HCDR3的序列分别如SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3所示；LCDR1、LCDR2、LCDR3的序列分别如SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9所示。
优选的，重链可变区的序列如SEQ ID NO.19所示，轻链可变区的序列如SEQ IDNO.20所示。
所述的单克隆抗体重链为1gG1亚型，轻链为Kappa型。
本发明还提供核苷酸序列，所述的核苷酸序列编码前述的抗禽流感病毒神经氨酸酶N2单克隆抗体。所述的核苷酸序列包括编码抗禽流感病毒神经氨酸酶N2单克隆抗体重链可变区的SEQ ID NO.14以及编码抗禽流感病毒神经氨酸酶N2单克隆抗体轻链可变区的SEQID NO.13。
一种表达载体，该表达载体含有前述的核苷酸分子。
一种宿主细胞或非人生物体，含有前述的表达载体。
一种抗禽流感病毒神经氨酸酶N2单克隆抗体的制备方法，该制备方法包含如下步骤：
步骤1：制备含有表达所述的抗禽流感病毒神经氨酸酶N2单克隆抗体的核苷酸分子的表达载体；
步骤2：用步骤1的表达载体转染真核宿主细胞；
步骤3：培养步骤2转染的真核宿主细胞；
步骤4：分离纯化，获得所述的抗体。
本发明还公开抗禽流感病毒神经氨酸酶N2单克隆抗体在制备抑制神经氨酸酶活性功能的药物中的用途；以及抗禽流感病毒神经氨酸酶N2单克隆抗体在制备用于中和N2的禽流感病毒的药物或N2的禽流感诊断试剂中的用途。
相对于现有技术，本发明的有益效果：
1、天然病毒免疫小鼠，增强小鼠对病毒神经氨酸酶四聚体蛋白的反应性。
2、利用免疫荧光(IFA)验证了N2-QW单克隆抗体可以特异性识别H9N2和H1N2禽流感病毒神经氨酸酶蛋白。
3、神经氨酸酶抑制实验(ELLA实验)证明640倍稀释的N2-QW单克隆抗体具有很好的神经氨酸酶抑制效果。
4、微量中和实验(MN)结果显示3.12μg的N2-QW单克隆抗体即可完全抑制100个TCID50H9N2禽流感病毒的复制。
5、获得了N2-QW单克隆抗体轻重链可变区基因序列和氨基酸序列，确认了该单克隆抗体序列的唯一性。
N2-QW单克隆抗体特异性地识别禽流感病毒神经氨酸酶N2，具有抑制神经氨酸酶活性功能和中和病毒能力。本发明提供的具有抑制神经氨酸酶活性的抗N2的禽流感病毒单克隆抗体及其轻重链可变区基因序列和氨基酸序列，可以应用于神经氨酸酶抑制剂类的抗流感病毒药物的研发。
附图说明
图1为N2-QW单克隆抗体与转染pCAGGS-NA的COS-1细胞IFA结果，用于鉴定与神经氨酸酶N2的反应，图1A N2-QW单克隆抗体转染pCAGGS-NA的COS-1细胞IFA结果为阳性，图1B阴性对照(N2-QW单克隆抗体与无pCAGGS-NA的COS-1细胞的IFA结果为阴性)；
图2为ELLA实验检测N2-QW单克隆抗体的神经氨酸酶的抑制效果；
图3为抗体轻重链PCR扩增结果；
图4为轻链可变区基因IgBLAST结果；
图5为重链可变区基因IgBLAST结果。
具体实施方式
下面结合具体的实施例，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。此外应理解在阅读了本发明讲授的内容之后，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申请所附权利要求书所限定的范围。
1、小鼠源抗禽流感病毒神经氨酸酶N2单克隆抗体的制备
1.1小鼠免疫
以含有H9N2流感病毒(A/Chicken/Jiangsu/X1/2004(X1))的鸡胚尿囊液腹腔注射免疫BALB/c小鼠，每两周免疫一次，每次0.3mL。三免10天后测定抗体滴度，细胞融合前三天加强免疫小鼠一次，腹腔免疫0.3mL，尾静脉免疫0.1mL。
1.2小鼠骨髓瘤细胞sp2/0的准备
用无血清培养基DMEM吹下已生长良好的小鼠骨髓瘤细胞sp2/0，待融合用。
1.3小鼠脾细胞和腹腔巨噬细胞的准备
加强免疫小鼠(即1.1免疫后的小鼠)致死后，浸于70％酒精。于超净台无菌取出小鼠脾脏，置于少量DMEM中，用1mL注射器针头扎刺释放脾细胞，至脾脏组织从血红色变为灰白色。
另取一只ICR小鼠，致死后浸于70％酒精。于超净台内剪开腹部皮肤，用10ml注射器吸取HAT培养基注入小鼠腹腔。按摩小鼠腹部，吸出含有腹腔巨噬细胞的HAT培养基注入90mlHAT培养基中待用。
1.4 PEG诱导细胞融合
将上述准备的小鼠骨髓瘤细胞和脾细胞混合后，1000rpm离心10min。弃尽上清，拍打混匀细胞后于1min内缓缓加入37℃预热的PEG溶液。然后，以先慢后快原则加入30mL无血清DMEM培养基，终止细胞融合。静置10min后，1000rpm离心10min，弃上清，重悬后加入上述含有腹腔巨噬细胞的90mlHAT培养基中。将细胞分装于6块96孔板中，于37℃,5％CO2的培养箱中培养。5天后更换HAT培养基，10天后吸出上清检测。
2、抗神经氨酸酶单克隆抗体筛选与杂交瘤亚克隆
取生长状态良好的鸡胚成纤维细胞(CEF)，0.005％胰酶消化后，分装于96孔细胞板，每孔5万CEF细胞。24h后加入含2μg/mLTPCK胰酶和1％血清的维持液稀释的H9N2的病毒液100μL。24h后用丙酮乙醇固定，晾干作为IFA检测细胞板，同时固定转染表达禽流感病毒神经氨酸酶N2的pCAGGS-NA质粒的COS-1细胞，筛选抗神经氨酸酶单克隆抗体。
2.1 IFA检测杂交瘤上清
向固定的检测板加入100μL N2-QW杂交瘤(能分泌本发明的N2-QW单克隆抗体)上清，于37℃恒温水浴锅孵育30min后，PBS洗3遍，加入1:100稀释的FITC羊抗鼠二抗100μL/孔，37℃孵育30min后PBS洗5遍，加入50％甘油，荧光显微镜下观察荧光。N2-QW杂交瘤克隆上清可见与转染pCAGGS-NA质粒的COS-1细胞特异性反应(图1A)。
2.2杂交瘤亚克隆与亚类鉴定
将N2-QW杂交瘤克隆扩大培养，取10μL细胞悬液稀释至50个/mL，然后定容到5mL，然后以100μL/孔分装到半块96孔板中。培养第6天检测细胞上清，对阳性孔再进行同样的两次亚克隆，至检测阳性率达90-100％。
采用Thermo公司的小鼠免疫球蛋白亚类鉴定ELISA试剂盒进行亚类鉴定，具体步骤见Thermo说明书。N2-QW单克隆抗体的重链鉴定为IgG1类型，轻链鉴定为kappa类型。
3、单克隆抗体纯化与单克隆抗体中和效价检测
3.1腹水制备与纯化
取10只8周龄BALB/C小鼠于提前一周每只小鼠腹腔注射0.5mL石蜡油。取生长状态良好的阳性克隆杂交瘤细胞，弃上清，用DMEM培养基吹打细胞，800rpm离心5min，去上清。用DMEM重悬细胞并稀释至500万/mL，每只小鼠腹腔注射0.5mL。四到五天后，待小鼠腹腔膨胀明显开始采集腹水。采取腹水5000rpm离心5min，取上清于-70℃冻存。采用Protein G抗体纯化柱纯化，具体步骤参考说明书71-7001-00 AR(GE医疗)。
3.2 ELLA实验
(1)以包被液将胎球蛋白(fetuin)过夜包被，所有包被酶标板板均4℃静置12h以上。
(2)包被板经PBST洗3遍后，加入梯度稀释抗体与最佳稀释度的病毒的混合液，每稀释度100μL/孔，再37℃作用16-18h。同时稀释抗新城疫F蛋白的单抗F-1D10作为阴性对照。
(3)PBST洗6遍后，加入1：1000稀释的HRP-PNA，100μL/孔，37℃作用2h。
(4)PBST洗6遍后，加入TMB显色液，100μL/孔，37℃显色15min。
(5)每孔加入100μL1％SDS终止显色，酶标仪OD650读值。
(6)抗体抑制率的计算方法如下：
实验结果显示，N2-QW单克隆抗体在20-640倍稀释度(稀释640倍后抗体浓度为0.0003ug/ml)时保持较好的神经氨酸酶抑制能力，在大于640倍稀释时抑制能力降低，而阴性对照抗体(抗新城疫F蛋白的单抗F-1D10)无神经氨酸酶抑制能力(图2)。
3.3中和实验
取生长状态良好的犬肾细胞系(MDCK)细胞，2万/孔上96孔板。24h后，另取96孔板，每孔加入50μL的含2μg/mL TPCK胰酶的opti-MEM，向第一孔中加入200μg/ml的纯化N2-QW单克隆抗体，然后倍比稀释至第11孔，最后一孔弃去50μL。以TPCK胰酶浓度为2μg/mL的opti-MEM将H9N2流感病毒稀释至2000个TCID50/mL，以50μL/孔加入倍比稀释的抗体中，37℃作用30min。
弃去MDCK细胞培养基，PBS洗两次，加入上述抗体和病毒混合液37℃、5％CO2培养箱培养72h后测定各孔HA效价，HA阴性的最大抗体稀释度即为抗体中和效价。N2-QW单克隆抗体对H9N2禽流感病毒的最低抑制浓度为3.12μg/mL，说明N2-QW单克隆抗体可以很好地抑制H9N2禽流感病毒的复制。
4单克隆抗体Fab基因测序
4.1 RNA提取与cDNA合成
取一瓶生长状态良好的N2-QW杂交瘤细胞，用AxyPrep总RNA小量制备试剂盒提取杂交瘤细胞总RNA。获得的总RNA以Oligo(dT)18为引物用Thermo Scientific RevertAidReverse Transcriptase反转录成cDNA。同时提取sp2/0骨髓瘤细胞总RNA反转录成cDNA作为阴性对照模板。
4.2 PCR扩增与连接T载体测序
以表1中针对小鼠IgG1轻重链可变区的引物扩增抗体可变区基因，VLF和VLR为扩增轻链基因的上下游引物，VHF和VHR为扩增重链基因的上下游引物。PCR产物经琼脂糖凝胶电泳胶回收660bp左右目的轻链可变区片段(图3中泳道3)和重链可变区片段(图3中泳道4)。两片段分别连接T载体后，经蓝白斑筛选将阳性克隆质粒送测序。测序结果经IgBLAST(https://www.ncbi.nlm.nih.gov/igblast/)分析，获得轻、重链的可变区基因序列及CDR构成(图4和图5)。
表1轻、重链可变区基因扩增用引物
注：K＝G/T,W＝A/T,R＝A/G,Y＝C/T,M＝A/C。
以上引物序列依次为SEQ ID NO.15-18。
由图4和图5可见：
N2-QW单克隆抗体轻链可变区基因序列的3个互补决定区(CDR)的核苷酸序列为：
CDR1：CAGAGTGTGAATAATGAT(SEQ ID NO.10)
CDR2：TATGCATCC(SEQ ID NO.11)
CDR3：CAGCAGGATTATACCTCTCCATTCACG(SEQ ID NO.12)
对应的氨基酸序列分别为：
LCDR1：Gln-Ser-Val-Asn-Asn-Asp(SEQ ID NO.7)即：QSVNND
LCDR2：Tyr-Ala-Ser(SEQ ID NO.8)即：YAS
LCDR3：Gln-Gln-Asp-Tyr-Thr-Ser-Pro-Phe-Thr(SEQ ID NO.9)即：QQDYTSPFTN2-QW单克隆抗体重链可变区基因序列的3个互补决定区(CDR)的核苷酸序列为：
CDR1：GGCTACTACATCACCAGTGATTTTACC(SEQ ID NO.4)
CDR2：ATACACTACAATGGTAACAGT(SEQ ID NO.5)
CDR3：GCAAAATACTCGTTTGGTAACTACGAATTTTTCGATGTC(SEQ ID NO.6)
对应的氨基酸序列分别为：
HCDR1：Gly-Tyr-Tyr-Ile-Thr-Ser-Asp-Phe-Thr(SEQ ID NO.1)即：GYYITSDFT
HCDR2：Ile-His-Tyr-Asn-Gly-Asn-Ser(SEQ ID NO.2)即：IHYNGNS
HCDR3：Ala-Lys-Tyr-Ser-Phe-Gly-Asn-Tyr-Glu-Phe-Phe-Asp-Val(SEQ IDNO.3)即：AKYSFGNYEFFDV
轻链可变区基因
5’-AGTATTGTGATGACCCAGATTCCCGAATTCCTGCTTGTATCAGCTGGAGACAGGGTAACCATAACCTGCAAGGCCAGTCAGAGTGTGAATAATGATGTAACTTGGTTCCAACAGAAGCCAGGGCAGTCTCCTAAACTGCTGATATACTATGCATCCAATCGCTACACTGGAGTCCCTGATCGCTTCACTGGCAGTGGATTTGGGACGTATTTCACTTTCACCATCAACACTGTGCAGGCTGAGGACCTGGCAGTTTATTTCTGTCAGCAGGATTATACCTCTCCATTCACGTTCGGCTCGGGGACAAAATTGGAAATAAAA-3’(SEQ ID NO.13)
重链可变区基因
5’-CAGGTGCAGCTGAAGGAGTCAGGACCTGACCTGGTGAAACCTTCTCAGTCACTTTCACTCACCTGCACTGTCTCTGGCTACTACATCACCAGTGATTTTACCTGGCACTGGATCCGACAATTTCCAGGAAACAAACTGGAATGGATGGGCTACATACACTACAATGGTAACAGTTACTACAACCCATCTCTCGAAAGTCGAATCTCTATCACTCGAGACACATCCAAGAATCAGTTCTTCCTGCAGTTGAGTTCTGTGACTGCTGAGGACACAGCCACATATTACTGTGCAAAATACTCGTTTGGTAACTACGAATTTTTCGATGTCTGGGGGGCAGGGATCACGGTCACCGTCTCCTCA-3’(SEQ IDNO.14)轻链可变区氨基酸序列
SIVMTQIPEFLLVSAGDRVTITCKASQSVNNDVTWFQQKPGQSPKLLIYYASNRYTGVPDRFTGSGFGTYFTFTINTVQAEDLAVYFCQQDYTSPFTFGSGTKLEIK(SEQ ID NO.20)
重链可变区氨基酸序列
QVQLKESGPDLVKPSQSLSLTCTVSGYYITSDFTWHWIRQFPGNKLEWMGYIHYNGNSYYNPSLESRISITRDTSKNQFFLQLSSVTAEDTATYYCAKYSFGNYEFFDVWGAGITVTVSS(SEQ ID NO.19)
综上所述，本发明公开了一种抗禽流感病毒神经氨酸酶N2单克隆抗体，利用病毒免疫小鼠，使用间接免疫荧光方法成功筛选获得一株特异性与禽流感病毒神经氨酸酶N2反应的N2-QW单克隆抗体。该单克隆抗体的重链鉴定为IgG1类型，轻链鉴定为kappa类型。神经氨酸酶抑制实验和体外抗体中和实验表面N2-QW单克隆抗体具有很好的神经氨酸酶抑制效果，从而限制病毒颗粒的释放抑制病毒的复制。本发明还克隆了N2-QW单克隆抗体轻、重链的可变区基因序列和氨基酸序列，确认了单克隆抗体基因序列与蛋白序列的唯一性。本发明涉及的单克隆抗体、可变区基因及氨基酸序列可用于N2的禽流感病毒的诊断和治疗药物研发等，具有广泛的临床应用价值。
本发明所述的实例是对本发明的说明而不能限制本发明，在与本发明相当的含义和范围内的任何改变和调整，都应认为是在本发明的范围内。
序列表
<110> 扬州大学
<120> 一种抗禽流感病毒神经氨酸酶N2单克隆抗体、编码基因及其应用
<130> xhx2019072201
<141> 2019-07-22
<160> 20
<170> SIPOSequenceListing 1.0
<210> 1
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Gly Tyr Tyr Ile Thr Ser Asp Phe Thr
1 5
<210> 2
<211> 7
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Ile His Tyr Asn Gly Asn Ser
1 5
<210> 3
<211> 13
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Ala Lys Tyr Ser Phe Gly Asn Tyr Glu Phe Phe Asp Val
1 5 10
<210> 4
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
ggctactaca tcaccagtga ttttacc 27
<210> 5
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
atacactaca atggtaacag t 21
<210> 6
<211> 39
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
gcaaaatact cgtttggtaa ctacgaattt ttcgatgtc 39
<210> 7
<211> 6
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 7
Gln Ser Val Asn Asn Asp
1 5
<210> 8
<211> 3
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 8
Tyr Ala Ser
1
<210> 9
<211> 9
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 9
Gln Gln Asp Tyr Thr Ser Pro Phe Thr
1 5
<210> 10
<211> 18
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
cagagtgtga ataatgat 18
<210> 11
<211> 9
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
tatgcatcc 9
<210> 12
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
cagcaggatt atacctctcc attcacg 27
<210> 13
<211> 321
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
agtattgtga tgacccagat tcccgaattc ctgcttgtat cagctggaga cagggtaacc 60
ataacctgca aggccagtca gagtgtgaat aatgatgtaa cttggttcca acagaagcca 120
gggcagtctc ctaaactgct gatatactat gcatccaatc gctacactgg agtccctgat 180
cgcttcactg gcagtggatt tgggacgtat ttcactttca ccatcaacac tgtgcaggct 240
gaggacctgg cagtttattt ctgtcagcag gattatacct ctccattcac gttcggctcg 300
gggacaaaat tggaaataaa a 321
<210> 14
<211> 360
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
caggtgcagc tgaaggagtc aggacctgac ctggtgaaac cttctcagtc actttcactc 60
acctgcactg tctctggcta ctacatcacc agtgatttta cctggcactg gatccgacaa 120
tttccaggaa acaaactgga atggatgggc tacatacact acaatggtaa cagttactac 180
aacccatctc tcgaaagtcg aatctctatc actcgagaca catccaagaa tcagttcttc 240
ctgcagttga gttctgtgac tgctgaggac acagccacat attactgtgc aaaatactcg 300
tttggtaact acgaattttt cgatgtctgg ggggcaggga tcacggtcac cgtctcctca 360
<210> 15
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
atgkccccwr ctcagytyct kgt 23
<210> 16
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
acactcattc ctgttgaagc tcttgac 27
<210> 17
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 17
caggtgcagc tkmaggagtc a 21
<210> 18
<211> 22
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 18
acaatccctg ggcacaattt tc 22
<210> 19
<211> 120
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 19
Gln Val Gln Leu Lys Glu Ser Gly Pro Asp Leu Val Lys Pro Ser Gln
1 5 10 15
Ser Leu Ser Leu Thr Cys Thr Val Ser Gly Tyr Tyr Ile Thr Ser Asp
20 25 30
Phe Thr Trp His Trp Ile Arg Gln Phe Pro Gly Asn Lys Leu Glu Trp
35 40 45
Met Gly Tyr Ile His Tyr Asn Gly Asn Ser Tyr Tyr Asn Pro Ser Leu
50 55 60
Glu Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Phe Phe
65 70 75 80
Leu Gln Leu Ser Ser Val Thr Ala Glu Asp Thr Ala Thr Tyr Tyr Cys
85 90 95
Ala Lys Tyr Ser Phe Gly Asn Tyr Glu Phe Phe Asp Val Trp Gly Ala
100 105 110
Gly Ile Thr Val Thr Val Ser Ser
115 120
<210> 20
<211> 107
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 20
Ser Ile Val Met Thr Gln Ile Pro Glu Phe Leu Leu Val Ser Ala Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Asn Asn Asp
20 25 30
Val Thr Trp Phe Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile
35 40 45
Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly
50 55 60
Ser Gly Phe Gly Thr Tyr Phe Thr Phe Thr Ile Asn Thr Val Gln Ala
65 70 75 80
Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln Asp Tyr Thr Ser Pro Phe
85 90 95
Thr Phe Gly Ser Gly Thr Lys Leu Glu Ile Lys
100 105
Claims (11)
1.一种抗禽流感病毒神经氨酸酶N2单克隆抗体，该抗体包含：重链和轻链；
所述的重链和轻链均包括可变区，该可变区包括互补决定区；
所述重链的互补决定区CDR1、CDR2和CDR3分别用HCDR1、HCDR2、HCDR3表示；
所述轻链的互补决定区CDR1、CDR2和CDR3分别用LCDR1、LCDR2、LCDR3表示，其特征在于，HCDR1、HCDR2、HCDR3的序列分别如SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3所示；LCDR1、LCDR2、LCDR3的序列分别如SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9所示。
2.根据权利要求1所述的一种抗禽流感病毒神经氨酸酶N2单克隆抗体，其特征在于，重链可变区的序列如SEQ ID NO.19所示，轻链可变区的序列如SEQ ID NO.20所示。
3.根据权利要求1所述的一种抗禽流感病毒神经氨酸酶N2单克隆抗体，其特征在于，所述单克隆抗体的重链为1gG1亚型，轻链为Kappa型。
4.一种核苷酸分子，其特征在于，所述的核苷酸分子编码权利要求1-3任意一项所述的抗禽流感病毒神经氨酸酶N2单克隆抗体。
5.根据权利要求4所述的核苷酸分子，其特征在于，所述的核苷酸分子包括编码抗禽流感病毒神经氨酸酶N2单克隆抗体重链可变区的SEQ ID NO.14以及编码抗禽流感病毒神经氨酸酶N2单克隆抗体轻链可变区的SEQ ID NO.13。
6.一种表达载体，其特征在于，该表达载体含有如权利要求5所述的核苷酸分子。
7.一种宿主细胞，其特征在于，该宿主细胞含有如权利要求6所述的表达载体。
8.一种权利要求1-3任意一项所述的抗禽流感病毒神经氨酸酶N2单克隆抗体的制备方法，其特征在于，该制备方法包含如下步骤：
步骤1：制备含有表达所述的抗禽流感病毒神经氨酸酶N2单克隆抗体的核苷酸分子的表达载体；
步骤2：用步骤1的表达载体转染真核宿主细胞；
步骤3：培养步骤2转染的真核宿主细胞；
步骤4：分离纯化，获得所述的抗体。
9.权利要求1-3任意一项所述的抗禽流感病毒神经氨酸酶N2单克隆抗体在制备抑制神经氨酸酶N2活性功能的药物中的用途。
10.权利要求1-3任意一项所述的抗禽流感病毒神经氨酸酶N2单克隆抗体在制备用于中和N2的禽流感病毒的药物中的用途。
11.权利要求1-3任意一项所述的抗禽流感病毒神经氨酸酶N2单克隆抗体在制备N2禽流感诊断试剂中的用途。
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| CN105985433A (zh) * | 2015-02-09 | 2016-10-05 | 复旦大学 | 针对h7n9亚型禽流感病毒的全人源单克隆抗体及应用 |
| CN107056938A (zh) * | 2017-05-23 | 2017-08-18 | 深圳普兰达科技有限公司 | 人源抗h7n9禽流感病毒高亲和力抗体10k及其应用 |
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| CN107056938A (zh) * | 2017-05-23 | 2017-08-18 | 深圳普兰达科技有限公司 | 人源抗h7n9禽流感病毒高亲和力抗体10k及其应用 |
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