CN109553680B - 一种单克隆抗体ZKns4B8及其应用 - Google Patents
一种单克隆抗体ZKns4B8及其应用 Download PDFInfo
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- CN109553680B CN109553680B CN201811520706.5A CN201811520706A CN109553680B CN 109553680 B CN109553680 B CN 109553680B CN 201811520706 A CN201811520706 A CN 201811520706A CN 109553680 B CN109553680 B CN 109553680B
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Abstract
本发明属于生物技术领域，具体涉及一种单克隆抗体ZKns4B8及其应用。本发明提供的单克隆抗体ZKns4B8是一种针对寨卡病毒NS1蛋白的全人源单克隆抗体，包括具有SEQ ID NO.1所示氨基酸序列的重链可变区域和SEQ ID NO.2所示氨基酸序列的轻链可变区域。本发明提供的单克隆抗体ZKns4B8中的重链和轻链是自然配对，故抗体的亲和力高。与寨卡病毒NS1蛋白特异性结合，不与登革病毒NS1交叉，可以作为诊断或治疗性抗体。
Description
技术领域
本发明属于生物技术领域，具体涉及一种单克隆抗体ZKns4B8及其应用。
背景技术
2015年至2016年寨卡病毒（ZIKV）在世界范围尤其是美洲地区迅速传播，因其可导致严重的出生缺陷及成人神经系统并发症，已被WHO宣布为国际公共卫生紧急事件。
ZIKV同样经伊蚊传播，常出现在登革病毒（DENV）流行区。ZIKV与DENV常出现在同一地区，但临床特征、危害及处置不同。急需尽早区别这两种病毒感染，尤其是对怀孕的妇女而言。ZIKV和DENV同属黄病毒，亲缘关系相近。由于基因组和蛋白质序列及构象上的相似所导致的交叉反应，为区分这两种病毒感染带来了困难。
血清多克隆抗体及从感染者分离的单抗研究表明，ZIKV诱导宿主产生的针对病毒膜蛋白E抗体存在广泛的交叉反应。而针对病毒非结构蛋白1（NS1）的抗体呈现高度特异性反应，为鉴别这两种病毒感染提供了可能。Balmaseda等（2017年）用从ZIKV感染者分离仅结合ZIKV NS1单抗，建立了基于NS1的竞争ELISA法，用于ZIKV感染诊断，并区别其它黄病毒如登革4个血清型、西尼罗病毒及黄热病毒。
目前针对ZIKV尚无有效的疫苗，更缺乏早期诊断及有效的治疗方法。NS1蛋白在黄病毒生命周期中起着重要作用，包括病毒复制、免疫逃逸以及在致病机制中的作用。已明确NS1在登革热发病机制中起着重要作用，与疾病的严重性相关。因此针对寨卡病毒NS1蛋白的全人单抗可能成为寨卡病毒病治疗的一种策略。
单抗的制备技术经历了鼠源抗体的杂交瘤技术、鼠人嵌合抗体、鼠抗体人源技术、继而出现人单抗制备技术。目前可用于大规模特异性抗体筛选的主要为噬菌体展示技术及单个B细胞抗体技术。噬菌体展示技术可用于筛选分离针对任何靶抗原的抗体。然而从随机组合抗体的重链及轻链库中筛选出的特异性抗体，其重链和轻链配对不是自然产生抗体原有的，影响抗体的亲和力和功能。单个B细胞抗体技术直接从单个B细胞扩增抗体重链和轻链的可变区基因，构建抗体表达载体，继而在细胞培养体系中表达抗体。这种技术可分离主要存在感染者体内构象特异的功能性抗体。
中国专利申请CN107188935A公开了一种寨卡病毒NS1抗原突变体及其应用，该抗原突变体通过对同源性高的片段进行基因敲除突变，然后经基因合成，载体构建，原核表达，抗原纯化及免疫学功能性分析筛选获得，具有降低登革阳性样本检出率的作用。但是，这种抗原突变体突变率低，只是降低了寨卡病毒与登革病毒抗体检测的交叉，无法根除。
综上可知，现有技术中存在特异性低，无法彻底区分寨卡病毒与登革病毒，筛选过程繁琐等缺点。
发明内容
本发明的目的在于克服现有技术中存在的缺点与不足，提供一种单克隆抗体ZKns4B8及其应用。本发明提供的单克隆抗体ZKns4B8具有亲和力高，可用于区别寨卡病毒和登革病毒感染的优点。
目前，针对寨卡病毒病这一新发感染性疾病尚无有效的疫苗，更缺乏早期诊断及有效的治疗方法。本发明通过对有限数目记忆B细胞的培养，筛选并获得抗体重链和轻链的可变区基因，构建抗体表达载体，继而在细胞培养体系中表达抗体。本方法适用于构建全人源单克隆抗体。
为了达到上述目的，本发明提供了一种单克隆抗体ZKns4B8，包括重链和轻链，所述重链的可变区域的氨基酸序列信息如SEQ ID NO.1所示；所述轻链的可变区域的氨基酸序列信息如SEQ ID NO.2所示。
EVQLVESGGGLIQPGGSLRLSCAASGFTISSKYMSWVRQAPGKGLEWVSIIYSSGSTYYADSVKGRFTISRDISKNTLYLQMNSLRAEDTAVYYCASLGQGSAFGYWGQGTLVTVSS（SEQ ID NO.1）；
DIVMTQSPSSVSASVGDRVTITCRASQGISTWLAWYQQKPGKAPRLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANSFPFTFGPGTKVEIK（SEQ ID NO.2）。
本发明还提供了一种编码所述重链的可变区域或上述轻链的可变区域的氨基酸序列的多聚核苷酸序列；编码所述重链可变区域的多聚核苷酸序列如 SEQ ID NO.3所示，编码所述轻链可变区域的多聚核苷酸序列如 SEQ ID NO.4所示；
GAGGTACAGCTCGTGGAGTCTGGAGGAGGCTTGATCCAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCGTCTGGGTTCACCATCAGTAGCAAGTACATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAATTATTTATAGCAGTGGTAGCACATACTACGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACATTTCCAAGAACACGCTGTATCTTCAGATGAACAGCCTGAGAGCCGAGGACACGGCCGTGTATTACTGTGCGAGTCTGGGTCAGGGGAGTGCCTTCGGATATTGGGGCCAGGGAACCCTGGTCACTGTCTCCTCAG（SEQ ID NO.3）；
GACATCGTGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTTGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTAGCACCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAGGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAACAGTTTCCCATTCACTTTCGGCCCTGGGACCAAAGTGGAAATCAAAC（SEQ ID NO.4）。
所述单克隆抗体ZKns4B8的制备方法为：
S1、从感染者外周血单个核细胞利用荧光抗体进行筛选，获得记忆B细胞；
S2、培养步骤S1所得的记忆B细胞，加入细胞因子利用EBV进行转化，采用捕获ELISA筛选B细胞培养上清中抗体的存在，得含阳性抗体的B细胞；
S3、提取步骤S2所得的B细胞的RNA，逆转录成cDNA，经PCR扩增抗体的重链和轻链可变区，克隆至抗体表达载体上，得含单克隆抗体ZKns4B8的表达载体；所述PCR引物及PCR扩增程序见文献（J Immunol Methods. 2008 Jan 1;329(1-2):112-24.）；
S4、将步骤S3所得的含单克隆抗体ZKns4B8的表达载体转染至293T细胞，5-6天后，收集上清液，即得。
优选地，所述步骤S1中的荧光抗体包括IgD-FITC、CD19-ECD、CD27-PC7、CD38-APCA750、IgM-PB和CD45-KO。
优选地，所述步骤S2中的细胞生长因子包括CpG、IL21、IL2及放射照射过的健康人外周血单核的细胞（PBMC）。
本发明还提供了一种单克隆抗体ZKns4B8在制备临床诊断或治疗寨卡病毒病药物中的应用。
与现有技术相比，本发明提供的单克隆抗体ZKns4B8，具有以下优点：
（1）本发明提供的单克隆抗体ZKns4B8，是经B细胞培养结合抗体克隆技术获得的靶向寨卡病毒NS1蛋白的全人单抗ZKns4B8；
（2）本发明提供的单克隆抗体ZKns4B8，所包含的重链和轻链是自然配对的，故抗体的亲和力很高；
（3）本发明提供的单克隆抗体ZKns4B8具有较高的寨卡病毒特异性，可将寨卡病毒和登革病毒分离开；
（4）本发明提供的单克隆抗体ZKns4B8是一种可以分离在体内存在而在体外很难效仿的构象结构域的功能性抗体。
附图说明
图1为单克隆抗体ZKns4B8的重链表达载体的结构示意图；
图2为单克隆抗体ZKns4B8的轻链表达载体的结构示意图；
图3为单克隆抗体ZKns4B8与病毒NS1蛋白的结合反应结果图；
图4为ZKns4B8亲和力测定结果图。
具体实施方式
下面结合具体实施例对本发明作进一步解释，但是应当注意的是，以下实施例仅用以解释本发明，而不能用来限制本发明，所有与本发明相同或相近的技术方案均在本发明的保护范围之内。若未特别指明，实施例中所用的技术手段为本领域技术人员所熟知的常规手段，所用原料为市售商品。
荧光抗体，抗体表达载体及流式细胞仪均可购自美国贝克曼库尔特有限公司(Beckman Coulter, Inc.)；cDNA合成试剂盒(SuperScript III First Strand SynthesisSystem, invitrogen, #18080051)可购自上海玉博生物科技有限公司；抗体表达载体见文献（J Immunol Methods. 2008 Jan 1;329(1-2):112-24.）
实施例1单克隆抗体ZKns4B8的制备
1.记忆B细胞的识别和分选
1.1外周血单个核细胞分离：取寨卡感染者恢复期外周静脉EDTA抗凝血，利用密度梯度离心方法分离外周血单个核细胞，分装5×106/管，置于液氮中冻存；
1.2荧光标记抗体染色：外周血单个核细胞放置于37℃中水浴溶化，使用PBS缓冲液洗涤3次，接着加入抗体进行染色，室温避光孵育15min，使用PBS缓冲液洗涤后，加入400µL PBS缓冲液悬浮细胞上流式仪，所述流式仪为贝克曼库尔特MoFlo Astrios EQ超高速流式细胞分选系统；
设9个分析管，如表1，1-7管加入相应的单个荧光标记的抗体，第9管加入7种混合的荧光标记的抗体，第8管为只有细胞的空白管。样品管同第9管一样染色；
1.3记忆B细胞的分选：样品管上机分析，依据7-AAD及CD45圈出活的CD45阳性的白细胞。依据CD19圈出B细胞。在IgM和IgD双阴的B细胞群体里定义IgD-IgM-CD27+CD38low群体为记忆B细胞。将记忆B细胞分选至含细胞培养液的96孔细胞培养板中，25-50细胞/每孔
表1 1-7管加入的荧光标记抗体的类别
| 管号 | 染色管设置 | 抗体及标记的荧光 | 体积（µL） | 荧光通道 |
| 1 | 单染 | IgD-FITC | 10 | FL1 |
| 2 | 单染 | CD19-ECD | 5 | FL3 |
| 3 | 单染 | 7-AAD | 10 | FL4 |
| 4 | 单染 | CD27-PC7 | 5 | FL5 |
| 5 | 单染 | CD38-APC A750 | 2 | FL8 |
| 6 | 单染 | IgM-PB | 5 | FL9 |
| 7 | 单染 | CD45-KO | 5 | FL10 |
| 8 | 空白管 | 不加抗体 | ||
| 9 | 样品管 | 7种抗体混合液 |
2.记忆B细胞培养及培养上清抗体的筛选。
向含记忆B细胞的96孔细胞培养板中加入CpG、IL21、IL2、放射照射过的健康人PBMC及含有EBV的B95.8细胞培养上清培养7-10天。用捕获ELISA筛选B细胞培养上清中针对寨卡病毒NS1蛋白抗体的存在。
3.抗体的克隆
3.1cDNA合成：对于筛选出的上清中有针对寨卡病毒NS1蛋白抗体存在的B细胞，先提RNA再逆转录成cDNA。cDNA合成为试剂盒(SuperScript III First Strand SynthesisSystem, invitrogen, #18080051)。
3.2巢式PCR扩增抗体的重链和轻链可变区基因：PCR引物序列参考文献（JImmunol Methods. 2008 Jan 1;329(1-2):112-24.），反应使用Phusion 超保真DNA聚合酶（Phusion High-Fidelity PCR Master Mix with GC Buffer, NEB, #M0532s)。
3.3抗体的重链及轻链可变区PCR产物经酶切（VH, AgeI/SalI, VK, AgeI/Xhol,VL, AgeI/BsiWI）克隆到含有人IgG1恒定区的抗体表达载体上，重链表达载体见图1，人IgG1恒定区的抗体表达载体见图2。将测得的重链和轻链可变区基因序列经IMGT/V-Quest分析。。
实施例2抗体的特异性分析
1.抗体的生产：将构建的序列确定的抗体的重链和轻链载体共转染入293T细胞，5-6天后收获含抗体的培养上清；
2.抗体的纯化：将含抗体的培养上清进行protein A亲和柱纯化，并测定蛋白含量；
3.抗体的反应：抗体与寨卡病毒（ZIKA）及登革病毒1-4型（DENV1、DENV2、DENV3、DENV4）NS1蛋白的结合反应，采用捕获NS1抗原ELISA法，反应结果见图3；
4.抗体亲和力测定（BLI）：应用Fortebio公司的Octet RED384系统，选择Anti-Human IgG Fc(AHC)传感器，亲和力检测结果见图4；以抗体作为Ligand，先固化抗体到传感器上（浓度20μg/mL）；抗原作为分析物，检测抗体与抗原的结合和解离能力。
最后应当说明的是，以上实施例仅用以说明本发明的技术方案而非对本发明保护范围的限制，尽管参照较佳实施例对本发明做了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的实质和范围。。
序列表
<110> 广州市第八人民医院
<120> 一种单克隆抗体ZKns4B8及应用
<130> 2018.11.29
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 117
<212> PRT
<213> 寨卡病毒抗体重链氨基酸序列(Zika virus antibody light chainamino acid sequence)
<400> 1
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ile Ser Ser Lys
20 25 30
Tyr Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ser Ile Ile Tyr Ser Ser Gly Ser Thr Tyr Tyr Ala Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Ile Ser Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ser Leu Gly Gln Gly Ser Ala Phe Gly Tyr Trp Gly Gln Gly Thr Leu
100 105 110
Val Thr Val Ser Ser
115
<210> 2
<211> 107
<212> PRT
<213> 寨卡病毒抗体轻链氨基酸序列(Zika virus antibody light chainamino acid sequence)
<400> 2
Asp Ile Val Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Thr Trp
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro Phe
85 90 95
Thr Phe Gly Pro Gly Thr Lys Val Glu Ile Lys
100 105
<210> 3
<211> 352
<212> DNA
<213> 编码寨卡病毒抗体重链氨基酸的多聚核苷酸序列(Polynucleotidesequences encoding Zika virus anti-weight chain amino acids)
<400> 3
gaggtacagc tcgtggagtc tggaggaggc ttgatccagc ctggggggtc cctgagactc 60
tcctgtgcag cgtctgggtt caccatcagt agcaagtaca tgagctgggt ccgccaggct 120
ccagggaagg ggctggagtg ggtctcaatt atttatagca gtggtagcac atactacgca 180
gactccgtga agggccgatt caccatctcc agagacattt ccaagaacac gctgtatctt 240
cagatgaaca gcctgagagc cgaggacacg gccgtgtatt actgtgcgag tctgggtcag 300
gggagtgcct tcggatattg gggccaggga accctggtca ctgtctcctc ag 352
<210> 4
<211> 322
<212> DNA
<213> 编码寨卡病毒抗体轻链氨基酸的多聚核苷酸序列(Polynucleotidesequences encoding Zika virus anti-weight chain amino acids)
<400> 4
gacatcgtga tgacccagtc tccatcttcc gtgtctgcat ctgttggaga cagagtcacc 60
atcacttgtc gggcgagtca gggtattagc acctggttag cctggtatca gcagaaacca 120
gggaaagccc ctaggctcct gatctatgct gcatccagtt tgcaaagtgg ggtcccatca 180
aggttcagcg gcagtggatc tgggacagat ttcactctca ccatcagcag cctgcagcct 240
gaagattttg caacttacta ttgtcaacag gctaacagtt tcccattcac tttcggccct 300
gggaccaaag tggaaatcaa ac 322
Claims (4)
1.一种针对寨卡病毒NS1蛋白的单克隆抗体ZKns4B8，包括重链和轻链，其特征在于，所述重链的可变区域的氨基酸序列为SEQ ID NO.1；所述轻链的可变区域的氨基酸序列为SEQID NO.2。
2.编码如权利要求1所述的单克隆抗体ZKns4B8的多聚核苷酸序列，其特征在于，编码所述重链的可变区域或轻链的可变区域的氨基酸序列的多聚核苷酸序列。
3.如权利要求2所述的多聚核苷酸序列，其特征在于，编码所述重链的可变区域的多聚核苷酸序列如SEQ ID NO.3所示，编码所述轻链的可变区域的多聚核苷酸序列如SEQ IDNO.4所示。
4.如权利要求1所述的单克隆抗体ZKns4B8在制备诊断或治疗寨卡病毒病药物中的应用。
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