CN109503712B - 一种单克隆抗体ZKns2E11及其应用 - Google Patents
一种单克隆抗体ZKns2E11及其应用 Download PDFInfo
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- CN109503712B CN109503712B CN201811519065.1A CN201811519065A CN109503712B CN 109503712 B CN109503712 B CN 109503712B CN 201811519065 A CN201811519065 A CN 201811519065A CN 109503712 B CN109503712 B CN 109503712B
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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ZKns2E11及其应用。本发明提供的单克隆抗体ZKns2E11是一种针对寨卡病毒NS1蛋白的全人源单克隆抗体，包括具有如SEQ ID NO.1所述氨基酸序列的重链可变区域和如SEQ ID NO.2所述氨基酸序列的轻链可变区域。本发明提供的单克隆抗体ZKns2E11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ZKns2E11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 表达质粒，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ZKns2E11及其应用。本发明提供的单克隆抗体ZKns2E11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ZKns2E11，包括重链和 轻链，所述重链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；
SVELIESGGGVVQPGRSLRLSCAASGFTFSNYAMHWVRQAPGKGLEWVA FISYDGSNKYYADSVKGRFTISRDNSKNTLSLQMNSLRAEDTALYYCARVQN GYEGDYWGQGTLVTVSS(SEQ ID NO.1)；
LPVLTQPPSVSVSPGQTARITCSGDALPKKYAYWYQQKSGQAPVLVIYED SKRPSGIPERFSGSSSGTMATLTISGAQVEDEADYYCYSTDSSGNLYVFGTGTK VTVL(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；
TCCGTGGAGCTGATAGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAG GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAACTATGC TATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCAT TTATATCATATGATGGAAGCAATAAATACTACGCAGACTCCGTGAAGGGC CGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTCTCTGCAGAT GAACAGCCTGAGAGCTGAGGACACGGCTCTATATTACTGTGCGAGAGTGC AGAATGGCTACGAGGGGGACTACTGGGGCCAGGGAACCCTGGTCACCGT CTCCTCAG(SEQ IDNO.3)；
CTGCCTGTGCTGACTCAGCCACCCTCGGTGTCAGTGTCCCCAGGACAA ACGGCCAGGATCACCTGCTCTGGAGATGCATTGCCAAAAAAATATGCTTAT TGGTACCAGCAGAAGTCAGGCCAGGCCCCTGTGCTGGTCATCTATGAGGAC AGCAAACGACCCTCCGGGATCCCTGAGAGATTCTCTGGCTCCAGCTCAGG GACAATGGCCACCTTGACTATCAGTGGGGCCCAGGTGGAGGATGAAGCTG ACTACTACTGTTACTCAACAGACAGCAGTGGTAACCTTTATGTCTTCGGAA CTGGGACCAAGGTCACCGTCCTAG(SEQ ID NO.4)。
所述单克隆抗体ZKns2E11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ZKns2E11的 表达载体；所述PCR引物及PCR扩增程序见文献(J Immunol Methods.2008Jan 1；329(1-2):112-24.)；
S4、将步骤S3所得的含单克隆抗体ZKns2E11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ZKns2E11在制备临床诊断或治疗寨卡病毒 病中的应用。
与现有技术相比，本发明提供的单克隆抗体ZKns2E11，具有以下优点：
(1)本发明提供的单克隆抗体ZKns2E11，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ZKns2E11；
(2)本发明提供的单克隆抗体ZKns2E11，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ZKns2E11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ZKns2E11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ZKns2E11的重链表达载体的结构示意图；
图2为单克隆抗体ZKns2E11的轻链表达载体的结构示意图；
图3为单克隆抗体ZKns2E11与病毒NS1蛋白的结合反应的结果图；
图4为单克隆抗体ZKns2E11亲和力测定结果图；
图5为单克隆抗体ZKns2E11制备胶体金试纸检测重组病毒NS1蛋白的结果 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.2008Jan 1；329(1-2):112-24.)。
实施例1单克隆抗体ZKns2E11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MoFloAstrios 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11-7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-APCA750 | 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.2008Jan 1；329(1-2):112-24.)，反应使用Phusion超保真DNA 聚合酶(Phusion High-Fidelity PCRMaster 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单克隆抗体ZKns2E11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proteinA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NS1蛋白(DENV1、 DENV2、DENV3、DENV4)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，选择 Anti-Human IgG Fc(AHC)传感器，亲和力检测结果见图4，其中，亲和力大小常 数KD为4.62E-10，结合常数Ka为5.57E+0.5，解离常数Kd为2.57E-0.4；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，检测重组病毒NS1蛋白结果见图5。
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ZKns2E11及其应用
<130> 2018.11.29
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 118
<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
Ser Val Glu Leu Ile Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr
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Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Phe Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Ser
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Leu Tyr Tyr Cys
85 90 95
Ala Arg Val Gln Asn Gly Tyr Glu Gly Asp Tyr Trp Gly Gln Gly Thr
100 105 110
Leu Val Thr Val Ser Ser
115
<210> 2
<211> 108
<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
Leu Pro Val Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln
1 5 10 15
Thr Ala Arg Ile Thr Cys Ser Gly Asp Ala Leu Pro Lys Lys Tyr Ala
20 25 30
Tyr Trp Tyr Gln Gln Lys Ser Gly Gln Ala Pro Val Leu Val Ile Tyr
35 40 45
Glu Asp Ser Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser
50 55 60
Ser Ser Gly Thr Met Ala Thr Leu Thr Ile Ser Gly Ala Gln Val Glu
65 70 75 80
Asp Glu Ala Asp Tyr Tyr Cys Tyr Ser Thr Asp Ser Ser Gly Asn Leu
85 90 95
Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu
100 105
<210> 3
<211> 355
<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
tccgtggagc tgatagagtc tgggggaggc gtggtccagc ctgggaggtc cctgagactc 60
tcctgtgcag cctctggatt caccttcagt aactatgcta tgcactgggt ccgccaggct 120
ccaggcaagg ggctggagtg ggtggcattt atatcatatg atggaagcaa taaatactac 180
gcagactccg tgaagggccg attcaccatc tccagagaca attccaagaa cacgctgtct 240
ctgcagatga acagcctgag agctgaggac acggctctat attactgtgc gagagtgcag 300
aatggctacg agggggacta ctggggccag ggaaccctgg tcaccgtctc ctcag 355
<210> 4
<211> 325
<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
ctgcctgtgc tgactcagcc accctcggtg tcagtgtccc caggacaaac ggccaggatc 60
acctgctctg gagatgcatt gccaaaaaaa tatgcttatt ggtaccagca gaagtcaggc 120
caggcccctg tgctggtcat ctatgaggac agcaaacgac cctccgggat ccctgagaga 180
ttctctggct ccagctcagg gacaatggcc accttgacta tcagtggggc ccaggtggag 240
gatgaagctg actactactg ttactcaaca gacagcagtg gtaaccttta tgtcttcgga 300
actgggacca aggtcaccgt cctag 325
Claims (4)
1.一种针对寨卡病毒NS1蛋白的单克隆抗体ZKns2E11，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SEQ ID NO.2。
2.编码如权利要求1所述的单克隆抗体ZKns2E11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ZKns2E11在制备诊断或治疗寨卡病毒病药物中的应用。
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