CN113150132A - 一种抗SARS-CoV-2的重组抗体及其应用 - Google Patents
一种抗SARS-CoV-2的重组抗体及其应用 Download PDFInfo
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Abstract
本发明涉及一种抗SARS‑CoV‑2的重组抗体及其应用,所述重组抗体包含抗SARS‑CoV‑2S蛋白抗体可变区域的氨基酸序列和抗SARS‑CoV‑2N蛋白抗体可变区域的氨基酸序列。本发明的重组抗体,极大地提高了原料的筛选效率,批间差异小且稳定性高;作为SARS‑CoV‑2中和抗体检测试剂盒的质控品及SARS‑CoV‑2检测试剂原料,在使用过程中可节约存储成本、减小配制工作量、提高工作效率,并减少了试剂死体积,降低原料成本。
Description
技术领域
本发明属于抗体工程技术领域,具体涉及一种抗SARS-CoV-2重组抗体及其应用。
背景技术
SARS-CoV-2是一类单股正链RNA冠状病毒,其主要结构蛋白有刺突蛋白(spikeprotein, S蛋白),核衣壳蛋白(nucleocapsid,N蛋白),膜蛋白(membrane protein,M蛋白),包 膜蛋白(envelope protein,E蛋白)。SARS-CoV-2表面的同源三聚体刺突糖蛋白S(Spike, S)包含S1和S2两个亚基,其中S1亚基分为N-端区(N-terminal domain,NTD)和含受体 结合域(receptor binding domain,RBD)的C-端区,S2亚基包含融合肽、2个7肽重复序列 HR(hepeat repeat region,HR)和跨膜区等膜融合过程所需基本元件。与SARS-CoV相似, SARS-CoV-2进入细胞经过了RBD与宿主细胞表面的血管紧张素酶2(angiotensinconverting enzyme 2,ACE2)结合、S2蛋白介导膜融合两个过程。为了更好地监测感染率、群体免疫 和保护性免疫,以及评价临床试验期间和大规模接种后的疫苗效力,2020年7月杜克-新加坡 国立大学医学院团队开发了一种可以评估靶向新冠病毒刺突蛋白受体结合域(RBD)的中和 抗体。而目前研发的大部分检测试剂盒中SARS-CoV-2中和抗体的靶点为S蛋白。
核衣壳蛋白(N,Nucleocapsid Protein)作为病毒核衣壳内最重要的蛋白之一,主要负责 RNA的复制功能。核衣壳蛋白(N蛋白)常作为冠状病毒诊断检测工具,是免疫学快速诊断 试剂的核心原料。目前能够同时靶向新冠病毒刺突蛋白及核衣壳蛋白中和抗体几乎没有,已 公开的SARS-CoV-2中和抗体检测试剂盒中配有仅针对SARS-CoV-2 S蛋白中和抗体和/或仅 针对SARS-CoV-2 N蛋白中和抗体的质控品,需要分别存储,存储成本较高,使用时需要分 别调试、配制,工作量大、效率低且批间差异较大。
发明内容
本发明的目的是提供一种能够同时针对SARS-CoV-2S蛋白和N蛋白的重组抗体。
为达到上述目的,本发明采用的技术方案是:
本发明第一方面提供一种抗SARS-CoV-2重组抗体,其特征在于:所述重组抗体包含抗SARS-CoV-2S蛋白抗体可变区域的氨基酸序列和抗SARS-CoV-2N蛋白抗体可变区域的氨基酸序列。
优选地,所述抗SARS-CoV-2S蛋白抗体可变区域的氨基酸序列包括抗SARS-CoV-2S蛋白抗体重链可变区和抗SARS-CoV-2S蛋白抗体轻链可变区,所述抗SARS-CoV-2S蛋白抗体重链可变区包含
VH CDR1:氨基酸序列为WRPRP(SEQ ID NO:5)或与其具有至少85%的同源性,优选具有至少90%的同源性,进一步优选具有至少95%的同源性,再优选至少具有96%、97%、98%、99%的同源性,和/或,
VH CDR2:氨基酸序列为SPGLTALSPSIQTMKRR(SEQ ID NO:6)或与其具有至少 85%的同源性,优选具有至少90%的同源性,进一步优选具有至少95%的同源性,再优选至少具有96%、97%、98%、99%的同源性,和/或,
VH CDR3:氨基酸序列为ETARLGS(SEQ ID NO:7)或与其具有至少85%的同源性,优选具有至少90%的同源性,进一步优选具有至少95%的同源性,再优选至少具有96%、97%、98%、99%的同源性,
所述抗SARS-CoV-2S蛋白抗体轻链可变区包含
VL CDR1:氨基酸序列为CADSVSMKRRLLARDY(SEQ ID NO:8)或与其具有至少 85%的同源性,优选具有至少90%的同源性,进一步优选具有至少95%的同源性,再优选至少具有96%、97%、98%、99%的同源性,和/或,
VL CDR2:氨基酸序列为GDSTYLQ(SEQ ID NO:9)或与其具有至少85%的同源性,优选具有至少90%的同源性,进一步优选具有至少95%的同源性,再优选至少具有96%、97%、98%、99%的同源性,和/或,
VL CDR3:氨基酸序列为EFGLGQMNSLRAEDTEVH(SEQ ID NO:10)或与其具有至少85%的同源性,优选具有至少90%的同源性,进一步优选具有至少95%的同源性,再优选至少具有96%、97%、98%、99%的同源性,。
优选地,所述抗SARS-CoV-2N蛋白抗体可变区域的氨基酸序列包括抗SARS-CoV-2N蛋白抗体重链可变区和抗SARS-CoV-2N蛋白抗体轻链可变区,所述抗SARS-CoV-2N蛋白抗体重链可变区包含
VH CDR1:氨基酸序列为QTRER(SEQ ID NO:21)或与其具有至少85%的同源性,优选具有至少90%的同源性,进一步优选具有至少95%的同源性,再优选至少具有96%、97%、 98%、99%的同源性,和/或,
VH CDR2:氨基酸序列为RRLRFALSMPGTELRDY(SEQ ID NO:22)或与其具有至少85%的同源性,优选具有至少90%的同源性,进一步优选具有至少95%的同源性,再优选至少具有96%、97%、98%、99%的同源性,和/或,
VH CDR3:氨基酸序列为VRHVTKL(SEQ ID NO:23)或与其具有至少85%的同源性,优选具有至少90%的同源性,进一步优选具有至少95%的同源性,再优选至少具有96%、97%、98%、99%的同源性,
所述抗SARS-CoV-2N蛋白抗体轻链可变区包含
VL CDR1:氨基酸序列为VQLLESEISRDNSKNT(SEQ ID NO:24)或与其具有至少 85%的同源性,优选具有至少90%的同源性,进一步优选具有至少95%的同源性,再优选至少具有96%、97%、98%、99%的同源性,和/或,
VL CDR2:氨基酸序列为IDVCHVQ(SEQ ID NO:25)或与其具有至少85%的同源性,优选具有至少90%的同源性,进一步优选具有至少95%的同源性,再优选至少具有96%、97%、98%、99%的同源性,和/或,
VL CDR3:氨基酸序列为QCGFDLFPPKPKDTLMIS(SEQ ID NO:26)或与其具有至少85%的同源性,优选具有至少90%的同源性,进一步优选具有至少95%的同源性,再优选至少具有96%、97%、98%、99%的同源性。
优选地,所述抗SARS-CoV-2重组抗体为人源化IgG抗体。
本发明第二方面提供一种重组核苷酸分子,所述核苷酸分子编码所述S蛋白抗体可变区域的氨基酸序列和所述N蛋白抗体可变区域的氨基酸序列。
优选地,所述重组核苷酸分子包含编码所述抗SARS-CoV-2S蛋白抗体重链可变区的氨基酸序列的片段、编码所述抗SARS-CoV-2S蛋白抗体轻链可变区的氨基酸序列的片段、编码所述抗SARS-CoV-2N蛋白抗体重链可变区的氨基酸序列的片段、及编码所述抗 SARS-CoV-2N蛋白抗体轻链可变区的氨基酸序列的片段。
具体地,所述编码所述抗SARS-CoV-2S蛋白抗体重链可变区的氨基酸序列的片段包含
编码VH CDR1的片段:核苷酸序列为tggaggcctcggcca(SEQ ID NO:11)或与其具有至少 85%的同源性,优选具有至少90%的同源性,进一步优选具有至少95%的同源性,再优选至少具有96%、97%、98%、99%的同源性,和/或,
编码VH CDR2的片段:核苷酸序列为 tcaccaggactaaccgcgttgagtccttctatccagacaatgaagcgacga(SEQ ID NO:12)或与其具有至少85%的同源性,优选具有至少90%的同源性,进一步优选具有至少95%的同源性,再优选至少具有96%、97%、98%、99%的同源性,和/或,
编码VH CDR3的片段:核苷酸序列为gaaacggcacgtttgggttct(SEQ ID NO:13)或与其具有至少85%的同源性,优选具有至少90%的同源性,进一步优选具有至少95%的同源性,再优选至少具有96%、97%、98%、99%的同源性;
所述编码所述抗SARS-CoV-2S蛋白抗体轻链可变区的氨基酸序列的片段包含
编码VH CDR1的片段:核苷酸序列为 tgcgcggactcggtctcaatgaagcgacgattgctggcccgcgattat(SEQ ID NO:14)或与其具有至少85%的同源性,优选具有至少90%的同源性,进一步优选具有至少95%的同源性,再优选至少具有96%、 97%、98%、99%的同源性,和/或,
编码VH CDR2的片段:核苷酸序列为ggagactccacctaccttcaa(SEQ ID NO:15)或与其具有至少85%的同源性,优选具有至少90%的同源性,进一步优选具有至少95%的同源性,再优选至少具有96%、97%、98%、99%的同源性,和/或,
编码VH CDR3的片段:核苷酸序列为gaatttggcctcggtcaaatgaactcact(SEQ IDNO:16)或与其具有至少85%的同源性,优选具有至少90%的同源性,进一步优选具有至少95%的同源性,再优选至少具有96%、97%、98%、99%的同源性。
具体地,所述编码所述抗SARS-CoV-2N蛋白抗体重链可变区的氨基酸序列的片段包含
编码VH CDR1的片段:核苷酸序列为cagacccgtgaacgc(SEQ ID NO:27)或与其具有至少 85%的同源性,优选具有至少90%的同源性,进一步优选具有至少95%的同源性,再优选至少具有96%、97%、98%、99%的同源性,和/或,
编码VH CDR2的片段:核苷酸序列为 cgacggttaagattcgccttgagtatgccaggaacggagcttcgggattat(SEQ ID NO:28)或与其具有至少85%的同源性,优选具有至少90%的同源性,进一步优选具有至少95%的同源性,再优选至少具有 96%、97%、98%、99%的同源性,和/或,
编码VH CDR3的片段:核苷酸序列为gttaggcatgtgaccaagctg(SEQ ID NO:29)或与其具有至少85%的同源性,优选具有至少90%的同源性,进一步优选具有至少95%的同源性,再优选至少具有96%、97%、98%、99%的同源性;
所述编码所述抗SARS-CoV-2S蛋白抗体轻链可变区的氨基酸序列的片段包含
编码VH CDR1的片段:核苷酸序列为 gttcagcttcttgagtcggaaatctctcgtgataattctaagaatact(SEQ ID NO:30)或与其具有至少85%的同源性,优选具有至少90%的同源性,进一步优选具有至少95%的同源性,再优选至少具有96%、 97%、98%、99%的同源性,和/或,
编码VH CDR2的片段:核苷酸序列为atcgacgtatgccatgtccaa(SEQ ID NO:31)或与其具有至少85%的同源性,优选具有至少90%的同源性,进一步优选具有至少95%的同源性,再优选至少具有96%、97%、98%、99%的同源性,和/或,
编码VH CDR3的片段:核苷酸序列为 caatgtggcttcgatctcttccccccaaaacccaaggacaccctcatgatctcc(SEQ ID NO:32)或与其具有至少85%的同源性,优选具有至少90%的同源性,进一步优选具有至少95%的同源性,再优选至少具有96%、97%、98%、99%的同源性。
进一步优选地,所述重组核苷酸分子各序列之间的排列顺序为:真核KOZAK-信号肽- 抗SARS-CoV-2S蛋白抗体CDR碱基序列-Linker-抗SARS-CoV-2N蛋白CDR碱基序列。
再进一步优选地,所述重组核苷酸分子各序列之间的排列顺序为:真核KOZAK-信号肽-抗SARS-CoV-2S蛋白抗体VL CDR1碱基序列-Linker-抗SARS-CoV-2S蛋白抗体VL CDR2碱基序列-Linker-抗SARS-CoV-2S蛋白抗体VL CDR3碱基序列-Linker-抗 SARS-CoV-2S蛋白抗体DL CDR1碱基序列-Linker-抗SARS-CoV-2S蛋白抗体DL CDR2 碱基序列-Linker-抗SARS-CoV-2S蛋白抗体DL CDR3碱基序列-Linker-抗SARS-CoV-2N 蛋白抗体VL CDR1碱基序列-Linker-抗SARS-CoV-2N蛋白抗体VL CDR2碱基序列-Linker- 抗SARS-CoV-2N蛋白抗体VL CDR3碱基序列-Linker-抗SARS-CoV-2N蛋白抗体DL CDR1碱基序列-Linker-抗SARS-CoV-2N蛋白抗体DL CDR2碱基序列-Linker-抗 SARS-CoV-2N蛋白抗体DL CDR3碱基序列。
更进一步优选地,所述真核KOZAK的碱基序列为GCCACC,真核KOZAK氨基酸序列为AT。
更进一步优选地,所述信号肽为蜂毒信号肽,其碱基序列为 AAGTTCCTGGTCAACGTCGCTCTCGTGTTCATG,其氨基酸序列为KFLVNVALVFM。
更进一步优选地,所述Linker的碱基序列为GGAGGCGGCGGATCT,其氨基酸序列为GGGGS,和/或,GAAGCCGCTGCCAAG,其氨基酸序列为EAAAK。
进一步优选地,所述重组核苷酸分子还包括人IgG Fc片段。
根据一种实施方式,所述重组核苷酸分子的碱基序列如SEQ ID NO:33所示。
本发明第三方面提供一种重组表达载体,所述重组表达载体包含所述的重组核苷酸分子。
优选地,所述表达载体为pFUSE-hIgG1-Fc2质粒。
本发明第四方面提供一种宿主细胞,用所述的重组表达载体转染哺乳动物细胞获得所述宿主细胞。
优选地,所述哺乳动物细胞为HEK293哺乳细胞和/或CHO(GS缺陷型)细胞。
本发明第五方面提供一种所述的抗SARS-CoV-2重组抗体的制备方法,所述制备方法包括如下步骤:
(1)人工合成编码权利要求1至3中任一项所述的可变区基因序列并构建在载体pFUSE-hIgG1-Fc2上制备重组质粒;
(2)将步骤(1)的重组质粒转染哺乳动物细胞表达重组蛋白。
(3)对步骤(2)的重组蛋白进行纯化处理,即得所述抗SARS-CoV-2重组抗体。
具体地,所述制备方法如下:
(a)人工合成针对抗SARS-CoV-2S蛋白抗体和抗SARS-CoV-2N蛋白抗体的单克隆抗体的CDR的碱基序列,插入刚性linker连接,N端加入蜂毒信号肽序列,在序列上游加入限制性核酸内切酶位点EcoRI,下游序列插入限制性核酸内切酶位点NcoI,将合成后的序列构建在pFUSE-hIgG1-Fc2质粒载体上。全基因合成时各序列之间的排列顺序为:真核KOZAK- 信号肽-抗SARS-CoV-2S蛋白抗体CDR碱基序列-Linker-抗SARS-CoV-2N蛋白CDR碱基序列。
(b)将含有目的基因的质粒载体pFUSE-hIgG-S-N转入大肠杆菌,通过抗生素筛选和菌落PCR鉴定重组克隆质粒,挑选阳性克隆进行大量培养提取重组质粒。
(c)将得到的含有目的基因的质粒转染哺乳动物细胞进行表达。大量培养细胞,收集细胞培养物,离心取细胞上清。
(d)将上个步骤中获取的细胞上清用Protein A亲和柱纯化。利用Gly-HCl进行梯度洗脱,去除杂蛋白,得到包含抗SARS-CoV-2S蛋白抗体可变区域的氨基酸序列和抗SARS-CoV-2 N蛋白抗体可变区域的氨基酸序列的人源化IgG。
本发明第六方面提供一种所述的抗SARS-CoV-2重组抗体在SARS-CoV-2病毒检测的中的应用。
本发明七方面提供一种所述的抗SARS-CoV-2重组抗体在SARS-CoV-2病毒检测的中的应用。
本发明第八方面提供一种所述的抗SARS-CoV-2重组抗体在SARS-CoV-2病毒中和抗体检测中的应用。
本发明第九方面提供一种SARS-CoV-2中和抗体检测复合质控品,所述复合质控品的原料包括所述的抗SARS-CoV-2重组抗体。
优选地,所述试剂盒采用磁微粒化学发光。磁微粒化学发光技术是以化学发光剂为底物的酶免疫技术,同时应用了纳米级磁性微粒做固相载体,增加了吸附面积,使抗原、抗体最大限度结合,并使其结合反应在近似液相的条件下进行,兼具化学发光与酶免疫技术的优点,是近几年快速发展的非放射性检测方法。其发光原理主要包含用辣根过氧化物酶 (HRP)、碱性磷酸酶(ALP)或吖啶酯直接标记抗体(抗原),与待测标本中相应的抗原(抗体)发生免疫反应后,形成固相包被抗体-待测抗原-HRP/ALP/吖啶酯标记复合物,然后在催化剂或氧化剂作用下,促使复合物分解发光。该方法具有灵敏度高、稳定性强、特异性强、线性范围宽、简单快速、安全无毒等优点,容易实现自动化定量检测患者自身抗体,其灵敏度和线性范围超过以往技术。质控品作为化学发光试剂的重要组成部分,主要作用是监控整个检测系统的状态,确认样本检测在检测系统正常状态下进行,对检测结果是否准确提供非常重要的参考作用。而现有的技术条件下的单项质控品批间差较难控制,且生产及储存成本更高。本发明的重组抗体可作为其质控品解决上述问题。
本发明通过用SARS-CoV-2重组抗原S蛋白和N蛋白免疫小鼠获得新型冠病毒抗S蛋白和抗N蛋白IgG抗体,通过序列设计和全基因合成得到这两种抗体的可变区碱基序列,并将序列引入含有人IgG Fc片段基因表达序列的pFUSE-hIgG1-Fc2载体中,制备一种含有SARS-CoV-2S蛋白和N蛋白IgG抗体的Fab段可变区及人IgG Fc片段序列的重组质粒,将该重组质粒转染哺乳动物细胞,获取抗SARS-CoV-2S蛋白和抗N蛋白IgG抗体的Fab 段可变区及人IgG Fc片段的重组抗体,其可用于新型冠病毒检测试剂盒,并可作为新型冠病毒S蛋白和N蛋白IgG抗体检测试剂盒的复合质控品对整合抗体检测进行质控。
通过重组核苷酸分子一次表达可获得同时针对S和N蛋白重组的抗体;重组技术使得质控品稳定性更好,利于控制质控品的批间差;同时细胞大规模培养技术能极大的降低生产成本;针对性的抗体设计实现质控品更加准确的监测检测系统,使质控品的调试、配制及批间差的控制难度都降低,提高了检测效率,节约了试剂盒成本。
由于上述技术方案运用,本发明与现有技术相比具有下列优点:
本发明的重组抗体,极大地提高了原料的筛选效率,批间差异小且稳定性高;作为SARS-CoV-2中和抗体检测试剂盒的质控品及SARS-CoV-2检测试剂原料,在使用过程中可节约存储成本、减小配制工作量、提高工作效率,并减少了试剂死体积,降低原料成本。
附图说明
图1为本发明使用的pFUSE-hIgG1-Fc2载体质粒质谱图。
图2为本发明的抗SARS-CoV-2在重组抗体的Western blotting图。
具体实施方式
为更好地说明本发明的目的、技术方案和优点,下面将结合具体实施例对本发明作进一步说明。
实施例1
1.1将分别用SARS-CoV-2S蛋白、N蛋白抗原免疫小鼠,获得可表达抗SARS-CoV-2S蛋白和抗SARS-CoV-2N蛋白IgG抗体的小鼠脾细胞。
1.2提取脾细胞与Sp2/0细胞融合,通过筛选、验证得到可高效表达抗SARS-CoV-2S蛋白单克隆抗体和抗SARS-CoV-2N蛋白单克隆抗体的细胞株。
1.3将获得的单克隆抗体进行测序,获取抗SARS-CoV-2S蛋白和抗SARS-CoV-2N蛋白 IgG抗体的Fab段可变区基因序列。
实施例2
2.1根据基因测序获得SARS-CoV-2S蛋白和N蛋白IgG抗体的Fab段可变区基因序列。
(1)抗SARS-CoV-2S蛋白IgG抗体CDR区氨基酸序列,针对测序的氨基酸序列,根据哺乳动物密码子偏爱性,获得抗体可变区域的碱基序列。
重链氨基酸序列:
EVQLVESGGDRFSLEGPRNSPVQPPDSLSVWRPRPGFGRLRRGNWSGSQSPGLTALS PSIQTMKRRDSPSPETMPRTPYTCRLAVRGLRTRPYITVRDETARLGSGPKGLWSLSL (SEQ ID NO:1)。
重链核苷酸序列:
gaagtacaactggtggagtctgggggagaccgattcagcctggagggtcccagaaactctcctgtgcagcctccggattcactttca gtatggaggcctcggccagggttcggcagactccggagaggaaattggagtgggtcgcaatcaccaggactaaccgcgttgagtccttcta tccagacaatgaagcgacgagattcaccgtctccagagacaatgccaagaacaccctatacctgcagattagcagtcagaggtctgaggac acggccatatattactgtgcgagacgaaacggcacgtttgggttctgggcccaagggactctggtcactgtctctg(SEQ ID NO: 2)。
轻链氨基酸序列:
DIVLTQAAFSNPVTLGTSASISCCADSVSMKRRLLARDYWYLQRPGQSPQLLISGDS TYLQGVPDRFSSSGSGTDFTLRISRVEEFGLGQMNSLRAEDTEVHFGGGTKLEIKR(SEQ ID NO:3)。
轻链核苷酸序列:
gatattgtgctgacgcaggctgccttctccaatccagtcactcttggaacatcagcttccatctcctgctgcgcggactcggtctcaatg aagcgacgattgctggcccgcgattattggtatctgcagaggccaggccagtctcctcagctcctgatttctggagactccacctaccttcaag gagtcccagacaggttcagtagcagtgggtcaggaactgatttcacactgagaatcagcagagtggaggaatttggcctcggtcaaatgaa ctcactacgagcagaagataccgaagtgcacttcggaggggggaccaagctggaaataaaacgg(SEQ ID NO:4)。
其CDR氨基酸序列为:
VH CDR1:WRPRP(SEQ ID NO:5),编码VH CDR1的核苷酸片段:核苷酸序列为tggaggcctcggcca(SEQ ID NO:11);
VH CDR2:SPGLTALSPSIQTMKRR(SEQ ID NO:6),编码VH CDR2的核苷酸片段:核苷酸序列为tcaccaggactaaccgcgttgagtccttctatccagacaatgaagcgacga(SEQ ID NO:12);
VH CDR3:ETARLGS(SEQ ID NO:7),编码VH CDR3的核苷酸片段:核苷酸序列为gaaacggcacgtttgggttct(SEQ ID NO:13);
VL CDR1:CADSVSMKRRLLARDY(SEQ ID NO:8),编码VL CDR1的核苷酸片段:核苷酸序列为tgcgcggactcggtctcaatgaagcgacgattgctggcccgcgattat(SEQ ID NO:14);
VL CDR2:GDSTYLQ(SEQ ID NO:9),编码VL CDR2的核苷酸片段:核苷酸序列为ggagactccacctaccttcaa(SEQ ID NO:15);
VL CDR3:EFGLGQMNSLRAEDTEVH(SEQ ID NO:10),编码VL CDR3的核苷酸片段:核苷酸序列为gaatttggcctcggtcaaatgaactcact(SEQ ID NO:16)。
(2)抗SARS-CoV-2N蛋白IgG抗体CDR区氨基酸序列,针对测序的氨基酸序列,根据哺乳动物密码子偏爱性,获得抗体可变区域的碱基序列。
重链氨基酸序列:
EVQLVESGGDRFSLEGPRNSPVQPPDSLSVQTRERGFGRLRRGNWSGSQRRLRFALS MPGTELRDYDSPSPETMPRTPYTCRLAVRGLRTRPYITVRDVRHVTKLGPKGLWSLSL (SEQ ID NO:17)。
重链核苷酸序列:
gaagtacaactggtggagtctgggggagaccgattcagcctggagggtcccagaaactctcctgtgcagcctccggattcactttca gtacagacccgtgaacgcgggttcggcagactccggagaggaaattggagtgggtcgcaacgacggttaagattcgccttgagtatgcca ggaacggagcttcgggattatgattcaccgtctccagagacaatgccaagaacaccctatacctgcagattagcagtcagaggtctgaggac acggccatatattactgtgcgagacgttaggcatgtgaccaagctggggcccaagggactctggtcactgtctctg(SEQ ID NO: 18)。
轻链氨基酸序列:
DIVLTQAAFSNPVTLGTSASISCVQLLESEISRDNSKNTWYLQRPGQSPQLLISIDVCH VQGVPDRFSSSGSGTDFTLRISRVEQCGFDLFPPKPKDTLMISFGGGTKLEIKR(SEQ ID NO:19)。
轻链核苷酸序列:
gatattgtgctgacgcaggctgccttctccaatccagtcactcttggaacatcagcttccatctcctgcgttcagcttcttgagtcggaaa tctctcgtgataattctaagaatacttggtatctgcagaggccaggccagtctcctcagctcctgatttctatcgacgtatgccatgtccaaggag tcccagacaggttcagtagcagtgggtcaggaactgatttcacactgagaatcagcagagtggagcaatgtggcttcgatctcttccccccaa aacccaaggacaccctcatgatctccttcggaggggggaccaagctggaaataaaacgg(SEQ ID NO:20)。
其CDR氨基酸序列为:
VH CDR1:QTRER(SEQ ID NO:21),编码VH CDR1的核苷酸片段:核苷酸序列为cagacccgtgaacgc(SEQ ID NO:27);
VH CDR2:RRLRFALSMPGTELRDY(SEQ ID NO:22),编码VH CDR2的核苷酸片段:核苷酸序列为cgacggttaagattcgccttgagtatgccaggaacggagcttcgggattat(SEQ ID NO:28);
VH CDR3:VRHVTKL(SEQ ID NO:23),编码VH CDR3的核苷酸片段:核苷酸序列为gttaggcatgtgaccaagctg(SEQ ID NO:29);
VL CDR1:VQLLESEISRDNSKNT(SEQ ID NO:24),编码VL CDR1的核苷酸片段:核苷酸序列为gttcagcttcttgagtcggaaatctctcgtgataattctaagaatact(SEQ ID NO:30);
VL CDR2:IDVCHVQ(SEQ ID NO:25),编码VL CDR2的核苷酸片段:核苷酸序列为atcgacgtatgccatgtccaa(SEQ ID NO:31);
VL CDR3:QCGFDLFPPKPKDTLMIS(SEQ ID NO:26),编码VL CDR3的核苷酸片段:核苷酸序列为caatgtggcttcgatctcttccccccaaaacccaaggacaccctcatgatctcc(SEQ ID NO:32)。
2.2将得到的针对抗S蛋白抗体和抗N蛋白抗体的单克隆抗体的CDR的碱基序列合成,插入刚性linker连接,N端加入蜂毒信号肽序列,在序列上游加入限制性核酸内切酶位点 EcoRI(-GAATTC-),下游序列插入限制性核酸内切酶位点NcoI(-CCATGG-),将合成后的序列构建在pFUSE-hIgG1-Fc2质粒载体上(质谱图见附图1)。合成时各序列之间的排列顺序为:真核KOZAK-信号肽-抗SARS-CoV-2S蛋白抗体CDR碱基序列-Linker-抗 SARS-CoV-2N蛋白CDR碱基序列。
功能基因氨基酸序列:
Linker碱基序列:GGAGGCGGCGGATCT;Linker氨基酸序列:GGGGS
Linker碱基序列:GAAGCCGCTGCCAAG;Linker氨基酸序列:EAAAK
真核KOZAK碱基序列:GCCACC;真核KOZAK氨基酸序列:AT
蜂毒信号肽序列碱基序列:AAGTTCCTGGTCAACGTCGCTCTCGTGTTCATG;
蜂毒信号肽序氨基酸序列:KFLVNVALVFM
2.3重组抗体核苷酸序列如SEQ ID NO:33所示。
实施例3
3.1使用实施例2中构建的载体转化大肠杆菌细胞,挑选阳性克隆鉴定pFUSE-hIgG-S-N 质粒。
3.2将含有目的基因的pFUSE-hIgG-S-N质粒转染HEK293哺乳细胞进行表达,大量培养细胞,收集细胞培养物,离心取细胞上清。
3.3将含有目的基因的pFUSE-hIgG-S-N质粒,通过Lipofectamine 2000试剂将转染至CHO (GS缺陷型)细胞中,通过GS筛选体系获得单克隆细胞株。
3.4大量培养细胞,收集细胞培养物,10000rpm、15min条件下离心取细胞上清。
实施例4
4.1将实施例3中获取的细胞上清用Protein A亲和柱纯化。利用Gly-HCl进行梯度洗脱,去除杂蛋白,得到包含抗SARS-CoV-2S蛋白抗体可变区域的氨基酸序列和抗SARS-CoV-2N 蛋白抗体可变区域的氨基酸序列的人源化IgG。
4.2将纯化后的抗体用Western blotting鉴定,结果如图2所示;
4.3用紫外可见分光光度计测定蛋白在波长280nm处的吸光值,用吸光值除以1.35得到抗体浓度为295.86mg/L。
4.4用自产新型冠病毒S蛋白和N蛋白检测试剂盒(磁微粒化学发光法)测试重组抗体的浓度:
将重组抗体按1/10、1/50、1/100稀释梯度小样,分别使用新型冠病毒S蛋白和N蛋白试剂盒测试,根据计算软件反算出重组抗体原液浓度,结果如下:
表1
稀释比例 | S蛋白(ng/mL) | N蛋白(ng/mL) |
1/10 | 27.95 | 27.22 |
1/50 | 5.68 | 5.32 |
1/100 | 2.91 | 2.67 |
反算原浓度 | 284.83 | 268.40 |
实施例5:重组抗体的应用
一种新型冠病毒抗S蛋白和抗N蛋白IgG抗体检测试剂的复合质控品
5.1本试剂的主要成分见表2
表2:
5.2本试剂的性能评估:
稳定性:将待检测的复合质控品试剂和单项质控品试剂分别分为2组试剂(共4组),分别置于4℃、37℃,放置7天,再分别使用公司自产研发用新型冠病毒S蛋白和N蛋白试剂盒测定质控品原料的信号保留率。
信号保留率=置于37℃试剂测试所得发光值/置于4℃试剂测试所得发光值
5.2.1复合质控品稳定性
复合质控品测试结果见表3:
表3
5.2.2单项质控品稳定性
单项质控品测试结果见表4:
表4
由表3、4可知,复合质控品试剂于4℃、37℃分放置7天后,用于新型冠病毒S蛋白和N蛋白的测试信号保留率均>90%,表明质控品稳定性好,且优于单项质控品稳定性,符合临床要求。
经验证该重组抗体可作为新型冠病毒S蛋白和N蛋白检测试剂盒(磁微粒化学发光法) 的质控品使用。可作为新型冠病毒检测试剂盒的试剂研发及优化的备选原料。
上述实施例只为说明本发明的技术构思及特点,其目的在于让熟悉此项技术的人士能够了解本发明的内容并据以实施,并不能以此限制本发明的保护范围。凡根据本发明精神实质所作的等效变化或修饰,都应涵盖在本发明的保护范围之内。
序列表
<110> 苏州携创生物技术有限公司
<120> 一种抗SARS-CoV-2重组抗体及其应用
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Arg Arg Asp Ser Pro Ser Pro Glu Thr Met Pro Arg Thr Pro Tyr Thr
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gaagtacaac tggtggagtc tgggggagac cgattcagcc tggagggtcc cagaaactct 60
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tgcagattag cagtcagagg tctgaggaca cggccatata ttactgtgcg agacgaaacg 300
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Asp Ile Val Leu Thr Gln Ala Ala Phe Ser Asn Pro Val Thr Leu Gly
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Arg Leu Leu Ala Arg Asp Tyr Trp Tyr Leu Gln Arg Pro Gly Gln Ser
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Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile
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Arg
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Trp Arg Pro Arg Pro
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Arg
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Glu Thr Ala Arg Leu Gly Ser
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Gly Asp Ser Thr Tyr Leu Gln
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<210> 10
<211> 18
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Glu Phe Gly Leu Gly Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Glu
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Val His
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<212> DNA
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<212> DNA
<213> 人工序列(rengongxulie)
<400> 12
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<400> 13
gaaacggcac gtttgggttc t 21
<210> 14
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<212> DNA
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<400> 14
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<212> DNA
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<400> 15
ggagactcca cctaccttca a 21
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<212> DNA
<213> 人工序列(rengongxulie)
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gaatttggcc tcggtcaaat gaactcact 29
<210> 17
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<213> 人工序列(rengongxulie)
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Glu Val Gln Leu Val Glu Ser Gly Gly Asp Arg Phe Ser Leu Glu Gly
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Pro Arg Asn Ser Pro Val Gln Pro Pro Asp Ser Leu Ser Val Gln Thr
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Gln Arg Arg Leu Arg Phe Ala Leu Ser Met Pro Gly Thr Glu Leu Arg
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Asp Tyr Asp Ser Pro Ser Pro Glu Thr Met Pro Arg Thr Pro Tyr Thr
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Arg Asp Val Arg His Val Thr Lys Leu Gly Pro Lys Gly Leu Trp Ser
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115
<210> 18
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gaagtacaac tggtggagtc tgggggagac cgattcagcc tggagggtcc cagaaactct 60
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cggagaggaa attggagtgg gtcgcaacga cggttaagat tcgccttgag tatgccagga 180
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catgtgacca agctggggcc caagggactc tggtcactgt ctctg 345
<210> 19
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<212> PRT
<213> 人工序列(rengongxulie)
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Asp Ile Val Leu Thr Gln Ala Ala Phe Ser Asn Pro Val Thr Leu Gly
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Pro Gln Leu Leu Ile Ser Ile Asp Val Cys His Val Gln Gly Val Pro
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Asp Arg Phe Ser Ser Ser Gly Ser Gly Thr Asp Phe Thr Leu Arg Ile
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Ser Arg Val Glu Gln Cys Gly Phe Asp Leu Phe Pro Pro Lys Pro Lys
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Asp Thr Leu Met Ile Ser Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys
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Arg
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<213> 人工序列(rengongxulie)
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tatctgcaga ggccaggcca gtctcctcag ctcctgattt ctatcgacgt atgccatgtc 180
caaggagtcc cagacaggtt cagtagcagt gggtcaggaa ctgatttcac actgagaatc 240
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<210> 21
<211> 5
<212> PRT
<213> 人工序列(rengongxulie)
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Gln Thr Arg Glu Arg
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Arg Arg Leu Arg Phe Ala Leu Ser Met Pro Gly Thr Glu Leu Arg Asp
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Tyr
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Val Arg His Val Thr Lys Leu
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Val Gln Leu Leu Glu Ser Glu Ile Ser Arg Asp Asn Ser Lys Asn Thr
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Ile Asp Val Cys His Val Gln
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<213> 人工序列(rengongxulie)
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<210> 29
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<212> DNA
<213> 人工序列(rengongxulie)
<400> 29
gttaggcatg tgaccaagct g 21
<210> 30
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<212> DNA
<213> 人工序列(rengongxulie)
<400> 30
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<211> 21
<212> DNA
<213> 人工序列(rengongxulie)
<400> 31
atcgacgtat gccatgtcca a 21
<210> 32
<211> 54
<212> DNA
<213> 人工序列(rengongxulie)
<400> 32
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<210> 33
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<212> DNA
<213> 人工序列(rengongxulie)
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gaattcgcca ccaagttcct ggtcaacgtc gctctcgtgt tcatgtgcgc ggactcggtc 60
tcaatgaagc gacgattgct ggcccgcgat tatggaggcg gcggatctgg aggcggcgga 120
tctggagact ccacctacct tcaaggaggc ggcggatctg gaggcggcgg atctgaattt 180
ggcctcggtc aaatgaactc actacgagca gaagataccg aagtgcacgg aggcggcgga 240
tctggaggcg gcggatcttg gaggcctcgg ccaggaggcg gcggatctgg aggcggcgga 300
tcttcaccag gactaaccgc gttgagtcct tctatccaga caatgaagcg acgaggaggc 360
ggcggatctg gaggcggcgg atctgaaacg gcacgtttgg gttctgaagc cgctgccaag 420
gaagccgctg ccaaggaagc cgctgccaag gttcagcttc ttgagtcgga aatctctcgt 480
gataattcta agaatactgg aggcggcgga tctggaggcg gcggatctat cgacgtatgc 540
catgtccaag gaggcggcgg atctggaggc ggcggatctc aatgtggctt cgatctcttc 600
cccccaaaac ccaaggacac cctcatgatc tccggaggcg gcggatctgg aggcggcgga 660
tctcagaccc gtgaacgcgg aggcggcgga tctggaggcg gcggatctcg acggttaaga 720
ttcgccttga gtatgccagg aacggagctt cgggattatg gaggcggcgg atctggaggc 780
ggcggatctg ttaggcatgt gaccaagctg ggaggcggcg gatctggagg cggcggatct 840
ccatggctga caaaactcac acatgcccac cgtgcccagc acctgaactc ctggggggac 900
cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc cggacccctg 960
aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag ttcaactggt 1020
acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag cagtacaaca 1080
gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg aatggcaagg 1140
agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa accatctcca 1200
aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc cgggaggaga 1260
tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc agcgacatcg 1320
ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg cctcccgtgc 1380
tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag agcaggtggc 1440
agcaggggaa cgtcttctca tgctccgtga tgcacgaggc tctgcacaac cactacacgc 1500
agaagagcct ctccctgtct ccgggtaaat gagtgctagc tgg 1543
Claims (13)
1.一种抗SARS-CoV-2重组抗体,其特征在于:所述重组抗体包含抗SARS-CoV-2S蛋白抗体可变区域的氨基酸序列和抗SARS-CoV-2N蛋白抗体可变区域的氨基酸序列。
2.根据权利要求1所述的抗SARS-CoV-2重组抗体,其特征在于:所述抗SARS-CoV-2S蛋白抗体可变区域的氨基酸序列包括抗SARS-CoV-2S蛋白抗体重链可变区和抗SARS-CoV-2S蛋白抗体轻链可变区,所述抗SARS-CoV-2S蛋白抗体重链可变区包含
VH CDR1:氨基酸序列为WRPRP或与其具有至少85%的同源性,和/或,
VH CDR2:氨基酸序列为SPGLTALSPSIQTMKRR或与其具有至少85%的同源性,和/或,
VH CDR3:氨基酸序列为ETARLGS或与其具有至少85%的同源性,
所述抗SARS-CoV-2S蛋白抗体轻链可变区包含
VL CDR1:氨基酸序列为CADSVSMKRRLLARDY或与其具有至少85%的同源性,和/或,
VL CDR2:氨基酸序列为GDSTYLQ或与其具有至少85%的同源性,和/或,
VL CDR3:氨基酸序列为EFGLGQMNSLRAEDTEVH或与其具有至少85%的同源性;
所述抗SARS-CoV-2N蛋白抗体可变区域的氨基酸序列包括抗SARS-CoV-2N蛋白抗体重链可变区和抗SARS-CoV-2N蛋白抗体轻链可变区,所述抗SARS-CoV-2N蛋白抗体重链可变区包含
VH CDR1:氨基酸序列为QTRER或与其具有至少85%的同源性,和/或,
VH CDR2:氨基酸序列为RRLRFALSMPGTELRDY或与其具有至少85%的同源性,和/或,
VH CDR3:氨基酸序列为VRHVTKL或与其具有至少85%的同源性,
所述抗SARS-CoV-2N蛋白抗体轻链可变区包含
VL CDR1:氨基酸序列为VQLLESEISRDNSKNT或与其具有至少85%的同源性,和/或,
VL CDR2:氨基酸序列为IDVCHVQ或与其具有至少85%的同源性,和/或,
VL CDR3:氨基酸序列为QCGFDLFPPKPKDTLMIS或与其具有至少85%的同源性。
3.根据权利要求1或2所述的抗SARS-CoV-2重组抗体,其特征在于:所述抗SARS-CoV-2重组抗体为人源化IgG抗体。
4.一种重组核苷酸分子,其特征在于,所述核苷酸分子编码权利要求1至3中任一项所述S蛋白抗体可变区域的氨基酸序列和所述N蛋白抗体可变区域的氨基酸序列。
5.根据权利要求4所述的重组核苷酸分子,其特征在于,其序列之间的排列顺序为:真核KOZAK-信号肽-抗SARS-CoV-2S蛋白抗体可变区域碱基序列-Linker-抗SARS-CoV-2N蛋白可变区域碱基序列。
6.根据权利要求5所述的重组核苷酸分子,其特征在于,所述真核KOZAK的碱基序列为GCCACC;所述信号肽的碱基序列为AAGTTCCTGGTCAACGTCGCTCTCGTGTTCATG;所述Linker的碱基序列为GGAGGCGGCGGATCT和/或GAAGCCGCTGCCAAG。
7.根据权利要求4所述的重组核酸分子,其特征在于,所述重组核苷酸分子还包括人IgG Fc片段。
8.根据权利要求7所述的重组核苷酸分子,其特征在于,所述重组核苷酸分子的碱基序列如SEQ ID NO:33所示。
9.一种重组表达载体,其特征在于:所述重组表达载体包含权利要求4至8中任一项所述的重组核苷酸分子。
10.一种宿主细胞,其特征在于:用权利要求9所述的重组表达载体转染哺乳动物细胞获得所述宿主细胞。
11.一种如权利要求1至3中任一项所述的抗SARS-CoV-2重组抗体的制备方法,其特征在于:所述制备方法包括如下步骤:
(1)人工合成编码权利要求1至3中任一项所述的可变区基因序列并构建在载体pFUSE-hIgG1-Fc2上制备重组质粒;
(2)将步骤(1)的重组质粒转染哺乳动物细胞表达重组蛋白。
(3)对步骤(2)的重组蛋白进行纯化处理,即得所述抗SARS-CoV-2重组抗体。
12.一种如权利要求1至3中任一项所述的抗SARS-CoV-2重组抗体在SARS-CoV-2病毒和/或SARS-CoV-2病毒中和抗体检测中的应用。
13.一种SARS-CoV-2中和抗体检测复合质控品,其特征在于:所述复合质控品的原料包括权利要求1至3中任一项所述的抗SARS-CoV-2重组抗体。
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