CN117088968A - 抗新型冠状病毒的抗体、检测新型冠状病毒的试剂和试剂盒 - Google Patents
抗新型冠状病毒的抗体、检测新型冠状病毒的试剂和试剂盒 Download PDFInfo
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Classifications
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- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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- C12N5/0602—Vertebrate cells
- C12N5/0681—Cells of the genital tract; Non-germinal cells from gonads
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- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56983—Viruses
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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- C07K2317/565—Complementarity determining region [CDR]
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
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Abstract
本发明公开了一种抗新型冠状病毒的抗体、检测新型冠状病毒试剂和试剂盒,涉及抗体技术领域。本发明通过将抗新型冠状病毒的IgG抗体的重链恒定区部分地替换为IgM抗体的重链恒定区,制备得到亲和力提升的抗新型冠状病毒的类IgM抗体,为新型冠状病毒的检测提供了更为丰富的抗体选择。
Description
技术领域
本发明涉及抗体技术领域,具体而言,涉及一种抗新型冠状病毒的抗体、检测新型冠状病毒的试剂和试剂盒。
背景技术
呼吸道飞沫和密切接触传播是新型冠状病毒肺炎主要的传播途径,在相对封闭的环境中长时间暴露于高浓度气溶胶情况中存在经气溶胶传播的可能。2019-nCoV具有很强的传染性,多数患者感染后出现临床症状,但也有部分患者为无症状感染者。人感染了冠状病毒后常见体征有呼吸道症状、发热、咳嗽、气促和呼吸困难等。在较严重病例中,感染可导致肺炎、严重急性呼吸综合征、肾衰竭,甚至死亡。虽然目前对于新型冠状病毒所致疾病没有特异治疗方法,但是轻症、或者无症状患者通过治疗可以大大的提高治愈率,缩短治疗时间。因此,对患者的检测或者鉴定变的尤为重要。
新型冠状病毒目前主要以核酸检测和病毒基因测序为病原学确诊证据,由于采样、操作以及试剂等多方面因素影响,核酸检测会出现假阴性结果。高度疑似2019新型冠状病毒(2019-nCoV)感染患者的病毒核酸阳性检出率仅为30%~50%。另外,核酸检测对仪器设备、检测场地及环境条件要求高,存在检测时间长、通量低等缺点,不便于目前疫情下的人群大规模检测。与核酸检测方法相比,抗体检测样本为血清或血浆,受样本采样的影响较小,有利于早期诊断和排除可疑病例,同时检测快速、方便、适合大规模筛查。因此,抗体检测试剂盒变得更为重要。
因此,迫切需要研发出一种快速便捷检测试剂盒用于临床检测,从而尽快隔离感染人群以阻断病毒传播。
新型冠状病毒2019-nCoV的结构蛋白分为:刺突糖蛋白(S蛋白)、包膜糖蛋白(E蛋白)、膜糖蛋白(M蛋白)和核衣壳蛋白(N蛋白),这些蛋白包括多个抗原表位。N蛋白与病毒基因组RNA 相互缠绕形成病毒核衣壳,在病毒RNA的合成过程中发挥着重要的作用。同时,N蛋白相对保守,在病毒的结构蛋白中所占比例最大,感染早期机体就能产生抗N蛋白的高水平抗体。最后,N蛋白是新型冠状病毒重要的标志蛋白,利用抗原与抗体特异性结合的原理,可通过N蛋白单克隆抗体检测抗原的存在,从而直接证明样本中含有新型冠状病毒,实现新型冠状病毒的检测。
目前2019-nCoV N蛋白单克隆抗体产品较少,性能存在差异。
鉴于此,特提出本发明。
发明内容
本发明旨在提供一种抗新型冠状病毒的抗体、检测新型冠状病毒的试剂和试剂盒。本发明所述的抗新型冠状病毒的抗体为经由IgG抗体恒定区改造得到的类IgM抗体。所述抗体对新型冠状病毒的N蛋白具有改善的亲和力,为新型冠状病毒的检测提供了更为丰富的抗体选择。
为了实现上述目的,根据本发明的一个方面,提供了一种抗新型冠状病毒或其N蛋白的抗体,所述抗体包括:
(i)氨基酸序列如SEQ ID NO:1-6所示的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3;
(ii)重链恒定区和轻链恒定区,所述重链恒定区C端含有IgM抗体重链恒定区的CH3-CH4 区氨基酸序列。
为了实现上述目的,根据本发明的第二个方面,提供了一种抗新型冠状病毒或其N蛋白的抗体,所述抗体包含氨基酸序列如SEQ ID NO:17所示的重链和氨基酸序列如SEQ IDNO:18所示的轻链。
为了实现上述目的,根据本发明的第三个方面,提供了一种抗体偶联物,所述抗体偶联物包括上述的抗体。
为了实现上述目的,根据本发明的第四个方面,提供了一种检测新型冠状病毒或其N蛋白的试剂或试剂盒,所述试剂或试剂盒包括上述的抗体或抗体偶联物。
为了实现上述目的,本发明还提供了一种载体、一种细胞及一种制备上述抗体的方法。
附图说明
为了更清楚地说明本发明实施例的技术方案,下面将对实施例中所需要使用的附图作简单地介绍,应当理解,以下附图仅示出了本发明的某些实施例,因此不应被看作是对范围的限定,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他相关的附图。
图1为实施例1的抗新型冠状病毒的IgG抗体9B54的还原性SDS-PAGE的结果。
图2为实施例2的抗新型冠状病毒的类IgM抗体8C21的还原性SDS-PAGE的结果。
具体实施方式
本发明提供了一种抗新型冠状病毒或其N蛋白的抗体,所述抗体包括:
(i)氨基酸序列如SEQ ID NO:1-6所示的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3;
(ii)重链恒定区和轻链恒定区,所述重链恒定区C端含有IgM抗体重链恒定区的CH3-CH4 区氨基酸序列。
在本发明中,术语“抗体”在最广义上使用,其可以包括全长单克隆抗体,双特异性或多特异性抗体,以及嵌合抗体,只要它们展示所需的生物学活性。
在本发明中,术语“互补性决定区”、“CDR”或“CDRs”是指免疫球蛋白的重链和轻链的高度可变区,指包含一种或多种或者甚至全部的对抗体或抗原结合片段与其识别的抗原或表位的结合起作用的主要氨基酸残基的区域。在本发明具体实施方式中,CDRs是指所述抗体的重链和轻链的高度可变区。
在本发明中,重链互补决定区用HCDR表示,其包括HCDR1、HCDR2和HCDR3;轻链互补决定区用LCDR表示,其包括LCDR1、LCDR2和LCDR3。本领域常用的CDR标示方法包括:Kabat编号方案、IMGT编号方案、Chothia和Lesk编号方案以及1997年Lefranc等人为免疫球蛋白超家族的所有蛋白质序列引入的新的标准化编号系统。Kabat等人是第一个为免疫球蛋白可变区提出标准化编号方案的人。在过去的几十年中,序列的积累导致了KABATMAN数据库的创建,Kabat编号方案通常被认为是编号抗体残基广泛采用的标准。本发明采用Kabat注释标准标示CDR区,但其他方法标示的CDR区也属于本发明的保护范围。
在本发明中,“框架区”或“FR”包括重链框架区和轻链框架区,是指抗体重链可变区和轻链可变区中除CDR之外的区域;其中,重链框架区可以被进一步细分成被CDR分隔开的毗邻区域,包含HFR1、HFR2、HFR3和HFR4;轻链框架区可以被进一步细分成被CDR分隔开的毗邻区域,包含LFR1、LFR2、LFR3和LFR4。
在本发明中,重链可变区由以下编号的CDR与FR按如下组合排列连接获得:HFR1-HCDR1-HFR2-HCDR2-HFR3-HCDR3-HFR4;轻链可变区由以下编号的CDR与FR按如下组合排列连接获得:LFR1-LCDR1-LFR2-LCDR2-LFR3-LCDR3-LFR4。
在可选的实施方式中,所述抗体具有氨基酸序列如SEQ ID NO:7-10所示的HFR1、HFR2、HFR3、 HFR4,和氨基酸序列如SEQ ID NO:11-14所示的LFR1、LFR2、LFR3和LFR4,或与所述HFR或 LFR具有至少80%同一性的氨基酸序列。
需要说明的是,在其他的实施例中,本发明提供的抗体的框架区氨基酸序列可以与上述对应框架区(SEQ ID NO:7、8、9、10、11、12、13或14)具有至少80%、81%、82%、83%、84%、85%、 86%、87%、88%、89%、90%、91%、92%、93%、94%、95%、96%、97%、98%或99%的同一性。
在本发明中,所述抗体恒定区包括重链恒定区和轻链恒定区。抗体重链类型决定了抗体的类型,哺乳动物抗体重链可分为五类,分别以希腊字母γ、α、μ、δ、和ε表示,据此将抗体相应地分为IgG、 IgA、IgM、IgD和IgE。在自然状态下IgM抗体在人体内以五聚体或者六聚体形式存在,IgM抗体是在免疫系统接触免疫原后最早表达的抗体。一个IgM抗体的重链有4个恒定区和1个可变区,轻链与其它型的抗体相同。在本发明中,所述“类IgM抗体”为非天然获得的IgM抗体,而是经由IgG 抗体恒定区改造,即,既保留了IgG抗体可变区,又具有IgM聚体形成能力的类似于自然状态下的 IgM抗体。
在可选的实施方式中,所述抗体重链恒定区含有CH1-铰链区-CH2-CH3-CH4的序列结构,所述CH1来自IgG抗体,所述铰链区来自IgG抗体铰链区的上铰链区,所述CH2-CH3-CH4来自 IgM抗体。
在可选的实施方式中,所述铰链区序列为VPRDC。
在可选的实施方式中,所述恒定区的种属来源为牛、马、乳牛、猪、绵羊、山羊、大鼠、小鼠、狗、猫、兔、驴、鹿、貂、鸡、鸭、鹅、火鸡、斗鸡或人。
在可选的实施方式中,所述恒定区的种属来源为小鼠。
在可选的实施方式中,所述重链恒定区序列如SEQ ID NO:15所示,所述轻链恒定区序列如SEQ ID NO:16所示。
需要说明的是,在其他的实施例中,所述恒定区序列可以与上述恒定区(SEQ IDNO:15或16) 具有至少80%、81%、82%、83%、84%、85%、86%、87%、88%、89%、90%、91%、92%、93%、 94%、95%、96%、97%、98%或99%的同一性。
在可选的实施方式中,所述抗体以KD≤10-9M的亲和力结合新型冠状病毒或其N蛋白。
在可选的实施方式中,所述抗体以KD≤10-10M的亲和力结合新型冠状病毒或其N蛋白。
在可选的实施方式中,所述抗体以KD≤3.10×10-11M的亲和力结合新型冠状病毒或其N蛋白。
KD的检测参考本发明实施例中的方法进行。
另一方面,本发明提供一种抗新型冠状病毒或其N蛋白的抗体,所述抗体包含氨基酸序列如 SEQ ID NO:17所示的重链和氨基酸序列如SEQ ID NO:18所示的轻链。
另一方面,本发明提供一种抗体偶联物,所述抗体偶联物包括上述的抗体。其中,所述抗体直接或间接共价偶联至待偶联物。或者,所述抗体以非共价吸附的方式偶联至待偶联物。
在可选的实施方式中,上述抗体偶联物中所述抗体偶联至生物素或其衍生物。
在可选的实施方式中,上述抗体偶联物中所述抗体偶联至标记物。
在可选的实施方式中,上述标记物是指具有能够被肉眼直接观察或被仪器检测或探测到的特性例如发光、显色、放射性等特性的一类物质,通过该特性可以实现对相应目标物的定性或定量检测。
在可选的实施方式中,所述标记物包括但不限于荧光染料、酶、放射性同位素、化学发光试剂和纳米颗粒类标记物。
在实际的使用过程中,本领域技术人员可以根据检测条件或实际需要选择合适的标记物,无论使用何种标记物,其均属于本发明的保护范围。
在可选的实施方式中,所述荧光染料包括但不限于荧光素类染料及其衍生物(例如包括但不限于异硫氰酸荧光素(FITC)羟基光素(FAM)、四氯光素(TET)等或其类似物)、罗丹明类染料及其衍生物(例如包括但不限于红色罗丹明(RBITC)、四甲基罗丹明(TAMRA)、罗丹明B(TRITC) 等或其类似物)、Cy系列染料及其衍生物(例如包括但不限于Cy2、Cy3、Cy3B、Cy3.5、Cy5、Cy5.5、 Cy3等或其类似物)、Alexa系列染料及其衍生物(例如包括但不限于AlexaFluor350、405、430、488、 532、546、555、568、594、610、33、647、680、700、750等或其类似物)和蛋白类染料及其衍生物(例如包括但不限于藻红蛋白(PE)、藻蓝蛋白(PC)、别藻蓝蛋白(APC)、多甲藻黄素-叶绿素蛋白(preCP)等)。
在可选的实施方式中,所述酶包括但不限于辣根过氧化物酶、碱性磷酸酶、β-半乳糖苷酶、葡萄糖氧化酶、碳酸酐酶、乙酰胆碱酯酶以及6-磷酸葡萄糖脱氧酶。
在可选的实施方式中,所述放射性同位素包括但不限于212Bi、131I、111In、90Y、186Re、211At、125I、188Re、153Sm、213Bi、32P、94mTc、99mTc、203Pb、67Ga、68Ga、43Sc、47Sc、110mIn、97Ru、62Cu、64Cu、67Cu、68Cu、86Y、88Y、121Sn、161Tb、166Ho、105Rh、177Lu、172Lu和18F。
在可选的实施方式中,所述化学发光试剂包括但不限于鲁米诺及其衍生物、光泽精、甲壳动物荧光素及其衍生物、联吡啶钌及其衍生物、吖啶酯及其衍生物、二氧环乙烷及其衍生物、洛粉碱及其衍生物和过氧草酸盐及其衍生物。
在可选的实施方式中,所述纳米颗粒类标记物包括但不限于纳米颗粒、胶体、有机纳米颗粒、磁性纳米颗粒、量子点纳米颗粒和稀土络合物纳米颗粒。
在可选的实施方式中,所述胶体包括但不限于胶体金属、分散型染料、染料标记的微球和乳胶。
在可选的实施方式中,所述胶体金属包括但不限于胶体金、胶体银和胶体硒。
在可选的实施方式中,所述胶体金属为胶体金。
在可选的实施方式中,上述抗体偶联物中所述抗体偶联至固相。
在可选的实施方式中,所述固相选自微球、板和膜。
在可选的实施方式中,所述固相包括但不限于磁性微球、塑料微球、塑胶微粒、微孔板、玻璃、毛细管、尼龙和硝酸纤维素膜。
在可选的实施方式中,所述固相为硝酸纤维素膜。
另一方面,本发明提供一种检测新型冠状病毒或其N蛋白的试剂或试剂盒,所述试剂或试剂盒包括上述的抗体或上述的抗体偶联物,包含上述的抗体的试剂或试剂盒具有更优的检测活性。
另一方面,本发明提供上述试剂或试剂盒在新型冠状病毒或其N蛋白检测中的用途。
另一方面,本发明提供一种编码上述抗体的核酸分子。
另一方面,本发明提供含有上述核酸分子的载体。
另一方面,本发明提供含有上述核酸或载体的细胞。
另一方面,本发明提供一种制备抗体的方法,其包括:培养如上所述的细胞。
在本发明公开了抗体或其功能性片段的氨基酸序列的基础上,本领域技术人员容易想到采用基因工程技术或其他技术(化学合成、重组表达)制备得到该抗体,例如从能够重组表达如上任一项所述的抗体的重组细胞的培养产物中分离纯化得到该抗体或其功能性片段,这对本领域技术人员来说是容易实现的,基于此,无论采用何种技术制备本发明的抗体或其功能性片段,其均属于本发明的保护范围。
为使本发明实施例的目的、技术方案和优点更加清楚,下面将对本发明实施例中的技术方案进行清楚、完整地描述。实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行。所用试剂或仪器未注明生产厂商者,均为可以通过市售购买获得的常规产品。
除非另有定义,否则本文使用的所有技术和科学术语具有与本公开内容所属领域的普通技术人员通常理解的含义相同的含义。尽管与本文描述的那些方法和材料类似或等同的任何方法和材料都可用于本文的制剂或单位剂量的实践或测试,但现在描述一些方法和材料。除非另有说明,否则本文采用或考虑的技术是标准方法。材料、方法和实例仅是说明性而非限制性的。
除非另外指明,否则实践本发明将采用细胞生物学、分子生物学(包含重组技术)、微生物学、生物化学和免疫学的常规技术,所述常规技术在本领域技术人员的能力范围内。文献中充分解释了这种技术,如《分子克隆:实验室手册(Molecular Cloning:ALaboratory Manual)》,第二版(Sambrook 等人,1989);《寡核苷酸合成(OligonucleotideSynthesis)》(M.J.Gait编,1984);《动物细胞培养(Animal Cell Culture)》(R.I.Freshney编,1987);《酶学方法(Methods in Enzymology)》(学术出版社有限公司(Academic Press,Inc.);《实验免疫学手册(Handbook of Experimental Immunology)》(D.M.Weir 和C.C.Blackwell编);《哺乳动物细胞用基因转移载体(Gene Transfer Vectors forMammalian Cells)》 (J.M.Miller和M.P.Calos编,1987);《当代分子生物学方法(CurrentProtocols in Molecular Biology)》 (F.M.Ausubel等人编,1987);《PCR:聚合酶链反应(PCR:The Polymerase Chain Reaction)》(Mullis 等人编,1994);以及《当代免疫学方法(Current Protocols in Immunology)》(J.E.Coligan等人编, 1991),所述文献中的每个文献均通过引用明确并入本文中。
以下结合实施例对本发明的特征和性能作进一步的详细描述。
本实施例中限制性内切酶、Prime Star DNA聚合酶购自Takara公司。MagExtractor-RNA提取试剂盒购自TOYOBO公司。BD SMARTTM RACE cDNA AmplificationKit试剂盒购自Takara公司。 pMD-18T载体购自Takara公司。质粒提取试剂盒购自天根公司。引物合成和基因测序由Invitrogen 公司完成。
实施例1:IgG抗体9B54的制备
1.重组质粒的构建
(1)抗体基因制备
从分泌抗新型冠状病毒N蛋白的抗体的杂交瘤细胞株中提取mRNA,通过RT-PCR方法获得 DNA产物,该产物用rTaq DNA聚合酶进行加A反应后插入到pMD-18T载体中,转化到DH5α感受态细胞中,长出菌落后分别取Heavy Chain及Light Chain基因克隆,各4个克隆送基因测序公司进行测序。
(2)9B54抗体可变区基因的序列分析
将上述测序得到的基因序列放在Kabat抗体数据库中进行分析,并利用VNTI11.5软件进行分析确定重链和轻链引物对扩增出的基因都是正确的,其中Light Chain扩增出的基因片段中,VL基因序列为321bp,其前方有57bp的前导肽序列;Heavy Chain引物对扩增出的基因片段中,VH基因序列为369bp,属于VH1基因家族,其前方有57bp的前导肽序列。9B54重、轻链的序列分别如SEQ ID NO:23、18所示。
(3)重组抗体表达质粒的构建
pcDNATM 3.4vector为构建的重组抗体真核表达载体,该表达载体已经引入HindIII、 BamHI、EcoRI等多克隆酶切位点,并命名为pcDNA3.4A表达载体,后续简称3.4A表达载体;根据上述pMD-18T中抗体可变区基因测序结果,设计该抗体的VL和VH基因特异性引物,两端分别带有HindIII、EcoRI酶切位点和保护碱基,通过PCR扩增方法扩出0.72kb的Light Chain基因片段和1.40kb的Heavy Chain基因片段。
Heavy Chain和Light Chain基因片段分别采用HindIII/EcoRI双酶切,3.4A载体采用HindIII/EcoRI 双酶切,将片段和载体纯化回收后Heavy Chain基因和Light Chain基因分别连接3.4A表达载体中,分别得到Heavy Chain和Light Chain的重组表达质粒。
2.稳定细胞株筛选
(1)重组抗体表达质粒瞬时转染CHO细胞,确定表达质粒活性
质粒用超纯水稀释至40ug/100ul,调节CHO细胞1.43×107cells/ml于离心管中,100μL质粒与 700μL细胞混合,转入电转杯,电转,第3、5、7天取样计数,第7天收样检测。
包被液稀释2019-nCoV N蛋白抗原到1μg/ml,每孔100μL,4℃过夜;次日,洗涤液清洗2次,拍干;加入封闭液(20%BSA+80%PBS),每孔120μL,37℃,1h,拍干;加入稀释后的细胞上清, 100μL/孔,37℃,30min(部分上清1h);洗涤液清洗5次,拍干;加入羊抗鼠IgG-HRP,每孔100μL, 37℃,30min;洗涤液清洗5次,拍干;加入显色液A液(50μL/孔,含柠檬酸+醋酸钠+乙酰苯胺+ 过氧化脲),加入显色液B液(50μL/孔,含柠檬酸+EDTA·2Na+TMB+浓HCL),10min;加入终止液(50μL/孔,含EDTA·2Na+浓H2SO4);酶标仪上450nm(参考630nm)处读OD值。结果显示细胞上清稀释1000倍后反应OD仍大于1.0,未加细胞上清孔反应OD小于0.1,表明质粒瞬转后产生的抗体对2019-nCoV N蛋白抗原有活性。
(2)重组抗体表达质粒线性化
准备下述试剂:Buffer 50μL、DNA 100μg/管、PuvⅠ酶10μL、无菌水补至500μL,37℃水浴酶切过夜;先用等体积酚/氯仿/异戊醇(下层)25:24:1,再用氯仿(水相)依次进行抽提;0.1倍体积(水相)3M醋酸钠和2倍体积乙醇冰上沉淀,70%乙醇漂洗沉淀,去除有机溶剂,待乙醇挥发完全用适量的灭菌水进行复融,最后进行浓度的测定。
(3)重组抗体表达质粒稳定转染,加压筛选稳定细胞株
质粒用超纯水稀释至400ng/ml,调节CHO细胞1.43×107cells/ml于离心管中,100μL质粒与700μL 细胞混合,转入电转杯,电转,次日计数;25umol/L MSX 96孔加压培养约25天。
显微镜下观察标记长有细胞的克隆孔,并记录汇合度;取培养上清,送样检测;挑选抗体浓度、相对浓度高的细胞株转24孔,3天左右转6孔;3天后保种批培,调整细胞密度0.5×106cells/ml,2.2ml 进行批培养,细胞密度0.3×106cells/ml,2ml进行保种;7天6孔批培上清送样检测,挑选抗体浓度及细胞直径较小的细胞株转TPP保种传代。
3.重组抗体生产
(1)细胞扩培
细胞复苏之后先在125ml规格的摇瓶中培养,接种体积为30ml,培养基为100%Dynamis培养基,放置于转速120r/min,温度为37℃,二氧化碳为8%的摇床中。培养72h,以50万cells/ml接种密度接种扩培,扩培体积根据生产需求进行计算,培养基为100%Dynamis培养基。之后每72h扩培一次。当细胞量满足生产需求时,严格控制接种密度为50万cells/ml左右进行生产。
(2)摇瓶生产及纯化
摇瓶参数:转速120r/min,温度为37℃,二氧化碳为8%。流加补料:在摇瓶中培养至72h时开始每天补料,HyCloneTM Cell BoostTM Feed 7a每天流加初始培养体积的3%,Feed 7b每天流加量为初始培养体积的千分之一,一直补到第12天(第12天补料)。葡萄糖在第六天补加3g/L。第 13天收样。用proteinA亲和层析柱进行亲和纯化。取2μg纯化的抗体进行还原性SDS-PAGE,2μg 外来对照抗体作为对照,电泳图如下图1所示,在还原性SDS-PAGE后显示两条带,1条Mr为50KD,另一条Mr为28KD。
实施例2:类IgM抗体8C21表达载体的构建及制备
1.编码IgM重链恒定区的多核苷酸的制备
本申请中,编码鼠抗体CH区的多核苷酸可以通过PCR的方式从鼠外周血细胞DNA中获取,也可以通过PCR的方式从分泌相应类型的单克隆抗体的杂交瘤细胞株获取。
2.类IgM抗体8C21表达载体的构建
将本实施例1中制备得到的IgG抗体9B54的恒定区部分替换为IgM抗体的恒定区,构建得到的含有9B54可变区的新的类IgM抗体,命名为8C21。
IgM本身序列具有的多聚体特点。首先将编码9B54的重链区段1的多核苷酸通过桥式PCR的方式和编码IgM CH区段2的多核苷酸进行连接。9B54的重链区段1的氨基酸序列如下:
EVLLQQSGPELVNPGASVKIPCKASGYTFTDYNMGWVKQSHGKSLEWIGAIIPNNGGTIYNQ KFKGKATLTVDESSSTAYMELRSLTSEDTAVYYCAREAYRDYDVKTWFAYWGQGTLVTVSAAK TTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSV TVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDC(SEQ ID NO:21所示)
所述IgM CH区段2的氨基酸序列如下:
PAVAEMNPNVNVFVPPRDGFSGPAPRKSKLICEATNFTPKPITVSWLKDGKLVESGFTTDPVTI ENKGSTPQTYKVISTLTISEIDWLNLNVYTCRVDHRGLTFLKNVSSTCAASPSTDILTFTIPPSFADIF LSKSANLTCLVSNLATYETLNISWASQSGEPLETKIKIMESHPNGTFSAKGVASVCVEDWNNRKEF VCTVTHRDLPSPQKKFISKPNEVHKHPPAVYLLPPAREQLNLRESATVTCLVKGFSPADISVQWLQ RGQLLPQEKYVTSAPMPEPGAPGFYFTHSILTVTEEEWNSGETYTCVVGHEALPHLVTERTVDKST GKPTLYNVSLIMSDTGGTCY(SEQ ID NO:22所示)
通过PCR获得的DNA片段,经HindIII/EcoRI双酶切,连接至pcDNATM 3.4vector中,简称pcDNA3.4A-8C21TB2VCH。
3.类IgM抗体8C21的表达制备
IgM抗体8C21的表达制备同实施例1中的2(2)至3(2)。取纯化的抗体进行还原性SDS-PAGE,外来对照抗体作为对照,电泳图如下图2所示,在还原性SDS-PAGE后显示两条带,1条Mr为60-75kD (重链),另一条Mr为20-35kD(轻链)。
实施例3:抗体的性能检测
1.亲和力检测
利用AMC传感器,将纯化出来的抗体用PBST稀释到10μg/mL,2019-nCoV N蛋白抗原用PBST 进行梯度稀释。
运行流程:缓冲液1(PBST)中平衡60s,抗体溶液中固化抗体300s,缓冲液2(PBST)中孵育180s,抗原溶液中结合420s,缓冲液2中解离1200s,用10mM pH 1.69GLY溶液及缓冲液3进行传感器再生,输出数据。KD表示平衡解离常数即亲和力;kon表示结合速率;kdis表示解离速率。结果见下表。
表1亲和力检测数据
样品名称 | KD(M) | kon(1/Ms) | kdis(1/s) |
对照 | 2.43E-08 | 4.52E+03 | 1.10E-04 |
9B54 | 1.02E-10 | 1.11E+06 | 1.13E-04 |
8C21 | 3.10E-11 | 2.11E+06 | 6.56E-05 |
2.活性检测
包被液(主要成分NaHCO3)稀释2019-nCoV N蛋白抗原到3μg/ml进行微孔板包被,每孔100μl,4℃过夜;次日,洗涤液清洗2次,拍干;加入封闭液(20%BSA+80%PBS),每孔120μl,37℃,1h,拍干;加入稀释后的实施例2中的单克隆抗体,100μl/孔,37℃,30min-60min;洗涤液清洗5次,拍干;加入HRP标记的羊抗鼠二抗,每孔100μl,37℃,30min;洗涤液(PBS)清洗5次,拍干;加入显色液A液(50μl/孔,含2.1g/L柠檬酸、12.25g/L柠檬酸、0.07g/L乙酰苯胺和0.5g/L过氧化脲),加入显色液B液(50μl/孔,含1.05g/L柠檬酸、0.186g/LEDTA·2Na、0.45g/L TMB和0.2ml/L浓 HCl),10min;加入终止液(50μl/孔,含0.75g/EDTA·2Na和10.2ml/L浓H2SO4);酶标仪上450nm (参考630nm)处读OD值。结果见下表。
表2活性数据
浓度(ng/ml) | 100 | 50 | 25 | 12.5 | 6.25 | 0.000 |
8C21 | 2.182 | 2.089 | 1.491 | 0.844 | 0.408 | 0.011 |
3.稳定性考核
将8C21抗体置于4℃(冰箱)、-80℃(冰箱)、37℃(恒温箱)放置21天,取7天、14天、21 天样品进行状态观察,并对21天样品进行活性检测,结果显示三种考核条件下抗体放置21天均未见明显蛋白状态变化,活性也未随考核温度的升高呈下降趋势,说明上述抗体稳定。下表为8C21 抗体考核21天的酶免活性检测OD结果。
表3
样品浓度(ng/ml) | 25 | 12.5 | 0 |
4℃,21天样品 | 1.501 | 0.882 | 0.012 |
-80℃,21天样品 | 1.498 | 0.853 | 0.011 |
37℃,21天样品 | 1.487 | 0.843 | 0.011 |
以上所述仅为本发明的优选实施例而已,并不用于限制本发明,对于本领域的技术人员来说,本发明可以有各种更改和变化。凡在本发明的精神和原则之内,所作的任何修改、等同替换、改进等,均应包含在本发明的保护范围之内。
本申请涉及的部分氨基酸序列如下所示:
序列编号 | 序列片段 |
SEQ ID NO:1 | DYNMG |
SEQ ID NO:2 | AIIPNNGGTIYNQKFKG |
SEQ ID NO:3 | EAYRDYDVKTW |
SEQ ID NO:4 | SASQGIRNYLN |
SEQ ID NO:5 | YTSTLHS |
SEQ ID NO:6 | QQYSKFP |
SEQUENCE LISTING
<110> 东莞市朋志生物科技有限公司
<120> 抗新型冠状病毒的抗体、检测新型冠状病毒的试剂和试剂盒
<130> P2022061CN01
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Ala Arg Glu Gln Leu Asn Leu Arg Glu Ser Ala Thr Val Thr Cys Leu
340 345 350
Val Lys Gly Phe Ser Pro Ala Asp Ile Ser Val Gln Trp Leu Gln Arg
355 360 365
Gly Gln Leu Leu Pro Gln Glu Lys Tyr Val Thr Ser Ala Pro Met Pro
370 375 380
Glu Pro Gly Ala Pro Gly Phe Tyr Phe Thr His Ser Ile Leu Thr Val
385 390 395 400
Thr Glu Glu Glu Trp Asn Ser Gly Glu Thr Tyr Thr Cys Val Val Gly
405 410 415
His Glu Ala Leu Pro His Leu Val Thr Glu Arg Thr Val Asp Lys Ser
420 425 430
Thr Gly Lys Pro Thr Leu Tyr Asn Val Ser Leu Ile Met Ser Asp Thr
435 440 445
Gly Gly Thr Cys Tyr
450
<210> 16
<211> 106
<212> PRT
<213> Artificial
<220>
<223> 人工序列
<400> 16
Ala Asp Ala Ala Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln
1 5 10 15
Leu Thr Ser Gly Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr
20 25 30
Pro Lys Asp Ile Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln
35 40 45
Asn Gly Val Leu Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr
50 55 60
Tyr Ser Met Ser Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg
65 70 75 80
His Asn Ser Tyr Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro
85 90 95
Ile Val Lys Ser Phe Asn Arg Asn Glu Cys
100 105
<210> 17
<211> 576
<212> PRT
<213> Artificial
<220>
<223> 人工序列
<400> 17
Glu Val Leu Leu Gln Gln Ser Gly Pro Glu Leu Val Asn Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Pro Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Asn Met Gly Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile
35 40 45
Gly Ala Ile Ile Pro Asn Asn Gly Gly Thr Ile Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Val Asp Glu Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Ala Tyr Arg Asp Tyr Asp Val Lys Thr Trp Phe Ala Tyr
100 105 110
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Ala Lys Thr Thr Pro
115 120 125
Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser
130 135 140
Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val
145 150 155 160
Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe
165 170 175
Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr
180 185 190
Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala
195 200 205
His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp
210 215 220
Cys Pro Ala Val Ala Glu Met Asn Pro Asn Val Asn Val Phe Val Pro
225 230 235 240
Pro Arg Asp Gly Phe Ser Gly Pro Ala Pro Arg Lys Ser Lys Leu Ile
245 250 255
Cys Glu Ala Thr Asn Phe Thr Pro Lys Pro Ile Thr Val Ser Trp Leu
260 265 270
Lys Asp Gly Lys Leu Val Glu Ser Gly Phe Thr Thr Asp Pro Val Thr
275 280 285
Ile Glu Asn Lys Gly Ser Thr Pro Gln Thr Tyr Lys Val Ile Ser Thr
290 295 300
Leu Thr Ile Ser Glu Ile Asp Trp Leu Asn Leu Asn Val Tyr Thr Cys
305 310 315 320
Arg Val Asp His Arg Gly Leu Thr Phe Leu Lys Asn Val Ser Ser Thr
325 330 335
Cys Ala Ala Ser Pro Ser Thr Asp Ile Leu Thr Phe Thr Ile Pro Pro
340 345 350
Ser Phe Ala Asp Ile Phe Leu Ser Lys Ser Ala Asn Leu Thr Cys Leu
355 360 365
Val Ser Asn Leu Ala Thr Tyr Glu Thr Leu Asn Ile Ser Trp Ala Ser
370 375 380
Gln Ser Gly Glu Pro Leu Glu Thr Lys Ile Lys Ile Met Glu Ser His
385 390 395 400
Pro Asn Gly Thr Phe Ser Ala Lys Gly Val Ala Ser Val Cys Val Glu
405 410 415
Asp Trp Asn Asn Arg Lys Glu Phe Val Cys Thr Val Thr His Arg Asp
420 425 430
Leu Pro Ser Pro Gln Lys Lys Phe Ile Ser Lys Pro Asn Glu Val His
435 440 445
Lys His Pro Pro Ala Val Tyr Leu Leu Pro Pro Ala Arg Glu Gln Leu
450 455 460
Asn Leu Arg Glu Ser Ala Thr Val Thr Cys Leu Val Lys Gly Phe Ser
465 470 475 480
Pro Ala Asp Ile Ser Val Gln Trp Leu Gln Arg Gly Gln Leu Leu Pro
485 490 495
Gln Glu Lys Tyr Val Thr Ser Ala Pro Met Pro Glu Pro Gly Ala Pro
500 505 510
Gly Phe Tyr Phe Thr His Ser Ile Leu Thr Val Thr Glu Glu Glu Trp
515 520 525
Asn Ser Gly Glu Thr Tyr Thr Cys Val Val Gly His Glu Ala Leu Pro
530 535 540
His Leu Val Thr Glu Arg Thr Val Asp Lys Ser Thr Gly Lys Pro Thr
545 550 555 560
Leu Tyr Asn Val Ser Leu Ile Met Ser Asp Thr Gly Gly Thr Cys Tyr
565 570 575
<210> 18
<211> 214
<212> PRT
<213> Artificial
<220>
<223> 人工序列
<400> 18
Asp Leu Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Ser Cys Ser Ala Ser Gln Gly Ile Arg Asn Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile
35 40 45
Tyr Tyr Thr Ser Thr Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Tyr Ser Lys Phe Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala
100 105 110
Pro Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly
115 120 125
Gly Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile
130 135 140
Asn Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu
145 150 155 160
Asn Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser
165 170 175
Ser Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr
180 185 190
Thr Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser
195 200 205
Phe Asn Arg Asn Glu Cys
210
<210> 19
<211> 123
<212> PRT
<213> Artificial
<220>
<223> 人工序列
<400> 19
Glu Val Leu Leu Gln Gln Ser Gly Pro Glu Leu Val Asn Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Pro Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Asn Met Gly Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile
35 40 45
Gly Ala Ile Ile Pro Asn Asn Gly Gly Thr Ile Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Val Asp Glu Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Ala Tyr Arg Asp Tyr Asp Val Lys Thr Trp Phe Ala Tyr
100 105 110
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala
115 120
<210> 20
<211> 108
<212> PRT
<213> Artificial
<220>
<223> 人工序列
<400> 20
Asp Leu Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly
1 5 10 15
Asp Arg Val Thr Ile Ser Cys Ser Ala Ser Gln Gly Ile Arg Asn Tyr
20 25 30
Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile
35 40 45
Tyr Tyr Thr Ser Thr Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Pro
65 70 75 80
Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Tyr Ser Lys Phe Pro Tyr
85 90 95
Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg
100 105
<210> 21
<211> 225
<212> PRT
<213> Artificial
<220>
<223> 人工序列
<400> 21
Glu Val Leu Leu Gln Gln Ser Gly Pro Glu Leu Val Asn Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Pro Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Asn Met Gly Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile
35 40 45
Gly Ala Ile Ile Pro Asn Asn Gly Gly Thr Ile Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Val Asp Glu Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Ala Tyr Arg Asp Tyr Asp Val Lys Thr Trp Phe Ala Tyr
100 105 110
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Ala Lys Thr Thr Pro
115 120 125
Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser
130 135 140
Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val
145 150 155 160
Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe
165 170 175
Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr
180 185 190
Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala
195 200 205
His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp
210 215 220
Cys
225
<210> 22
<211> 351
<212> PRT
<213> Artificial
<220>
<223> 人工序列
<400> 22
Pro Ala Val Ala Glu Met Asn Pro Asn Val Asn Val Phe Val Pro Pro
1 5 10 15
Arg Asp Gly Phe Ser Gly Pro Ala Pro Arg Lys Ser Lys Leu Ile Cys
20 25 30
Glu Ala Thr Asn Phe Thr Pro Lys Pro Ile Thr Val Ser Trp Leu Lys
35 40 45
Asp Gly Lys Leu Val Glu Ser Gly Phe Thr Thr Asp Pro Val Thr Ile
50 55 60
Glu Asn Lys Gly Ser Thr Pro Gln Thr Tyr Lys Val Ile Ser Thr Leu
65 70 75 80
Thr Ile Ser Glu Ile Asp Trp Leu Asn Leu Asn Val Tyr Thr Cys Arg
85 90 95
Val Asp His Arg Gly Leu Thr Phe Leu Lys Asn Val Ser Ser Thr Cys
100 105 110
Ala Ala Ser Pro Ser Thr Asp Ile Leu Thr Phe Thr Ile Pro Pro Ser
115 120 125
Phe Ala Asp Ile Phe Leu Ser Lys Ser Ala Asn Leu Thr Cys Leu Val
130 135 140
Ser Asn Leu Ala Thr Tyr Glu Thr Leu Asn Ile Ser Trp Ala Ser Gln
145 150 155 160
Ser Gly Glu Pro Leu Glu Thr Lys Ile Lys Ile Met Glu Ser His Pro
165 170 175
Asn Gly Thr Phe Ser Ala Lys Gly Val Ala Ser Val Cys Val Glu Asp
180 185 190
Trp Asn Asn Arg Lys Glu Phe Val Cys Thr Val Thr His Arg Asp Leu
195 200 205
Pro Ser Pro Gln Lys Lys Phe Ile Ser Lys Pro Asn Glu Val His Lys
210 215 220
His Pro Pro Ala Val Tyr Leu Leu Pro Pro Ala Arg Glu Gln Leu Asn
225 230 235 240
Leu Arg Glu Ser Ala Thr Val Thr Cys Leu Val Lys Gly Phe Ser Pro
245 250 255
Ala Asp Ile Ser Val Gln Trp Leu Gln Arg Gly Gln Leu Leu Pro Gln
260 265 270
Glu Lys Tyr Val Thr Ser Ala Pro Met Pro Glu Pro Gly Ala Pro Gly
275 280 285
Phe Tyr Phe Thr His Ser Ile Leu Thr Val Thr Glu Glu Glu Trp Asn
290 295 300
Ser Gly Glu Thr Tyr Thr Cys Val Val Gly His Glu Ala Leu Pro His
305 310 315 320
Leu Val Thr Glu Arg Thr Val Asp Lys Ser Thr Gly Lys Pro Thr Leu
325 330 335
Tyr Asn Val Ser Leu Ile Met Ser Asp Thr Gly Gly Thr Cys Tyr
340 345 350
<210> 23
<211> 447
<212> PRT
<213> Artificial
<220>
<223> 人工序列
<400> 23
Glu Val Leu Leu Gln Gln Ser Gly Pro Glu Leu Val Asn Pro Gly Ala
1 5 10 15
Ser Val Lys Ile Pro Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr
20 25 30
Asn Met Gly Trp Val Lys Gln Ser His Gly Lys Ser Leu Glu Trp Ile
35 40 45
Gly Ala Ile Ile Pro Asn Asn Gly Gly Thr Ile Tyr Asn Gln Lys Phe
50 55 60
Lys Gly Lys Ala Thr Leu Thr Val Asp Glu Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Glu Leu Arg Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Glu Ala Tyr Arg Asp Tyr Asp Val Lys Thr Trp Phe Ala Tyr
100 105 110
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ala Ala Lys Thr Thr Pro
115 120 125
Pro Ser Val Tyr Pro Leu Ala Pro Gly Ser Ala Ala Gln Thr Asn Ser
130 135 140
Met Val Thr Leu Gly Cys Leu Val Lys Gly Tyr Phe Pro Glu Pro Val
145 150 155 160
Thr Val Thr Trp Asn Ser Gly Ser Leu Ser Ser Gly Val His Thr Phe
165 170 175
Pro Ala Val Leu Gln Ser Asp Leu Tyr Thr Leu Ser Ser Ser Val Thr
180 185 190
Val Pro Ser Ser Thr Trp Pro Ser Glu Thr Val Thr Cys Asn Val Ala
195 200 205
His Pro Ala Ser Ser Thr Lys Val Asp Lys Lys Ile Val Pro Arg Asp
210 215 220
Cys Gly Cys Lys Pro Cys Ile Cys Thr Val Pro Glu Val Ser Ser Val
225 230 235 240
Phe Ile Phe Pro Pro Lys Pro Lys Asp Val Leu Thr Ile Thr Leu Thr
245 250 255
Pro Lys Val Thr Cys Val Val Val Asp Ile Ser Lys Asp Asp Pro Glu
260 265 270
Val Gln Phe Ser Trp Phe Val Asp Asp Val Glu Val His Thr Ala Gln
275 280 285
Thr Gln Pro Arg Glu Glu Gln Phe Asn Ser Thr Phe Arg Ser Val Ser
290 295 300
Glu Leu Pro Ile Met His Gln Asp Trp Leu Asn Gly Lys Glu Phe Lys
305 310 315 320
Cys Arg Val Asn Ser Ala Ala Phe Pro Ala Pro Ile Glu Lys Thr Ile
325 330 335
Ser Lys Thr Lys Gly Arg Pro Lys Ala Pro Gln Val Tyr Thr Ile Pro
340 345 350
Pro Pro Lys Glu Gln Met Ala Lys Asp Lys Val Ser Leu Thr Cys Met
355 360 365
Ile Thr Asp Phe Phe Pro Glu Asp Ile Thr Val Glu Trp Gln Trp Asn
370 375 380
Gly Gln Pro Ala Glu Asn Tyr Lys Asn Thr Gln Pro Ile Met Asp Thr
385 390 395 400
Asp Gly Ser Tyr Phe Val Tyr Ser Lys Leu Asn Val Gln Lys Ser Asn
405 410 415
Trp Glu Ala Gly Asn Thr Phe Thr Cys Ser Val Leu His Glu Gly Leu
420 425 430
His Asn His His Thr Glu Lys Ser Leu Ser His Ser Pro Gly Lys
435 440 445
Claims (11)
1.一种抗新型冠状病毒或其N蛋白的抗体,其特征在于,所述抗体包括:
(i)氨基酸序列如SEQ ID NO:1-6所示的HCDR1、HCDR2、HCDR3、LCDR1、LCDR2和LCDR3;和
(ii)重链恒定区和轻链恒定区,所述重链恒定区的C端含有IgM抗体重链恒定区的CH3-CH4区氨基酸序列。
2.根据权利要求1所述的抗体,其特征在于,所述抗体还包括:
(iii)氨基酸序列如SEQ ID NO:7-14所示的HFR1、HFR2、HFR3、HFR4、LFR1、LFR2、LFR3和LFR4,或与所述框架区具有至少80%同一性的氨基酸序列。
3.根据权利要求1所述的抗体,其特征在于,所述恒定区的种属来源为牛、马、猪、绵羊、山羊、大鼠、小鼠、狗、猫、兔、驴、鹿、貂、鸡、鸭、鹅或人;
可选地,所述恒定区的种属来源为小鼠;
可选地,所述重链恒定区序列如SEQ ID NO:15所示或与SEQ ID NO:15具有至少80%的同一性;
可选地,所述轻链恒定区序列如SEQ ID NO:16所示或与SEQ ID NO:16具有至少80%的同一性。
4.根据权利要求1所述的抗体,所述抗体以KD≤10-9M的亲和力结合新型冠状病毒或其N蛋白。
5.一种抗新型冠状病毒或其N蛋白的抗体,所述抗体包含氨基酸序列如SEQ ID NO:17所示的重链和氨基酸序列如SEQ ID NO:18所示的轻链。
6.一种抗体偶联物,其特征在于,所述抗体偶联物包括权利要求1-5任一项所述的抗体;
可选地,所述抗体偶联至生物素或其衍生物;
可选地,所述抗体偶联至固相;
可选地,所述抗体偶联至标记物;
可选地,所述标记物选自荧光染料、酶、放射性同位素、化学发光试剂和纳米颗粒类标记物;优选为胶体金。
7.一种检测新型冠状病毒或其N蛋白的试剂或试剂盒,其特征在于,所述试剂或试剂盒包括权利要求1-5任一项所述的抗体或权利要求6所述的抗体偶联物。
8.一种核酸,其特征在于,其编码权利要求1-5任一项所述的抗体。
9.一种载体,其特征在于,其含有权利要求8所述的核酸。
10.一种细胞,其特征在于,其含有权利要求8所述的核酸或权利要求9所述的载体。
11.一种制备如权利要求1-5任一项所述的抗体的方法,其特征在于,其包括:培养权利要求10所述的细胞。
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